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BIO 362
Exam 4 Study Guide
SBU
Spring 2014
Marcu

Exam 4 Review Sheet:

Marcu Lecture 1:

Most of THE REGULATION IS NOT MEDIATED BY ANYTHING ON

THIS SLIDE!!!!!! But you need all of this for Transcription.
Transcription InVIVO can be regulated before any of this binds,
Transcription In VIVO can be regulated at the level of Initiation of
Transcription. Initiation means that RNA Polymerase binds with TF11F,
and you begin to transcribe a gene, but you stop after 30-50 Nucleotides,
polymerase is paused, the next level of control is at that the release of the
Start Site!!!!And another level of Control Elongation!!!!!!!
Some genes are being transcribed, but only lay down 50 Nucleotides, many
genes are like that, What then really turns the Gene on to get real mRNA?
Its NOT initiation of transcription; it is release from the start site and
elongation control. (Many genes that are regulated this way for example in
Cell Growth do it this way, In which genes are already initiated but they cant
really go until you turn on the final switch which release polymerase from
the Start Site and now you get a rapidly elongating polymerase. This
Happens in VIVO, In Vitro this doesnt happen because you dont have
regulation there.
Some things that regulating transcription at the level of DNA BINDING,
TBP binds to the TATA Box

If a gene has TATA, the purpose of TBP and TATA is to give you a very
precise location of transcription at one precise Nucleotide. So many
genes that have TATA generally start transcription at one precise
Nucleotide.
What really TBP and TATA are is this Molecular Rule? This tells RNA
Polymerase to start transcribing at a certain place. Some genes in addition to
TATA also have Initiator Sites; this Initiator Sites binds part of RNA
Polymerase called TF11I.

Some genes have Initiator, and some genes have both Initiator and TATA
box, if you have Initiator Motif and TATA again you have a precise Start
Site.
If you have a gene that has No Initiator or No TATA, you get start site at
multiple places, these have an downstream element that binds important
transcription factors. Sometimes a Transcription factor needed for
Transcription binds these elements downstream of the start site of
Transcription or promoter may extend downstream of the start site.
Then you have genes that dont have any preferred Start Sites and you
typically find these Regions called CpG islands Upstream (these are motifs
of DNA that have C and Gs in them (Multiple Sites, Multiple Transcription
factors can bind them.) Have no TATA or InR.

Also have situation where these CpGs are interspersed between Start Sites
and you have other unique factors binding.
You need a lot of Mechnismhms to explain how you get diversity of
regulation of so many genes in the Mammalian Cell Nucleus.

Heterochromatin: No Initiation almost nothing bound to them.

EuChromatin: Which is transcriptionally active being transcribe by RNA
Polymerase which means you get released from the start site and there no
Nucleosomes at all or very few.
Important aspects of Nucleosomes that are regulated in Transcription are the
modifications of the Histone Tails specially H3, H4 in the nucleosome, and
how that influences the decision to turn a gene on or off. That type of control
is called Epigenetic Control.

Histone H1 IS NOT PART OF THE NUCLEOSOME CORE PARTICLE!!

It binds to DNA or located between Nucleosomes in an AT rich region in
spacer linker DNA.
Some genes have Histone H1 located between Nucleosomes, bound to
Linker DNA, Some genes dont.

Important Slide:

HMGB1 and HMGA1 binds where Histone H1 binds, when they bind
DNA, and if enough of them bind between Nucleosomes in DNA and DNA
in chromosomes, they do something special, the SPECIAL THING THEY
DO IS VERY IMPORTANT FOR ENHANCERS TO COMMUNICATE
WITH PROMOTERS!!!!!!!!
These bend DNA when it bends DNA, it puts a slight kink in the whole
DNA molecule, If you have a whole bunch of Nucleosomes and HmgB1,
thats a lot of kinks what happens is that you start to get loop structure, this
HMGB1 AND HMGA1 is a reason why you can get Long Distance
Communication!!!!

Histone H1 and Nucleosomes dont bend DNA, also when HMGB1 and
HMGA1 bind this they help some transcription factors to bind onto an
adjacent site, it helps because some transcription factors dont bind well to
their motifs on DNA, these are DNA BINDING TRANCRIPTION
FACTORS , these are not like General Transcription factors TF11D etc.
They might be single polypeptides or two where they bind very specifically
to a very specific DNA Sequence and are called DNA Binding
Transcriptional Activators, they are the most diversified way of
transcription regulation, and there are thousands and thousands of them.
One type of Transcriptional Activator called GR(GlucoCorticoid Receptor)
its a receptor that is in the Nucleus and it binds GlucoCorticoid Hormone.
When this Hormone binds Receptor it can be activated. Hormone bound to
receptor and bound to DNA, it can activate the Transcription of the
Glucocortcoid response of genes.
HMGB1 facilitates GR binding, it maybe increase the Km of DNA binding
of GR , GR will stay on DNA longer ,if its near an HMGB1 site. This is not
the only thing HMGB1 does, is acts in the regulation of inflammation.
HMGB1 regulates Chromatin Structure, transcription
Concept: If HMGB1 finds itself outside the cell, cells secrete HMGB1,
some do it more actively. When a cell dies by Necrosis (a cell is basically
aged and things start to leave out of the cell.) When you have tissue damage,
some tissues can become necrotic. HMGB1 is released by Necrotic Cells
and tissues, and also actively released by Immune effector cells like
Macrophages. When HMGB1 is outside the cell it initiates an inflammatory
response, it binds receptors on surface of cells and doesnt work like a
transcription factor at all, instead it works like a Cytokine that is regulating
an inflammatory reaction. It basically says Im a Necrotic tissue and deal
with ME!!!!!
HMGB1 functions like an Alarm signal it is released by necrotic tissue and
that causes cells to migrate to Necrotic tissue, because they migrate toward
HMGB1 and the cells that migrate are immunoeffector cells and also stem
cells that end up repairing the tissue.

The Mediator Complex, Cells use a large protein complex that regulates
transcription initiation and elongation,
Some of the proteins in the mediator complex you also find in TF11D, some
genes that dont have TATA Motif , they get around by using the Mediator
Complex. In experiments in Yeast it was proven that TBP(TATA BINDING
PROTEIN) is not required to sustain life of the cell, it is required for some
aspects of cell growth . But for Life it is not necessary. For a cell to be alive,
dont really need TBP , BUT YOU NEED THE MEDIATOR ,If you get rid
of the Mediator the cell dies.

ALL GENES IN THE NUCLEUS USE THIS MEDIATOR COMPLEX!!!!

The Mediator helps communicate with Polymerase, between the promoters
of transcription, which is near the start site, also communicates with the
activators that are bound to a very distinct enhancer region!!!!!!

You also probably have HMGB1 binding sites, as well as other things that
help bend or loop DNA. You need the Mediator to communicate with things
that are far away, and lets signals to be transmitted to one site to another!!!

Arrows in the figure above are designed to show you that, its almost like
electrical signals; one thing is bound that lets it communicate with
something else.
Enhancers: Have many factors bound to them, many more then
transcription factors that would be bound to a promoter.
Also have HMG proteins in this figure. The Mediator is communicating with
the Activators, also directly communicating with CTD of RNA pol 2, the
Mediator not only communicates with things that initiate Transcription, also
makes circuits with things that regulate transcription by release of start site
and elongation.
The Mediator as a general complex is regulating Initiation, Release from
start site and Elongation. Very Important, Release from start site regulatory
proteins that phosphorylate specific amino acids , protein kinases , the
mediator also regulates Kinases that regulate Cell Cycle. Depending on
phosphorylation of CTD you might have a gene that initiated transcription,
but has not been released from start site , or released and is now elongating.
A lot of regulation is what regulates the CTD of RNA pol 2. That is also
involved on how TF11H binds to polymerase, in order for TF11H to regulate
TF11E properly there has to be appropriate phosphorylation of the CTD.
BOTTON LINE: (Literally and Conceptually)

Is you start with things binding to DNA first, then start recruiting a lot of
other stuff, the other stuff is protein-protein interactions, and once you
recruit enough protein and make these mega complexes, now you need to
activate it, sometimes its activated from the start or you need to activate.
You need a signal to finally initiate, finally release from the start site.
So the Concepts, is first, you have to bind things to the enhancers and
promoters. ,
Interesting Questions:
What regulates who binds first, second is what starts the process and what
turns it on?
Pol 2 ,TF11H and other factors are not the signals they are required but they
are not interpreting the signals and they are not starting the process or
regulating the process.
How Enhancers work?
What are the proteins in the cell that allow an enhancer to communicate with
the promoter, its not just the mediator, the mediator is a communication
device that transfers the signal, what creates the signal of the enhancer and
how that regulates transcription itself.
Marcu Lecture 2:

All genes have promoters for Transcription, this is where RNA POL gets to
the start site, transcription factors are needed to recruit other things and a
number of genes (not all) have Transcriptional Enhancer Regions, can be
thousand or millions of base pairs away, 5 of gene start site, or even within
a genes Intron.
Enhancers: Bind a lot of DNA binding transcriptional activators, MUCH
HIGHER DENSITY OF ACTIVATORS BINDING TO ENHANCERS
THEN TRANSCRIPTION FACTORS BINDING TO PROMOTERS!!!!

Example of an Enhancer that is regulating Metallothioneine, A gene that

responds to a variety of signals coming from the outside of the cell, its a
gene that induces a protein that gets rid of unnessacary metal ions. This is a
stress type response that a number of things can activate. Enhancers has all
of these different Motifs, GRE, E BOX, MRE MOTIFS (These are DNA
Sequences inside the enhancer) around 300 nucleotides, other enhancers
may be very large around 4000 to 5000 nucleotides. So you have these
transcriptional activators that bind to these specific sequences within the
enhancer, Steroid Receptors binding to GRE, MTF1 binding to this MRE
Element. When all these Activators are bound then the Enhancer has full
activity, and then could get appropriate level of activity to the promoter!!!!
AND REMEMBER the Mediator, is in between this Enhancer and promoter
region. So the Mediator is doing communication between the promoter and
enhancer. Enhancers are much more complex then promoters, can also
respond to signals coming from the outside of the cell. Steroid Receptor is
activated by Hormone coming from the outside of the cell; MTF1 is also
activated by Metal Ions that the cell is taking up. Enhancers can
communicate far distances either Upstream or Downstream!!!!

All Enhancers have the same kind of features to them, they just bind
different transcriptional activators, depending on the promoter they are
activating, p50 and p65(members of the NfB family which only bind DNA
in a dimer. HMGB1s that bind to DNA (nucleosomes) they kink DNA and
put a bend to DNA, they are facilitating the formation of this bent structure,
where all other guys are binding and interacting with each other.
Spent a lot of time on this slide:

Experiment that tells you how to find an Enhancer, how do you identify the
active elements in the Enhancer.
What techniques do you use? A reverse genetic experiment, You first
identify enhancer, and want to find out where the important active elements
are, So you have DNA sequences, these are the guys that binding these sites,
Supaposition that different Transcriptional activators bind very specific

sequence, most of the sequences they bind have been identified. So lets say
you look at AP2 site , and see your DNA sequence is just like a AP2 site,
maybe AP2 binds there
How do you know AP2 binding site is important for activity of the
Enhancer? How do you know this octamer site is important for activity of
enhancer ?
Yaxis: Is the percent activity (which means if you mutate sequence to a
different sequence you lose activity, if you mutate a sequence with a high
bar, you lose less activity. These different drops in activity basically mean
that is the part of the enhancer that are important for activity, a number of
these drops overlap these binding sites. So how is this experiment done, so
you take the enhancer you have, and attached it to a reporter gene, a gene
that will report to you the activity of the enhancer? Its not the real gene, but
more like an Assay system, you can attach the enhancer to a gene that emits
light, a luciferase gene, also attach Enhancer to a Beta-Galactosidase gene,
when it binds Beta-gal, it will turn cells into a color. The Luciferase gene is
the BETTER ASSAY. BOTTOM LINE: Is that Luciferase is MORE
SENSITIVE assay. You have Luciferase gene with a promoter (MUST
HAVE) and insert this in a plasmid vector, what you do is take your punitive
enhancer, you wild type sequence and then you have site-directed mutations
and clone it into a plasmid vector that doesnt have the enhancer, can put it
upstream or downstream of promoter of Luciferase, now put this into the
cell, and the way you do it is by Chemical Transpection, which Nicks
DNA with Calcium phosphate, and make a precipitate with DNA, and cells
will take up the Calcium phosphate DNA CO-precipitate, DNA is mixed
with precipitate , cells need it, and then when its get into the Nucleus , it has
some degree of recombinant DNA activity, What happens is DNA at low
frequency , initially its in the circle of the plasmid in the nucleus, and then
sometimes it integrates , it doesnt have to integrate, but the important thing
is it has to get into the nucleus!! , Only genes in the Nucleus will be
transcribed. Dont get transcription outside the nucleus.
Another way to get it in, its you can have a plasmid that is much more
efficient, because in Calcium precipitate some DNA gets into low frequency
%1 or less, but Luciferase assay is very sensitive, so you are good with that.
Another way is to use a plasmid that you can transpect by Calcium
phosphate into a special kind of cell line. What this Cell Line does is it
recognizes some sequences in the plasmid, special plasmid in the cell line,
cell will express RNA from it and RNA will be packed as a Retrovirus and
the Retrovirus buds out of the cell, and the retrovirus (RNA), now you take
tissue culture medium and in the culture medium outside the cell you have

your virus, now you can infect any cell you want with this virus. Any target
cell you want, you infect with this special virus and your special Virus has
the reporter with enhancer, wild type or mutated. This efficiency of infection
can be 100%, every single cell, viral infection much more natural way of
getting sequences inside the cell, and once RNA gets into the cell, it is
converted back to DNA by Reverse Transcriptase an enzyme that converts
RNA to cDNA. That cDNA gets integrated into the genome randomly, and
now you might get expression and cell emits light. If do it with Wild-type
sequence get 100% activity.
How do you know its 100%?
You take the plasmid with No enhancer, just the promoter and then you want
to see what the enhancer did to promoter. Maximal activity difference
between No enhancer and Wild type enhancer, and now you introduce Sitedirected mutations and thats your Data. Everything is done by putting it
back to the cell and so it can integrate into random chromosomes. You are
really looking at activity of the enhancer in the chromosome, you cant do
that InVitro(Not possible)
It is called Reverse Genetics, because you are putting a gene back into the
cell!!! Need a technique that get your DNA into the Nucleus, so you can
test the activity of the enhancer, this is the same way activity of promoters
are defined, also Repression sequences. You can also have a situation, where
you mutate a sequence, and that activity goes up instead of down, that means
its some kind of repression sequence.

One-way in which you can tell if something is a puntitive enhancer!!! Dnase

1 hypersensitive experiment, idea behind this is if a gene is in
Heterochromatin it is decorated with a lot of Nucleosomes with almost no

space in between and therefore (Heterochromatic DNA is

Transcriptionally inactive). If a few Nucleosomes or some have
dissociated maybe the gene is active. Heterochromatic DNA is resistant to
DNase 1, its resistant because the Nucleosome is protecting DNA, you
isolate Cell Nuclei from Cells that expressing your gene of interest, If they
are not expressing it from the start maybe you can add inducer to culture
medium. You now take your Nuclei and incubate it with a small amount of
DNase 1. DNase 1 protein will get into the Nucleus by just passive diffusion
gets into Nucleus, and if a lot of Nucleosomes, there isnt enough DNase 1
to do anything. But if you have some space in between the Nucleosomes
maybe a transcription factor is bound, but this cant protect DNA like the
Nucleosome can. So transcription factors bound instead of Nucleosomes,
DNase 1 will cut the DNA rite there!!!!!!(Double strand break) Then
after you done DNase 1 incubation experiment, you extract DNA from
Nucleus and after extraction, you incubate DNA with Restriction enzyme ,
and you know from the beginning the sequence of your gene, and sequence
around gene and you know a choice of restriction enzyme , you can use this
to cut your gene. The restriction enzyme probably cuts a sequence of 6 or 8
base pairs. You know the sequence of your gene inside and out, after you do
this digest, first DNase 1 with Nuclei, extract DNA then cut DNA and you
then put this DNA on a agarose gel and you electrophoresis the DNA, and
then what you do is cleave with DNase 1, extract DNA, cleave with
Restriction enzyme, what you are trying to do is detect the presence of a
fragment, can detect the presence of a fragment because you can have a
radiolabeled DNA probe because you isolated the gene by molecular cloning
and radiolabeled with p32 and then you do a Southern Blot! In the
Southern Blot you have this band, then you do the experiment again and not
incubate Nuclei with Dnase 1. THE BAND IN THE SOUTHERN BLOT
ONLY APPEARS IF YOU PRETREATED NUCLEI WITH
DNASE1!!!!!!!
You know the Cut site, because you know the DNA sequence of your gene,
if you know the size of the cut, then you know other size of the cut. You can
tell where the NUCLEOSOME FREE REGION IS!!!! So now lets say
you identified this sequence and it is way downstream of the gene, then you
say that if this has anything to do with the activity of your gene, then what
you do is that you have two different types of cells, in one cell your gene its
activated and then in the other cell gene is NOT ACTIVE!!!. So you
compare if the gene is Transcribed or if its NOT. You then find your gene is
transcribed when this Hypersensitive site is present!!!!, then you know that
you have direct correlation with a Nucleosome free region appearing far

away from your gene but only when your gene is active saying this region
can be an Enhancer!!!!! Then you clone this region and do another
experiment and then you can identify regions that make it an active
enhancer. So you need to use Cloning techniques, and use transcription
techniques to get DNAs back in Cells, and you need to use techniques that
will map Dnase 1 Hypersensitive Sites in the genome that could be
Enhancers!!!!

Another Technique that is used, that will tell you WHO is binding to the
Enhancer?
The Luciferase experiment wont tell you who is bound to the sequence, will
only tell you if the sequence is important, but you dont know why it could
be important, it might just be for structural reasons, or maybe not even
bound to the sequence.
Chromatin ImmunoPrecipitation experiment ChIP, so you have a cell
where your gene is on, and a cell where your gene is off and you determine
where enhancers were by other experiments, so you extract DNA, but not
NAKED DNA(eww), you extract chromatin in which everything is bound
to DNA: transcription factors, nucleosomes etc You dont want to disrupt
the chromatin!! But you isolate the chromatin from the Nucleosome. What
you do is you sheered Chromatin with a technique that cause Random
double strand breaks in DNA, one of the best ways to do it is by Sonication,
DNA will break because its a long rigid rod and then break DNA based on
degree of sonication to any random size. This DNA is still associated with
Nucleosomes and whatever was bond to DNA, NOW what you do is you
take this and do an Immunoprecipitation experiment, with a very specific
antibody that only recognizes a specific protein. So lets say you have an
Antibody for AP2(specific for AP2) and you immunopreciptate your

fragment of DNA of AP2 with an Anti-AP2 Antibody. You have now an

Immunoprecipate and have an Anti-AP2 Antibody (Anti Transcription
factor) . (After you isolate the chromatin, youve sheered it , you have to
make sure its stable and things dont dissociate from DNA , so you Crosslink the broken DNA with formaldehyde, Before your Immunopreciptation.
Use formaldyde as a chemical cross-linker reaction that cross-links all the
proteins directly to DNA as they were inside you, it wont change their
locations, it will just fix it there, now you can do your immunoprecipitation,
then you can reverse the cross-links and you can do this by treated it with
light of a certain wavelength that will break the cross link. And now you can
purify the DNA that is in the Immunopreciptate , and you do a detection
experiment with PCR. , You know the sequence of your gene, and the
sequence of your enhancer, so you design two primers for exactly the
sequence you are looking for, then you ask yourself when doing the PCR
DO I RON OR YIGIT see my piece of DNA of the exactly the rite size in
my immunopreciptate and if we do AP4(JUST AN EXAMPLE) was bound
there, it WOULDNT HAVE BEEN IN THE PRECIPTATE UNLESS
AP4 WAS BOUND TO IT, because thats the only way the AntiBody would
of put it in the immunopreciptate, so you extract DNA identify it , so you
need to know the sequence and everything else( You need the sequence of
the gene, need to identify where the enhancer is, need to know activity of
regions in the enhancer. And now you ask what is bound here and because
the sequence that had activity looked like an AP4 site because you know
AP4 binds here that why you choose Anti-Ap4 Antibody, now maybe it
doesnt bind AP4, so then you would choose ANOTHER ANTIBODY that
binds sequences similar to AP4 sites, so basically you can find everyone that
binds to the enhancer regions in a gene!!!
Remember, all of these experiments are done with the Nuclei as they are
found in the cell, you didnt do anything InVitro. These Experiments DNase
1 Hypersensity and the basic Chip gives you the natural activity of the genes
in its normal location, you didnt even take the gene out, it is still in its
Normal Chromosome spot, regulated in Normal way, with all the same
sequences around it. Thats what you use for Hypersensitivity experiment
and thats what you use for Immunopreciptation. You only do reverse
genetics, when you clone a gene and put it back in, only after you know
these other things.

Other important things that are allowing Enhancers to communicate with

promoters, So lets say you have a promoter and enhancer interacting with
themselves (Enhancer is very far away from the promoter, need a way to
loop out DNA, and put it in 3D space the enhancer closer to the promoter, as
if they were next to each other, there are proteins one of them is called a
Cohesion (this binds and create this loop structure)
Cohesins are a class of proteins that allow chromosomes to interact with
each other. This protein is creating these loops but it turns out that this loop
that enhancer is interacting with promoter, its not going to do it all the time,
certainty will do it if the gene is active (being transcribed) might also loop
out if the genes is about to be transcribed (THIS WOULD MEAN
SOMETHING IS MISSING) Whats missing is not the promoter or
enhancer, but they are missing something that allows them to be fully active.
Maybe the Mediator is not there) Maybe specific transcription factor is not
bond there, Maybe its bound but not active and you need a signal from the
outside of the cell (post translation modifier protein) and now can be active.
So theres a lot of mechanisms in which you can turn a gene on!!!!!WE
NOW KNOW THAT IN SOME CASES (ENHANCERS AND
PROMOTERS are present in many genes that are transcriptionally silent,
they are not making mRNA, but the enhancer is still interacting with the
promoter). Maybe RNA POL 2 and mediator are not there generally. You

can prove they are not there by some experimental techniques (Chromatin
Immunopreciptatioin, DNase Hypersensitivity, you can immunopreciptate
the region where enhancer and promoter are interacting by appropriate
Antibodies.
Can ask if gene is on or off? Can ask if its in Euchromatin or
Heterochromatin. Lets say its in Euchromation but the gene is not active
then can ask why is not active, EVEN WHEN IN EUCHROMATIC DNA.
But gene is not being transcribed. Yigits starts to ask whats present and
whats missing ?
You can choose lots of different antibodies in Chromatin
Immunopreciptation experiment, antibodies against Histone H3 Lysine 27
acetylated etc
Chromatin Marks: (Modifications) they mark Chromatin, lets say Lysine
H3 acetylated, methylated, triple Methylated, these are marks of genes that
are almost active, its active in Chromatin, transcriptionally accessible
chromatin, but your gene isnt being transcribed yet, the mRNA is not
made, if these marks are present the gene is almost ready to go probably
missing one signal, being one factor needs to bind or be activated because
the gene is in active Chromatin. These marks tell you the gene is primed to
be transcribed. Even if gene is transcribed, these marks wont disappear.
And if a gene is in Heterochromatin and transcriptionally silent there are
other chromatin marks that tell it that it is in Heterochromatic DNA.
Example: Lets say this specific H3JK27 that is acetylated in Euchromatin
but now is triple Methylated on same Lysine that would now be
Heterochromatin. Can have an Antibody that will see if Lysine is
Methylated or triple methylated. You can use these marks to determine state
of gene.
Epigenetic Control: Chromatin Mediated Control to determine if a gene is
transcriptionally active or almost active state. Almost active mRNA is not
being made yet. Another thing is that you have genes that are
Transcriptionally accessible (its accessible for RNA Pol 2 to get there also
TF11B Mediator Complex etc..) you can use Antibodies against mediator or
so fourth and if they are there or not , and see if thats the reason your genes
is not being transcribed.
Then you ask if the mRNA is not made, DOES THAT MEAN THERE IS
NO TRANSCRIPTION INITIATION? You do an experiment to see if
theres a little RNA made rite on the start site, if a little piece of RNA is
made lets say 50-100 nts but then it stops dead in its tracks, this should
mean that there is a block in the release from the start site or a block in
Elongation and thats why you dont get mRNA. Thats why this gene is

in this accessible state, it mean RNA pol 2 is there, Mediator is there, but
some kind of signal is missing (to get polymerase off the start site) Thats
generally a signal coming from the outside of the cell or inside (Generally
those are post translational modifications) . Enhancers and promoters the
way they interact or when they interact determines the state of activity of a
gene, a gene can be silent, ready to go or being transcribed.

A. Gene is not being transcribed!!!! No marks present

B. Almost being transcribed!!! Marks present
C. Gene is being transcribed everyone is there; Mediator is there, RNA Pol
2 is there Enhancer and promoter are interacting.
This is showing that there are signals coming from the outside of the cell
and they are determine whether marks are present or absent. These
accessibility marks are altering the positions of the Nucleosomes, the
degree at which it grips DNA. These marks tell you that the nucleosome has
either been slightly moved or it is more relaxed. If DNA is free from the
Nucleosomes, the nucleosome is still there, but now you can have things
bind to the DNA. The Nucleosomes are impedance to activity and
transcription. If a nucleosome is bound to a critical positive element, start
site, you have to relax the Nucleosome or get rid of it entirely. These marks
tell you if is relaxed and Hypersensitivity experiment tells you if there is a
Nucleosome there or not.

Defined three different ways, you can consider the activity of an enhancer.
An Enhancer might be active, gene might be active, you are getting mRNA,
the arrow is rite on the start site (Gene is on).
In B the gene is off, here there is no mRNA Here the block is at the level of
initiation!!!!!
In C Gene is also off, but you do get a little something in this one, this
might be regulation at the release from the start site, Initiation has
happened.
Primed Enhancers: Because this does have some marks that say there is a
dissemble pack of chromatin, but it doesnt have polymerase bound, it
doesnt have Mediator, its missing a lot of other things, some nucleosomes
are missing, there are some transcription factors bound to a region where
there is no nucleosome, remember that you are looking at only this enhancer
region in this figure. When gene is primed there is no initiation, no mediator,
but you look at the enhancer and it does have some chromatin marks that tell
you its almost active but not active yet, most of the transcription factors that
bind enhancers are all missing, only one is bound!!! the binding of that one
factor is correlated with these marks being present . We called this one
factor that is bound before all the other ones the Pioneer transcription
factor. Its called the Pioneer because its like discovering a new world, its
saying I got here first and now all you other guys can hop on me. This tells
you how factors get to enhancers, one after the other. By binding Pioneer
Factor, this tells you that there is enough chromatin that is disassembles if
these marks are present.
For poised enhancer, now after pioneer binds you have an enhancer and the
gene is poised, means Pioneer factor has bound and all the other guys are

bound and now enhancer is active and RNA pol ,mediator is here and you
start transcription , get initiation and get a short RNA but it wont be released
from the start site. POISED: Means almost ready to make the mRNA, all
you need is one more signal and then get RNA.
All of these techniques especially ChIP ,DNASE 1 Hypersensitivity and
reverse genetic approach you can define classes of enhancers (totally active,
primed, poised) all of this is done inside the Nucleus , none of these
experiments are InVitro.

Pioneer Factors, Cooperative Binding, pioneer binds other guys bind also
there, also some particular kind of Histones H2A.Z , sometimes Histones
and Nucleosomes exchange with one another Histones!!, this Histone gives
the Nucleosome more relaxed properties , so you might even change
Histones in the nucleosomes. If its H2 A.Z instead of H2A that is more
relaxed.
In Cooperative Binding, the pioneer factor binds, its DNase 1 Hypersensitive
after this happens other factors start to bind, these guys SNI/SNF,
p400,Tip60 they cant bind DNA on their own they are transcriptional
regulators , need to piggy back on something to get to DNA, maybe they
interact with Pioneer Factors or may interact with one of these marks, might
bind to chromatin marks. Generally they get there by binding to a mark and
pioneer factor at the same time, when they are there they will be there for a
certain amount of time. These complexes t are things that remodel
chromatin, in which the Nucleosome will actually be released and that will
make it even more accessible to binding.

We call these factors Chromatin Remodeling factors they cant bind to

DNA directly they have to recruited by some recruiting mechanism.
Transcriptional CoActivators: p400 associates with Histone acetyl
transferase or Tip 60 and so if you recruit this CoActivator that will give
you even more active chromatin marks, directly recruiting something that
gives you more active chromatin marks and more accessibility, you recruit
co-activators, chromatin remodelers, other transcription factors and
finally you get an active gene. Its a recruitment thing, one guy has to get
there and other guy hops on. Side Note: Also can have weak enhancers,
Modest Enhancers, super enhancers!!!!
Marcu Lecture 3:

Enhancers: Is something that enhances the activity of promoters, many

genes have enhancers, Enhancers can determine Cell Identity, you can have
a Muscle cell, epithelial cell, these cells express very specific genes that
define their functions and fate.
So Every time you have a specific cell type, which over expresses particular
proteins, and define that differentiated cell type the reason is that gene is
regulated by a Super Enhancer. The enhancer is so powerful that in the
absence of the enhancer, the promoter essentially has no activity at all or
very little , the difference being several orders of magnitudes. If you use
PCR and you look at expression of B-Hemoglobin fibroblast, if look for
mRNA in fibroblast and you use the most powerful PCR , you can see BHemoglobin mRNA , this means that at most you have one or two molecules
of mRNA per cell . In Red blood cell that is making B-Hemoglobin protein

that expresses lots of mRNA , you have more then one thousand times that
amount , so thats why we call it a Super Enhancer( its not just enhancing
the promoter above Basal level, Enhancers can drive expression of Genes
thousand of times above the Normal level, the reason why is because there is
so many transcription factors bound to that enhancer and you get tremendous
amount of communication between Enhancer and promoter and make a heck
amount of mRNA. We know that in certain cancers this is part of reason for
cancer, there is a cancer-causing gene in some cancers, which is
tremendously overexpressed because it is turned on by a Super Enhancer. So
called Oncogenes, which encodes for an oncoprotien that alters cell growth
control, its generally always a normal protein, normal mRNA, cells are just
making too much of it, and as a consequence over expression deregulates
cell growth control and that will contribute to cancer. Some Super Enhancers
do this. Super Enhancers are normally in DNA they are NOT mutations.
Sometimes chromosomes can break and rejoin, sometimes this break and
rejoin process is abnormal in cancer cells, that is what is happening in
oncogenes. Other cases like the B-Globin genes, T-Cell genes, these super
enhancers that determine cell identity are not mutations . In the cell specific
lineages that determine identity, you are missing expression of certain key
transcription factors and this key transcription factor that binds DNA is
critical for activity of the enhancer, sometimes you may also be missing a
Transcriptional Co-Activator, where transcription factors that binds to
enhancer, needs a Co-Activator to bind it to communicate with the Mediator.
Generally you are dealing with the presence or absence of a protein, a
critical protein is missing and a differentiated cell expresses that protein but
other cells dont. Once certain proteins are expressed an Enhancer can
become a Super-Enhancer!!!!

The B-Globin case is very interesting, a specialized class of enhancers called

Locus Control Region. Some Loci in cells are occupied by a single gene, A
locus may have more then one gene, that locus may define the identity of a
cell, and then many genes are Co-Regulated,(important) if you need
expression of more than one gene, more than one protein, its easier for the
cell to coregulate at the same time, turn them on all at the same time. To turn
them on to the same degree, all at the same time, easiest way is to have a
Locus Control Region Enhancers (it means there are Enhancers in specific
chromosomes location , when they turn on, they turn on a whole Locus with
multiple genes in them, that means that more then one promoter is
responsive to that enhancer, each mRNA in the locus has its own promoter,
with its own transcription factors binding to it, and the enhancer turns on all
those promoters simultaneously!!!!! The Human B-Globin gene cluster is
regulated by an LCR. Human B-Globin gene locus has one unique aspect
that the LCR helps it regulate. During Fetal Life, you dont express BGlobin, you express the gamma form, so what happens is that in fetal life it
is the same enhancer, and what the enhancer does is that specific
transcription factors are associated with it that are only expressed in certain
stages of development, before you are born that enhancer will only now only
turn on those genes, it wont turn on the B gene, after you are born the fetal
globins go away, those proteins arent expressed anymore, now lets say the
B-Globin is expressed under the same enhancer, but regulated by
DIFFERENT TRANSCRIPTION FACTORS!!!!!!! So the enhancer not only
determines the Cell Identity, but they can also change their mind on how
they are going to work in development which is also something an enhancer
can do, no ones has ever seen promoters do things like that. You can see in
the picture the LCR enhancer which is defined by many Dnase 1
Hypersensitivity sites, discovered by Dnase 1 Hypersensivity analysis with

respect to most of the genes, its about 30 to 40 kbases away, the enhancer is
pretty big (20,000 bps long) the enhancer is spanning a large piece of DNA,
so transcription factors are binding all over that, its a very complicated
enhancer, the reasons its so big, because some things that bind are regulated
different promoters, the control is determine by the presence or absence of
factors that bind to these different promoters.

Next important thing to understand here is how is the enhancer regulating

transcription. Karzai give diagram with TF11B,TF11D,TF11F with RNA
pol 2 and told you about the Pre-Initiation complex then he said there is a
communication between the mediator and TF11H and then pre-Initiation
goes to Initiation and now you make this short little RNA and then you
release from the start site and now polymerase can elongate , there is levels
of control at the level of initiation and level of control at release from the
start site. So how is the enhancer of the B-Globin gene locus regulating these
promoters? Is it regulating it at the level of initiation, release from the start
site or elongation? DONT ASSUME THE ENHANCER IS REGULATING
THE PROMOTER AT THE LEVEL OF INITIATION Thats a bad
assumption. So in the diagram you have initial state where LCR is NOT
functional, so polymerase isnt there and no one has bound anything, then
you have PARTIAL LCR DEPENDENT state, some factors are bound
and as a consequence enhancer is engaged now get formation of Initiation
Complex with polymerase rite on the start site, and what happens is you
make some RNA but its short RNA, not full RNA.
LCR DEPEDNET: Fully dependent as a consequence, instead of initiation
complex , you now get phosphorylation of the CTD in POL 2 the reason

why you get phosphorylation, because the LCR binds a factor that wasnt
binding before and the name of that factor is NF-E2.
NF-E2 engages another protein which is a Kinase ( and one of its targets is
the CTD in polymerase , the kinase is part of a post translational mechanism.
Phosphorylation of the CTD kicks the polymerase off the start site.
What the Enhancer did when it was fully LCR Dependent is that its
regulating transcription by releasing polymerase from the start site, NOT by
regulating Initiation. You have to realize its not just proteins binding to
enhancer, it has to do with other proteins they recruit and those proteins they
recruit which couldnt bind DNA will modify the transcriptional apparatus
in a post translational way( Post translational Control or modifications of
proteins can turn a gene on or turn a gene off without binding anything, not a
DNA binding mechanism. But if NF-E2 werent bound to the site in the
enhancer, this wouldnt have happened. NF-E2 had to be there, this is the
guy that flags and says COME HERE KINASE bind to me and mediator is
also helping, and tells the Mediator to help us engage TF11H, as well as
other proteins are required for release from the start site. If you take away
one thing, the Mediator is not there,TF11H , NFE2 is not there, none of this
can happen. So essentially you are assembling these large complex of
proteins that communicate with each other and transfer this phosphorylation
signal, remember there isnt Nucleosomes in the way, the Chromatin
Remodeling complex was recruited before this happen, and just because
DNA is in Euchromatic state , it might be POISED, it could just be ready to
go, but you are missing this phosphorylation , but its still in active chromatin
Active Chromatin: Not a lot of Nucleosomes, RNA Pol ,a lot of guys here
but the gene isnt on yet.

Why is the enhancer selective for the gene its driving? Many enhancers can
up regulate different promoters, but InVivo that doesnt happen, part of the
reason is that chromosomes are built in a certain way, that they have these

brick wall stop signs in them, these brick wall stop signs are called
INSULATORS. Lets says you have Locus A then brick wall then Locus B
and the enhancer of Locus A cannot go across the insulator brick wall to
activate the other Lucus!!!!! Insulators protect Loci from Inappropriate
activation by the wrong enhancer, Insulators are DNA, loaded with
Nucleosomes, its Heterochromatic basically its very specialized loop
structures that form in chromosomes , you have a loop that allows enhancer
to communicate with promoters, then other types of loops in-between the
loci, and the loops lock it in the conformation in which the enhancer cant
communicate with another promoter!!!!!
There are loops that are activating loops that allow enhancers to
communicate and loops that are brick walls that prevent enhancer
communication. Research has asked if the proteins that create loops of
insulator are the same proteins that communicate with promoters? IN SOME
CASES YES, but may be functioning in a different way. Basically enhancers
only regulate specific loci , because Insulators which are loaded with
Nucleosomes create loops in DNA that block activity of an enhancer , and
prevent it from driving another locus.

These two slides show you cases when a gene is on, off, also situation where
gene is on in one direction and off in the other direction. Also a lock and
key situation, in which the enhancer is the key and the enhancer opens a
lock. If the lock is open the gene is on, if the key cant open the lock
MAYBE AN INSULATOR IS GUARDING IT. At the bottom of the slide
you have a key that fits very well into the first lock and get RNA but then

you get no RNA in the second lock, there is this CTCF protein which is
transcription factor that binds DNA, has a lot of Zinc Fingers and its believe
that this protein is one of the genes that allow Insulators to form!!!!
First enhancer in picture is working because of proximity, the second
enhancer is working because of compatibility( means that the factors that are
bound to the enhancer and the factors that are bound to this promoter are
incompatible for the first lock in the picture above, they cant interact and
the Mediator wont help, no proper communication between the mediator and
the rest of the complex, but the enhancer can bind perfectly with the second
promoter in the picture. So this is the Compatibility situation
So these are the various situations that you can find in the Mammalian
genome.

Some of these proteins that allow loops to form, Chromatin loop: which
allow promoters and enhancers to communicate, stabilized by a protein that
makes a barrel like structure (belong to a family of proteins called Cohesins,
these are the same family of proteins that allow Chromosomes to
interact with each other in Cell division and Meiosis, so these proteins
also facilitate appropriate interactions between chromosomes . Many of
these proteins are involved in these large structures that tie DNA together so
to speak, so its stabilized. Another protein CTCF sometimes help cohesin
do this. CTCF not just involved in Insulation, also involved in helping
Cohesin stabilize these loops.

Specific types of transcriptional activators, what kind of DNA binding

domains they have and on basics of these binding domains and activation
domains how they function differently from each other!!! There many
different types of Transcriptional activators encoded by the genome,
probably most of your genome, largest part has to do with the existence of
THOUSANDS OF TRANSCRIPTIONAL ACTIVATORS!!!!!
Example: One class of transcriptional activator alone, one that has a Zinc
finger DNA BINDING DOMAIN, there is at least a thousand of them
encoded in the genome, and thats just the one that has a Zinc Finger DNA
binding domain. Other types of binding domains, one called a
Homeodomain, Leucine Zipper domain, Helix-loop-Helix domain.
Concept here is that Transcriptional Activators give you diversity in gene
control, mainly because they have unique DNA BINDING DOMAINS!
THAT BIND unique DNA SEQEUNCES. There is a variety of different
DNA binding domains and each one constitutes a different family of
transcription factors. Leucine Zipper family, Zinc finger family etc.. and
each of these guys will bind a slightly different DNA Sequence inside the
family. In order for DNA binding to be tight, to have the highest possible
Km there is a core sequence of about 6 to 8 bases generally, DNA binding
site of a transcriptional activator is about 12 to 14 bases and the center is
usually important its making the contacts with specific amino acids in the
binding domain of the transcription factor. If you slightly alter the
nucleotides in that 12 base pair sequence , you might find a different
member of that family. Thats why when he showed the slide in determining
where the active DNA sequences are in the enhancer and then said ok this is

an active sequence because if you mutate it the enhancer doesnt work. Then
you do a Chromatin Immunopreciptation to see what binds there, just
because the DNA sequence is known lets say to bind the AP4 transcription
factor, there is an AP4alpha ,AP4 beta, etc. basically a family and you
dont know which one of these it is unless you have an Antibody in the
Immunoprecipatation that is going to distinguish them from each other. Its
that complicated. These guys are all in familys and are all binding to
slightly different sequences. The DNA binding domain is separate and is
functionally distant and works like a module , and the protein as a
transcriptional activator is modular because it has an activation domain,
and other flexible domains that separate them. What the slide is illustrating
is that there is a DNA binding domain in the transcriptional activator they all
have one!!!! if its a DNA binding transcriptional activator , this is not true
for TF11B,TF11F , not talking about general transcription factors , he is
talking about the DNA Binding ones in this slide. In addition all
transcriptional activators that bind DNA, if they are going to activate
transcription they need an activation domain, this domain is separate and
distinct from the DNA binding domain. The activation domain is the
domain that is maybe going to communicate with the mediator, its a domain
that might recruit a Histone acetylase, something that might modify
chromatin, and something that might recruit a Chromatin remodeling
complex, its something that allows the big complex of transcription to
assemble, and the DNA binding domain says I am bound here and my
activation domain is like a flag , my flag can recruit a remodeling complex.
Then there is another transcription factor bound to the enhancer nearby it has
an activation domain but its function is not to recruit remodeling complex its
let say to recruit the Histone Acetylase. GET THE IDEA, that these things
are all bound to specific sequences, the activation domains have different
functions in transcription by recruiting different proteins. Sometimes the
activation domains are recruiting the same proteins but they just enhance the
recruitment of that protein. SO THESE ACTIVATION DOMAINS ARE
CRITICAL!!!!!!!!! Lets say you take SP1s DNA BINDING DOMAIN and
cut it out by restriction enzymes and then if you take GAL 4S DNA binding
domain and fuse them together, guess what you have done to GAL 4, you
now made GAL4 transcription factor bind to SP1 DNA binding sites, they
are interchange , they are really modular , you can make a MONSTER
transcription factor that doesnt exist in nature , its something that basically
binds to the wrong sequence and activates somebody else, sometimes that
happens in cancer, basically have chromosome translocations. Where a
chromosome breaks and then another chromosome breaks then the

chromosomes cross fuse with each other and you get a hybrid chromosome.
What they have done is that where the fusions occur you have put the
activation domain of one transcription factor attached to the DNA binding
domain of another one. And you created a new transcription factor that
would never exist in nature and that contributes to cancer!!! This can all
happen with chromosome abnormal breakages in cancer cells. MYC
oncogene was discovered in chromosomes translocations.

Here the Zinc fingers are form by alpha helices, and rite at the tip of the
fingers are the amino acids that are binding to the DNA, you have a
conformation in the proteins DNA binding domain and rite at the tip of the
fingers you have some basic charged amino acids!!! Basic charged amino
acids are very important to bind to DNA because DNA has a lot of negative
phosphopdiester backbone. This is one type of DNA binding domain but
there many other types.

The Leucine Zipper domain, transcription factors only work as dimers!!!!

Here you have an activator that will either dimerize with itself, so it makes a

Homodimer, or a Hetero dimer (with another protein). So you either have

Homodimers or Heterodimers and sometimes you get proteins that are
ACDC proteins (they dont know which school they belong to). So a
Homodimer looks like the following Leucine Zipper domain that holds the
proteins together, because Leucine Zippers contact each other , the zipper is
a periodic row of leucines , every seventh residue in the protein is a Leucine
and this Leucine sticks out and then the Leucine interacts with a Leucine
through Hydrophobic interactions that exclude water , hydrophobic
interaction that stabilizes the structure, the zipper constitutes the DNA
binding domain, need some basic charged amino acids from both proteins
and then they bind to DNA., if theres only one protein that is not stable
binding to DNA, it dissociates it cant stably bind , you need two of these
proteins to stably bind. Then the Leucine Zipper INDIRECTLY creates the
DNA binding domain, the DNA binding domain is from both proteins
together ,sometimes what these guys do that have these Leucine Zippers if
they homodimerize they bind to a specific sequence , if they want to
Heterodimerize ( not faithful to themselves ;) ) theyll go to another protein
and bind a different sequence, and sometimes they bind exactly the same
sequence , it depends on the transcription factor and how they bind DNA
what the amino acids are . Sometimes these guys give you more diversity in
control in the following way, remember that these factors are in familys, in
regard to the specificity in binding to DNA. Sometimes you find in these
Leucine Zipper proteins that are very similar, except there is a major
difference between them, one of them has an activation domain and the other
one doesnt but they are Lecine Zipper domains and are allowed to dimerize,
you can bind DNA because they have the DNA binding domains but the
activation domain is somewhere else in the protein, one of the proteins has
an activation domain and the other one doesnt. If you dont use the other
protein with the activation domain when you make the zipper , if you dont
use it you cant recruit anyone else, if it was just the protein without the
activation domain it will bind DNA perfectly fine , it will bind the same
sequence as the Heterodimer EXCEPT there is no activation domain at all
and it wont recruit anything . If you have situation where this Leucine
Zipper is a Heterodimer one protein has an activation domain and the other
one doesnt it binds exactly the same DNA sequence but now you CAN
recruit something, because one of them has the activation domain. This
means that depending on who you dimerize to, you can short circuit
transcription or activate transcription. HE SAID TO CHOOSE
CAREFULLY WHO YOU MATE WITH IN REAL LIFE because if you
choose the wrong one it might short-circuit your life like the leucine zipper.

This protein CBP(CREB BINDING PROTEIN) , its very similar to

another protein p300 which is on another chromosome. CBP and p300
belong to a class of proteins called Transcriptional Co-Activators, very
special class of proteins, which are extremely large. P300 molecular weight
means its 300,000 daltons (BIG PROTEIN). Like a landing pad for many
things binding at the same time. One thing these proteins cant do is they
cant bind DNA. They are recruited by proteins that bind to DNA
transcriptional activators, enhancer of the B-interferon gene, you have the
activators that are bound to the B-interferone gene enhancer in the picture
above. Each of these activators has a binding site in this activation domain
that helps recruits this CO-ACTIVATOR!!! What this co-activator does for
transcription is many things at the same time. Many of these guys are needed
to help recruit it, one of them is not enough, each of them has a binding site
in this big protein that is separate and distinct in the protein and the protein
is more assured that it should be there . This protein another domain it has is
Histone acetlytransferse, what it will do is that it will acetylate histones in
nearby nucleosomes and relax the structure of the nucleosomes. After those
Histones get acetylated, this result in recruitment of other proteins that
remodel the nucleosome that dissociates it or relocate it. Its a stepwise
process in transcription dealing with genes that are in Chromatin!!!! Or
Nucleosomes. You need co-activators!!!! To alter Nucleosome structure
and tell transcription to proceed. You also need them to communicate with
the Mediator and communicate with Polymerase!!!! See that this coactivator has another domain that makes direct communication with the
general transcription factors associated with RNA Polymerase 2. This shows
you how subtle these linkages are made to create this apparatus, how does
the enhancer communicate, it communicates not just by Mediator but by also
these Co-Activators!!!!!. You call them Co-Activators because they dont

bind DNA but they coactivate and associated with the activators!!! If the
activator doesnt have an activation domain, you cant recruit this!!!!!

A special kind of protein called CREB is mediated by a transcripitional

activator, its a leuncine zipper , this leucine zipper protein binds to a
specific sequence in DNA , will bind DNA perfectly fine as a dimer. In
order for CREB to activate transcription it has to recruit CREB binding
protein co-activator (CBP) BIG GUY IN PREVIOUS SLIDE, in order to do
this you have to modify DNA bound CREB post translationally , you need
to phosphorylate this Serine !!!! , if serine is not phosphorylated this protein
cant even recruit CREB binding protein(CBP) , to phosphorylate that Serine
you need to activate a Kinase called PKA. Which is regulated by cAMP ,
this gives you an idea on how a signal coming from the outside of the cell
activates a membrane bound kinase that sets up a signal transduction
pathway that allows the signal from the outside of the cell via this pathway
through the cytoplasm to communicate with a protein bound to DNA in the
nucleus to modify this serine to activate transcription. You can then activate
transcription totally by a Posttranslational mechanism, it can be bound to
DNA
but you dont transcribe something because you are missing a
modification. This is a very important principle because this means you can
activate the transcription of some genes with a signal independent of any
new protein synthesis. You dont need to make another protein , its posttranslational modification and the cell is likes a ready to go kick but its
missing the signal to turn the gene on. Mechanishm in which you have

CREB bound to them, but the CREB is inactivated, and then posttranslationally modify it so its active, now it binds CBP and now you can
bind the rest of the machinery. One example on how a signal can turn genes
on independent of new protein synthesis by specialized aspects of
transcriptional control.
In Lecture 4 tell you about Histone code
MARCU LECTURE 4:
Ended with large protein complexes, that form as a result of enhancer,
promoter interactions, the transcription factors that bind to the enhancers are
many more then the ones that bind to the promoter. These factors bind by
specific DNA binding domains and they are in families, different
transcription factors, different binding domains that recognize different
DNA sequences. Then talk about the activation domains of the DNA bound
transcriptional activators then go about recruiting the rest of the transcription
machinery. Initially what they need to recruit, if the gene was in a primed
state, heterochromatic state,
Primed: No mRNA being made yet, not even a short RNA.
Poised: Next level, where you get the short mRNA made
Then gene gets turn on by releasing from the start site.
We have a situation where you are in this primed state, that means RNA pol
2 is not there, mediator is not there, none of the general machinery is there
but the activators are bound to the enhancers, bound to the promoters, who
do they recruit to the ball rolling, Obviously this means that if activators are
bound to the enhancers it means that before all those activators got there,
there must have been a pioneer factor that got there first. What they are
going to recruit is two kinds of transcription factors, these types of factors
they are going to recruit they are going to recruit by interacting with their
activation domains. The activation domains being a modular unit, inside
DNA bound transcriptional activators, one of the first things they recruit
is a large Co-Activator Complex, there are several genes that encode these
large Co-Activators one of them he told us was CBP/p300 told us that they
are very large proteins that have landing pads for the activation domains of
many transcription factors!!!! Why do you need transcriptional CoActivator? Whats the purpose? Why do you need to recruit this co-activator
before you go into Poised State and then the active state?

All these Activators bound to the enhancer, by recruiting these mega CoActivators complexes., there is different genes encoded it there is the CBP
gene, p300 which is homologous to the CBP but it is in a different gene, and
there is another one called p400 . These are extremely large proteins with
different domains in them with different functions, one of the functions that
some of the domain have is to interact with activators that are bound to the
enhancer and to interact with the mediator, one of this Co-Activators major
function is that its a MOLECULAR BRIDGE!!!! TRANSMIT SIGNALS
FROM THE ENHANCER TO THE MEDIATOR!!!! Remember the
enhancer could be very far away from the promoter, and you need to remove
nucleosomes and have a few in between and you need to loop that DNA so
you can get all these things in proximity, This protein in 3D Space is
extremely close to the Mediator, might only be an Angstrom away or portion
of an angstrom. In addition to provided connections it has other functions as
well. If other proteins piggy back along , these proteins are important for
transcriptional control also, they cant bind DNA and cant interact with the
activation domains of these other guys , but they are piggy backing on this
co-activator . And remember the co-activator cant bind DNA either, the only
way to get in here is to have these activators binds first, one of these guys
piggy backing is a Histone Acetyl transferees and what it will do is it will
acetylate Histones in nearby nucleosomes that are still there, and the
acetylations are occurring largely in Histone H3, the amino terminal tail of
Histone H3 extends outside Nucleosome barrier, what you are doing by
acetylating is you are altering the structure of the Nucleosome in a relaxation
type way , by acetylations what you are doing is that you are allowing the

grip of the nucleosomes on the DNA to be relaxed. And what you are
acetylated are LYSINES, SO when you acetylate a Lysine on a Histone, you
remove its positive charge ,if you remove positive charges from Histones
then they will find it more difficult to interact with the DNA which its
phosphodiester backbone that has a lot of negative charges!!!! And so the
Histones are then slightly repelled from the DNA, just enough that it relaxes
a bit. Now that it relaxes a bit, you need to remove the nucleosomes out of
the way, if you want to really begin transcription, the nucleosomes you need
to move out of the way are the ones that may be imparting on lets say
Mediator Function, or Nucleosomes that impacting RNA polymerase at the
Start site. Dont forget that you havent made a little RNA yet so you
havent even initiated transcription and one reason that may be true is that
you are in the chromosome and you still have nucleosomes in these regions
that you have to deal with, so you are relaxing these nucleosomes by
acetylations but now you to move them out of the way, now you recruit
another kind of complex called ATP DEPENDENT CHROMATIN
REMODELING COMPLEX you need ATP you need energy to drive the
function of this complex, it has multiple proteins in it , the way this is
recruited is that some of these guys that are bound to the enhancer in
addition to recruiting the co-activator(CBP) they can also facilitate the
recruitment of the ATP CHROMATIN REMODEDLING COMPLEX
They have other domains in their activation region that can help to recruit
ATP Chromatin remodeling complex. This protein CBP also has an
interaction with this Chromatin remodeling complex. And there is a third
way this is recruited, remember he said that the Histones are acetylated,
when you acetylated a Histone that provides new binding domains for
special kinds of proteins, acetlayed histones will bind to proteins with a so
call bromodomain, proteins that have a bromodomain , bromodomains
bind to acetylated histones , one of the proteins in the remodeling complex
has a bromodomain,you do things in a stepwise manner , well this is the
stepwise process that happens InVivo!!!! You need to relax the
Nucleosomes, move them out of the way, want to remove them out of the
way you recruit this ATP Chromatin remodeling Complex!!! This
remodeling complex literally with the help of ATP, the DNA unwinds
around the Nucleosomes and dissociate and now these Nucleosomes are free
from this region, now TF11D will be there if a TATA , maybe TF11 I will
bind the Initiator, polymerase will get there and now you go from PreInitiation to Initiation and now make short RNA, and then you have to
release from the start site and other proteins are involved in release from the
start site. Show in a stepwise manner on how modifications of Histones

drive this process. Histone Code depending on the type of mark you had on
them, whether its an acetylation, methylation, monomethylation, double
methylation etc and it depends on which Lysine is Methylated involves
mainly Histone H3 and H4, as a consequences of these modification
increasing in number , also phosphorylation of Histones as well
You begin to have a progressive effect on transcriptional control, you go
through this process of binding one thing, interacting with another thing,
bringing in the remodeling complex, bringing in the mediator, releasing
from the start site, it depends very much on the types of modifications of
Histones and it occurs in a stepwise manner. Stepwise Manner means that
the first acetylation you get , and if you want to methylate a specific residue
involved in active transcription the acetylation has to be there FIRST , or the
methylation wont happen, want to phosphorylate Histones specific site the
acetylation and methylation BOTH need to be there and then the Kinase can
phosphorylate , the enzymes that are doing this are literally reading a
Histone code. Some of these enzymes read the code and others dont read
the code.

You recruit a pioneer factor, you get the ball rolling, got to have the
remodeling complex, and you dissociate and expose Nucleosomes. You can

see that this ATP chromatin remodeler has all these proteins in it.

Another summary slide, that shows you what can happens after you recruit
the remodeling complex, after you invoke the remodeling complex, DNA
binding protein , which was block to bind to these Nucleosomes , now it can
bind because you moved the nucleosomes out of the way , this is one
possibility but this is just repositioning (still same amount of Nucleosomes)
, second possibility you actually lost a nucleosome as a result of the
remodeling complex , third case you have unwrapped DNA around the
Nucleosome to allow the protein to see the sequence , sequence of DNA that
was inside the Nucleosome is now visible , you can also exchange different
types of Nucleosomes , like Histone Variants H2A.Z, basically a number of
things that can happen as a consequence of remodeling chromatin, so its
very critical to do that.

Remind us that all this STARTS WITH THE PIONEER FACTOR binding
to probably some place in the Enhancer also can be in the promoter BUT
YOU NEED ONE SINGLE FACTOR TO BIND FIRST. Thats the big
mystery in gene control if you start with Heterochromatin how does the first
factor ever get there, if the Nucleosomes are blocking everything, there must
be something that happens that allows the first factor to get in there, if its not
in there all the time. Pioneer factor as a starting process.

Here is the Histone Code, so you have an active chromatin nucleosome, and
inactive chromatin nucleosome. If the nucleosome is still there obviously the

Remodeling complex hasnt got here and removed the nucleosome. In the
slide this is before the remodling complex has done its job. It shows
situation where remodeling complex hasnt got here. You have the tails
Histone H3 and H4 , you can see that if its active chromatin specific
Lysines are acetylated , in inactive chromatin those same Lysines are
methylated be careful of the kind of methylations , also note the different
residues involved Lysine 4 is important, Lysine 9 , Serine 10 .Lysine 14 in
Histone H3. Also Lysine 27
In other Histone H4 Lysine 5, Lysine 12
You are basically dealing with modifications of different Lysines in
different positions in a Histones tails.
Histone Acetyl Transferase takes Acetyl CoA as a substrate, takes the
Lysine of Histone and with Acetyl CoA it acetylates the Lysine. 23:16

This shows Histone 3,some of the Lysine residues that being modified, here
is Histone H4 ,color code tells you if its a Arginine or Lysine and whether if
its acetylated or methylated, also phosphorylations.

What this slide is designed to show you, IF YOU WANT THE TAKE
HOME MESSAGE: Depending on the modification present the key on the
left tells you the consequences of some of these modifications on gene
activity , you can see the modifications , you can see which residue it is lets
say Histone H3 , you have a certain modification and maybe the gene is
being expressed , other modifications may have Histones deposited in the
Nucleosomes, you have another modification that is a methylation it may be
involved in Gene Silencing , and gene is in Heterochromatin. Text box
shows some of these enzymes that are responsible for these modifications
and then their reading proteins that are in a complex with the actual enzyme
that does the process. Sometimes the same protein is an enzyme and a
reader, A reader means it sees the site, it doesnt mean that if it recognizes it,
its going to change it. You can have two proteins involved, one that binds
the site as the reader and another complex that actually does the
modifications, or two things built into an additional modification protein.

What this slide is designed to show you is that as a consequence of these

modifications , how does that help to recruit other proteins that are involved
in transcriptional control , how does it interpret these Histones codes ,
mechanistically how does these proteins use this Histone code that are
involved in transcription when these histones are modified. First toward
upper left corner you have an Acetylation that has happened, and as a
consequence of that acetylation, a protein with a BROMODOMAIN can
now be recruited because it binds the Acetylated Histone, where it cant bind
if the Histone wasnt Acetylated. Other side you have a Methylation instead
of a Acetylation, now you recruit a different type of protein called a
Chromodomain proteins, chromo domain binds a methylated histone, it
wont bind the acetylated one. It turns out that proteins with Bromodomains
are actively involved in putting Nucleosomes in different locations and
pushing them out of the way or maybe dissociating them if there is enough
energy and time to do that. Then there is another kind of remodeling
complex (DONT THINK REMODELING IS ONLY A POSTIVE THING
FOR TRANSCRIPTION), How do the nucleosomes get into the
Heterochromation in the first place? You also have to understand how an
active gene goes into Heterochromatin if you want to turn it off; there also a
reverse process of turning them off and putting the Nucleosomes back on.
Those particular Nucleosome remodelers will have a protein in them with a
Chromodomain, but the ones that displace the Nucleosomes wont have a

chromodomain!!!!!! The Chromodomain is going to bind a specific

methylated residue and that methylation mark, is going to bind a
chromadomain and a particular remodeling complex and a consequence of
that remodeling binding to the chromadomain , that REMODLER PUTS
NUCLEOSOME ON!!!! It doesnt take it off and thats why you may end up
in Heterochromatin!!!!! Or gene silencing if you modify that Histone with a
Methylation. So thats the Mechanism in general terms. The DNA is in the
Genome, you just need the Histones that created by these Histone octamers ,
the Histone associate with each other and as a complex they bind to the
DNA and the Nucleosomes forms , the Histone proteins are made in the
Cytoplasm.

So Bromodomain stabilizes Open chromatin, Chromadomain stabilizes

closed chromatin

A very specific example, the Beta-Interferon gene enhancer, you have

activators bound to enhancer, remember he said these activators can recruit
CBP or p300(Co-Activator) , he didnt tell us that before they bind
CBP/p300 they bind to this guy GCN5 first , GCN5 is a Histone
Acetyltransferase(HAT) , what happens is that this acetyltransferase already
starts the acetylation of the Histones, starts it going , you have a few
acetylations but not many .

Here you can see, exactly where the Lysines is acetylated , and you can see
that this Nucleosome is sitting rite on the start site , you have to relax that
nucleosome on the start site , or you are never to going to recruit the
machinery RNA Pol 2 , Mediator and so fourth.
And now as a consequence of the modifications of the Histones caused by

this Acetylase so this is a HAT (Histone Acetlytransferse), now this

Histone H3 at serine 10 is subject to be phosphorylated by a Kinase, but it
cant phosphorylate unless Histone H3 was previously acetylated by GCN5,
because other wise the Kinase cant bind , it cant be recruited , theres
something that changes the conformation and now the protein can bind and
the kinase phosphorylates , you can see the phosphorylation in Serine 10, all
the other acetylations stay.

Now CBP(Co-Activator) is there , now even TF11D can be there , and now
a remodeler can even be here , the reason why you actively recruit CBP now
and other proteins, you have lets say components in TF11D ( Has TBP and a
lot of other proteins that dont bind to DNA, TF11D is a complex of 12-14
proteins , TBP might be there or might not (only involved if there is a TATA
motif) , these guys are being recruited for several reasons now , the state of
the modifications of the Histones has changed , you have specific
acetylations and phosphorylations, as a consequences these proteins CBP
and this SWI/SNF remodeler have bromodomains!!!!!!!!!!!! And there
is a component of TF11D largest component there is a protein that has a
Bromodomain, so essentially you assemble this much bigger complex, now
the Nucleosome is in SAD SHAPE: (
As a consequence of this happening now, the remodeler will dissociate it and
now you will get transcription, now the transcription you get are you going
to get the small RNA? Or the small RNA is it already there now? And
maybe when that nucleosome goes away you will get release from the start

site, its not clear here if what turns the gene on at the level of getting the
mRNA is initiation or release from the start site, that WILL DEPEND ON
THE GENE, each gene will have different requirements for that whether you
need to modify a protein, recruit another protein, or maybe you dont,
depends on whether its Initiation control, release from the start site control
or elongation control, after this point.
Good Summary Slide:

Tell us a little about Gene Regulation, that stops transcription not activates
transcription. What kind of things are going to repress transcription, you
already know one, remodeling complexes with Chromodomains
recognizing specific types of Methylated Histones that are going to put
Nucleosomes on, but they are a lot of other things that are causing
repression. There could be proteins that are bound to the enhancer or bound
elsewhere and what these proteins do is when they bind instead of the

activator binding, they short circuit the whole process. There are some DNA
binding proteins that bind to the exact same sequences as the activator but
they dont activate they repress!!!!

This Slide gives you three different mechanisms InVivo, In (a ) it shows you
two proteins can bind to the same sequence , one activates and the other
represses the one that binds is whoever gets there first , the guy that gets
there first is he going to stay there all the time? No , everyone thats in the
nucleus is only there for a limited period of time (like we are on this earth) ,
so lets say a protein binds and it may dissociate very rapidly and maybe the
next time the other guy binds, depends on what is available the concentration
of the molecules and how long they stay bound .Shows us a technique on
how dynamic transcription really is . Many of the DNA bound activators are
coming on and off the DNA remarkly quickly and that is part of the
regulation. Another possibility for repression is two different DNA binding
sites an activator binds to one and the repressor protein binds to the other ,
so the repressor protein can block the activation domain of the other guy that
is the activator, and then the other guy cant do what he is really supposed to
do. Another repression possibility is you have a binding site for Repressor
and a binding site for an activator, the repressor might bind and give signal
to communicate with the mediator, CBP or somebody, but if this repressor is
bound its job is to do the opposite, here is communicating with the
polymerase Mediator complex. You get this different modalitys depending
on the gene or circuit.

Summary Slide so to speak, at the top you can have active chromatin, could
be a gene that is almost ready to go or a gene that is going to be transcribed
momentarily, you have an activating sequence bound to an activator, that
activator has an activation domain, activation domain recruits GCN 5 and
keeps the ball rolling toward activation. At the bottom the activator
sequence instead is bound to something that has a repression domain, that
repression domain recruits the OPPSOSITE OF A HISTONE ACETYLASE
it recruits a Histone DeAcetlylase Complex!!!!!! And in order to do that
you need a Co-Repressor!!!!! There are classes of protein that are bound to
Histone Deacetylases that have the opposite effect of a Histone
Acetylase!!!!! IT REMOVES THE ACETYLATION that the Histone
Acetylate put on, or keeps the HISTONE DEACYLATED!!!! If you keep
the Histone decetylated it retains its positive charge on the Lysine , and now
the nucleosome retains its grip on the DNA , so the active mechanisms is
these proteins have a domain that is going to recruit the opposite type of
protein to the activators , sort of like the Christ and Anti-Crist(He said this in
class) .

Summary of previous slides,

Coactivators and HATS: Activations!!
HDAC: Histone Deacetylase and co-repressors give you repression!!!!!!!
Remember when you see an arrow, it means process is progressing toward
an onset!!!
Slash Bar: A broken circuit, it means you are not getting transcription as
indicated in this diagram!!!!!
Based on what he told us so far can we assume an active gene, if its being
actively transcribed, there should be no Histone Deacetylase involved? He
told us that Histone Deacetlayse is needed to repress.
If gene is active can you assume that there are no Histone Deacetylase
associated with the chromatin? NO!!! Be a scientist!!!!!

Here the H stands for Histone Acetylase, in the active gene, but there are
still some Histone Deacetlyase there, what are they doing? If you keep the
Histones totally acetylated all the time and constantly being acetylated that
will result in a gene that has extreme transcriptional activity, and maybe you
want to use the Deacetylase to regulate the transcription of the gene slowly
not like a light switch that you turn on and off, you are using the Deacetylase
as a gradual switch, many genes are regulated in this way, NOT DIRECTLY
ON AND OFF, you still want the ability to Deacetlyate some of these
acetylations to allow the nucleosomes to grip, again part of that has to do
with the following ,when the RNA Polymerase leaves the Start site , maybe
the nucleosome that was on the start site , maybe it gets back on when RNA
polymerase leaves, dont assume that because one Polymerase left the start
site ,it means that next polymerase life is easy this is not true , unless the
gene is extremely transcriptionally active , maybe its so active that its one of
these enhancers regulating cell identity genes and there is thousands of
mRNAs per cell then maybe nucleosomes dont get back on the start site but
that its a unique situation. A more general situation would be is that
maybe the nucleosome gets back on the start site after the first polymerase
leaves or if its not totally reassemble then maybe it will slide from another
position for these things to start to happen you need to remove some of these
acetylations.
Primed Gene: The primed gene is not making an RNA yet, if you are not
making an RNA , HDACs inhibit RNA Polymerase recruitment, to

H3K4metylated primed promoters, this H3K4 methylation one single

methylation that is positive transcription. OMGG NOT ALL
METHYLATIONS HAVE A NEGATIVE IMPACT!!!!!!!!!!!!!! SOME OF
THEM HAVE A POSTIVE EFFECT. If its a single methylation at the K4
position in H3 thats positive, however if HDACs are present and you
remove acetylations even though you have this positive mark because you
lost the acetylations the nucleosomes stay on the start site and you cant
recruit the polymerase, so this thing is not even a pre-initiation complex, its
further way from being initiated and HDACs are involved in that. D: for
HDACs in the diagram!!!!!!
Silent Gene: A silent gene will have very special kinds of methylations on
specific Histone residues, HISTONE H3 Lysine K27(27th position from the
Amino terminus) , a triple methylation this will recruit a special complex
of proteins initially discovered in Drosophila a polycome complex this
complex of proteins is just a load of different types of transcriptional
repressors, what they do is recruit remodeling complexes that keep putting
nucleosomes on DNA and make sure the Nucleosomes stay put, and there
may not even be Pioneer Factors in this thing , so many Nucleosomes on it
that its in Heterochromatin. When you see this Histone mark and you seen
enough of them, which tells you this gene is probably in Heterochromatin. If
there is enough of these specific types of marks THATS NEVER
ASSOCIATED WITH AN ACTIVE GENE!!!!!!
H3K4me: Is associated with an active gene, just like acetylations are
Another type of summary slide: Shows you remodeling, this H3K9
DEMETYLATION is also associated with Activity, This one H3K27 that if

you methylate it, its Heterochromatin but if you DE methylate thats activity.
NCoR and SMRT are the names of other types of Co-Repressors!!!!! That
recruits the HDACs!!!!!!! There is more then one protein that will function
in repression.
Lecture 5 talks about Dynamic exchange stuff
Marcu Lecture 5:
Introduce technique that lets you look at how proteins bound to DNA
interact with each other InVivo in cells and this technique can be used not
just to look at interactions of proteins in DNA, but modifications of it, that
allow you to look at how rapidly transcription factors come on and off DNA,
even on Enhancers. Concept that Enhancers create this big complex of thing
s(factors) binding DNA that recruit many other proteins . Dont assume that
this complex is extremely stable in real time because part of the regulation in
involves Dynamic exchange on and off the DNA, another type of control he
gets into that regulates transcription!!!!
Introduce a study in which experiments were done to determine how
HMGB1 interacts with the Glucocorticoid receptor and other proteins on
DNA. The experiment is called Real time in situ FRET-pbFRET .

This just reminds you of HMGB1 binding to Nucleosomes, and that it

bends DNA !!!!!!

Using this technique as to show us how things interact on DNA. What you
do is that on the left side it shows Donor (which corresponds to a Fluorform)
, a fluroform in this context would be GFP(Green Fluorescent Protein)
,cyan protein, yellow protein. There are a variety of proteins that you could
put these genes encoding these proteins inside a cell, it will integrate into the
cells nucleus by the techniques he told us on prior lectures., so its in a

plasmid, expression vector, now the cells will shine green if you shine a
certain wavelength of light or another color, depending on the emission
wavelength of the fluroform. How do you get the fluorform to emit light?,
you have a laser and the laser is exciting the fluorform, if you use a very
powerful laser at a very high frequency (short wavelength) what happens is
that the length of time this is going to be green lets say GFP is extremely
short talking about seconds , the reason why is that the energy state of the
protein is so high with this very strong laser , almost all of the color is
rapidly bleached . SO that would be the normal situation, if you put the GFP
in the cell, However if you have two different Fluoroforms , you have a
donor and an acceptor fluorform , the acceptor can take energy from the
donor, two different fluoforms and two different colors at the same time
since the other fluoroform is there , depending on the distance and thats the
critical point here !!!!! The energy can transfer between fluoroform or the
energy CANT . Lets say one fluorform is fused in frame to a protein , in
this case its HMGB1, and lets say the other fluorform is fused inframe to
another protein that can bind DNA , If the distance between these two
fluorforms on DNA is an Angstrom or less then an Angstrom away and
other words the distance two proteins can be touching each other(haha) so
really no space between them, they are literally touching !!!! in order for this
energy to be transferred , if that energy is transferred and this original donor
will stay greener for a much longer period of time because you have reduced
its energy!!! So its doesnt totally bleach, What the technique is doing is , its
saying you have two fluorforms together , you make these two fusion
proteins in this expression vector , put them in a cell and then you shine light
, this really powerful laser and you look to see what is the effect on the
length of time that this guy can stay green if this other guy is present in your
experiment then do a lot of controls to see if they both have to bind DNA ,
those kind of experiment in which you can make mutations in the DNA
binding domain, it allows you to do a lot of things now , you have an assay
of two proteins that are close nearby.

So what happens is that, in this experiment you have Cyan protein fused to
Glucocorticoid receptor , FRET Together with photobleaching Fret analysis
reveals that the Glucocorticoid receptor transcriptional activator protein and
the HMGB1 chromatin architectural protein specifically interact with each
other BUT only if they are bound to DNA in chromatin!!!!!!!. First of two
slides that illustrates this. What you are looking at there is time in seconds,
looking at Normalized fluorescence intensity, camera is taking pictures very
quickly, and what you are looking at is CFP-GR fluorescent intensity, So its
not GFP, you are looking at Cyan protein fluorescent that is fused to
Glucocorticoid receptor, and if you put in the cells this one fusion protein.
look how fast you lose the Cyan color, its because you get very rapid
bleaching. However if you mix the Cyan protein glucocorticoid receptor
(CFP-GR) with HMGB1 fused to yellow fluorescent protein, its important
that one is cyan and one is yellow because the yellow effector can take some
energy off the first one and that has to do with the frequency of light emitted
by the first one , light coming from the Cyan proteins some of that can be
absorbed by the yellow fluorescent protein , you are not looking at yellow
color , you are looking at cyan color , now look how bright it is in the first
picture , but in the first experiment without the yellow protein the cyan color

is totally gone at the end. Now how important was it that this was HMGB1?
Now you can do more experiments for instance is just because HMGB1 was
in the Nucleus, it doesnt have to bound to DNA, if it was just in the Nucleus
was that enough for this to happen? So there is a very simple control
experiment you can do, What is NLS? Nuclear localization signal (yeah,
yeah cell bio fuckers I know you know this)
Every single protein that wants to go into the Nucleus, need to have a
NLS!!! These are signals that are built into proteins that tell them where to
traffic in the cell depending on where they function , so proteins that should
go to the nucleus should have a NLS!!!! Proteins that should go to RER
have some other kind of signal, versus proteins that go to the lysosome,
endosome, etc.. Versus proteins that go out to the cell membrane, this is
transcription so there are Nuclear Localization Signals, so you take a very
powerful NLS you fuse it to YFP doesnt do anything, it means NO its not
just the YFP has to be in the Nucleus. Do other experiment, and the kinds of
experiments you do are the following, here is Glucocortocoid receptor alone,
and you do these experiments many times, there is an extreme statistical
defect in how long you retain this signal, another experiment done with
Histone H3, if you use Histone H3 and fuse that Histone to YFP you also
get an effect there is some interaction between Glucocorticoid receptor and
Histone H3!!!!! But the effect you see even though statiscally significant
its more modest!!!!! Then the effect you get with HMGB1. Also do with
NLS signals and dont get any statiscal difference.

Now what about binding to DNA, want to interpret this Data, want to
understand what the shift is, this part where you are losing the Cyan protein
quickly, is if you only use the Cyan protein and no HMGB1 YFP. The shift

means you are seeing the Cyan color for a longer period of time, the
reason why there is these two different traces because you have these two
independent experiments, the cells have been transfected two different times
independent of each other, that whys there is error bars in the analysis.
Because you are doing the experiment many time, do the experiment at least
three times, four times would be better. Here is the critical experiment that
says DNA Binding is critical, so what you do is take HMGB1 and mutate
the part that lets it bind DNA, its DNA binding domain and you mutate those
amino acids only in HMGB1 that let it bind DNA, now fuse that mutant
HMGB1 to YFP AND YOU COMPLETELY LOSE THE EFFECT , it has
to be bound to DNA just like GR is bound to DNA , that essentially proves
that GR and HMGB1 sit nearby each other , in some place, in some
chromosomes. So this is an extremely useful technique and you can do this
lets say with all the transcription factors that bind to a specific enhancer,
likes say the B-Interferon enhancer and how it starts with the Histone Code,
recruit other proteins etc. You can do this experiment by putting these
colored fluroforms on different transcription factors and bind them to an
enhancer, and see how many factors in the enhancer directly interact with
each other and how many dont. All of these things are really possible to
look at and how many interactions they have specifically, all of the
techniques cannot do that BUT THIS ONE CAN!!!!!!
Marcu has selected specific kinds of transcriptional activators!!!! Talk about
what these activators do to cell physiology.
Transcriptional Activators that only work in response to a Hormone, so
these are hormone receptors, receptors not in the sense that they are on the
surface of a cell but receptors in the Nucleus!!!!!! Signals that come from the
outside of the cell and change the mind of the cell with regard in to what is
going to be transcribing. It shows how signal transduction pathways can
rapidly change transcriptional control, also illustrates a very IMPORTANT
CONCEPT!!!!! That you can turn genes ON and OFF and alter the function
of a cell pathway independent of any NEW protein synthesis, many genes
that can be turned on and off by signals with no protein synthesis being
required, you can add a protein synthesis inhibitor like cyclohexmide , dont
need any new proteins for any of this to happen, for these Hormones to
activate their receptors!!!! How do they do that is and how do these signals
work!!!

Here you have an Environmental signal, at the bottom you have your target
genes, can affect Transcription factors, can induce a MicroRNA, affect
chromatin regulators, and almost affect anything that will regulate gene
expression.

In the case of Steroid Hormone Receptors, they fall into two classes: Type 1
receptor and Type 2 Receptor with regard to the mechanism, they both still
need hormones but the mechanism are different by which they work. Type 1
receptor classic example the Glucocorticoids Receptor, in this case you can
assume hormone receptor is the Glucocortcoid receptor and naturally bound
on the cytoplasm to a heat shock protein HSP70 . In a complex with HSP 70

in the cytoplasm the glucocorticoid receptor cant get into the nucleus; its
Nuclear localization signal is blocked. If you have glucocorcoid hormone
outside the cell is passively goes through the cell membrane and it binds to
this complex and as a consequence of binding it binds to a domain in the
glucocorticoid receptor , the glucocorcoid receptor has multi-functional
domains like transcriptional activators, it has a DNA-binding domain,
activation domain and it has one more domain that other activators DONT
HAVE!!!! IT HAS A HORMONE-LIGAND BINDING DOMAIN!!!!!!
One additional domain!!! And when the Hormone binds to that domain in
the glucocorticoid receptor it changes the conformation of the protein and
now it kicks out Hsp70 , in which Hsp 70 is no longer able to bind to GR in
this altered conformation , now the Hormone bound to GR, goes in and GR
works as a dimer , its a dimer protein and it goes into the nucleus and binds
its response element and can now start transcription. Maybe in this case, this
was the one factor you were missing to activate your gene or maybe this was
the pioneer factor although very unlikely that its the pioneer factor, so
usually many other factors were already bound to the enhancer and maybe
this was the one you were missing to get the ball rolling to get transcription.
The reason why when you add hormone to the cell and you do your
experiment, you can see the GR target genes turn on in matter of minutes.
You can see the mRNA from GR within 15 to 20 minutes!!!! Start to
accumulate. Thats fast enough that its extremely unlikely that the
Glucocortcoid receptor in this case was a pioneer factor, because many
of the things were already there and you were just missing that to help
recruit co-activator to recruit the Mediator to recruit RNA Pol 2, he told us
that this is independent from Protein Synthesis, that means that you can
recruit co-activators, you can recruit the Mediator, can recruit polymerase
but you dont need any protein synthesis to do that, because the cell has all
these proteins in the Nucleus already!!!! They are just not assemble on that
gene to transcribe, so the assembly effect gets all these things there, you
dont need new protein synthesis!!!!!!!!!!!!!! You told us that for the
Remodeler you need ATP, you need energy but that doesnt mean you need
protein synthesis. Now Type 2 is more complicated and they have another
level of control that GR cant have. Type 2 receptors which would be
Androgen receptor for instance, progesterone receptor, these are not in the
cytoplasm they are already in the Nucleus, THEY ARE SITTING RITE ON
THEIR BINDING SITE, they are bound to DNA already in the absence of
their Hormone but what they are doing to the target gene, THEY ARE
ACTIVELY REPRESSING THEIR TARGET GENE in the absence of their
Hormone, the way they do that is , is that when they are bound to DNA they

recruit Co-Repressors and HDACs to deacylate the Histones , active

repression , not passive repression. Passive Repression in this context that
you are JUST MISSING THE FACTOR and its not bound to DNA, In this
case its not something that is missing, the thing is there and is actively
repressing the gene, if you add the Hormone, the Horomone goes through
the cell membrane, goes through the cytoplasm, enters the Nucleus, it will
bind this that is on the DNA and when it binds these co-repressors and
HDACs they cant bind to the estrogen receptor or the progesterone receptor
anymore , they leave!!!! And instead are replace by co-activators and now
they turn transcription on, its literally a switch the hormone switches the
mind of this protein it tells it NO you are not a repressor anymore, NOW
you are an Activator. This happens very quickly, this is now an explanation
physiologically on WHAT TURNS YOU ON when you look at someone
that looks interesting (HAHA Trancripition can explain sexual responses)

HE SAID THIS FOOLS!

Similar slide in which he showed us where activation domains and where
binding domains are, all these are steroid receptors have a DNA binding
domain that bind its unique DNA sequence , each one binds a different DNA
sequence , they have a variable region of different lengths that is an
activation domain ,so it depends on what the function of the receptor is and
what it recruits , some of them have a very big activation domain that does
many different things and some of them have a very short activation domain
that doesnt do that many things. But all these Steroid Receptors have this
Ligand-Hormone Binding domain that binds its private Hormone!!!!
Thyroid Hormone can only bind thyroid hormone receptor etc

The sequence these receptors bind to have some similarities but also have
very clear strong differences. So Androgen cannot turn on progesterone
target genes vice versa and thats why we are male and females.

What are the consequences if you bind Hormone, but to understand that you
need to know about this special kind of Co-Activator called Steroid
Receptor CoActivators, some of these Hormone receptors have their own

private Co-Activators that they need to recruit , these co-activators will then
modify the chromatin like he told us CBP does or p300 and also will
facilitate communication with the Mediator , what is the roll of the Hormone
binding here to the recruitment of the co-activator, mechanistically how does
that happen.

This shows you that the DNA binding domain is a Leucine Finger Domain

Now Estrogen, there is a Helix here in the protein, if you bind estrogen to
this estrogen receptor this Helix has a dramatic change on the conformation
of the protein. VERY DRAMTIC CHANGE

This is the structure of the Co-Activator Steroid Receptor Co-Activator

/SRC 1. These proteins cant bind to DNA, they are like other Co-Activators
like p300,CBP, they have various types of Helices, other types of Domains
,Nuclear Hormone Receptor Interaction Domain (CRITICAL) and they
have another Domain which allows them to recruit p300 and CBP, it doesnt
mean that p300 and CBP are not involve in the regulation it just means that
they are not recruited rite away. Like he told us when he explain the BInterferon enhancer GCN5 got here first its not just unique to the BInterferon Enhancer which is not regulated by a Hormone. Thats why this is
a CONCEPT!!!!!! The mechanisms are slightly different but the concept
stays the same, you still use the Histone Code and you still use it in a similar
way that he told us before. But you have to get this guy there first.

There is this motif here Leucine,Leucine XX Leucine , this is the interaction

of a nuclear receptor with its Co-Activator in the presence of Hormone, this
motif LLXXL has a binding site in the receptor BUT THAT BINDING

SITE IS ONLY VISBILE TO THE CO-ACTIVATOR , it could only bind to

the binding site if the Hormone is already bound to the receptor to alter its
conformation to make this binding site visible for this motif LLXXL BUT if
the receptor is not bound to its Hormone , this motif that is present in the
Co-Activator cant bind to its binding site because its buried in the protein ,
you need to alter the conformation of the steroid Receptor that then allows
this LLXXL motif in the co-activator to interact with the DNA bound
estrogen receptor that is bound to its hormone. 34:31

Here is the Hormone bound to DNA, first repression transcription, its

actively recruiting Repressors and HDACs, you are adding the Hormone ,
look at the dramatic change in this Helix , to the DNA bound receptor , you
then add the Co-Activator , now it can bind.

Top of the slide shows, if you are activating transcription, now at the bottom
of the slide the sequence could be the binding site for a Steroid Receptor,

this protein that is bound has a DNA binding domain or repression domain
this could be the steroid receptor and thats exactly how it works, the steroid
receptor recruits a Co-Repressor which recruits HDACs which remove the
Acetylations, tightens the grip of the Nucleosomes on DNA and suppresses
transcription, so this active repression by the DNA bound receptor in the
absence of its Hormone, now you add Hormone and it totally changes
because the activation domain is visible and thats because you altered the
conformation of the protein and now the Co-Activator can bind via LLXXL
motif to its binding site. This is one clear example how this works in
response to Hormones independent of any new protein synthesis.
Here you have the ligand bind the DNA bound receptor, you recruit SRC
and you recruit p300 and youll activate transcription.

What did the Pioneer factors have to do with this?

We know something more, we know a number of estrogen receptor DNA

binding sites find themselves nearby binding site for another transcription
factor and that might be the enhancer or the promoter that binding site binds
a class of proteins of the FOX A family of transcription factors. FOX A has
a specialized DNA binding domain it has this forkhead motif that binds to
specific sequences in DNA, what we know that is this FOX A guy functions
like a Pioneer and because its there, and because its there now the estrogen
receptor its binding is better or facilitating and if you want you can also do
experiments were you can make fusion proteins with different colored Fluor
forms and you can demonstrate this. So this was one signaling pathway
that has to do with how independent of new protein synthesis you can
activate genes that are regulated by Hormone receptors.!!!!!
Now a much more complicated family of transcription factors

That also do it with how independent of new protein synthesis, you can
activate genes that are regulated by Hormone receptors. These new ones

also use signaling pathways that are independent of new protein synthesis
sometimes and sometime not, some of these can activate transcription that
dont need protein synthesis and other ones you can activate that do need it.
These transcription factors are the nuclear factor cappa B family NF-kB
these transcription factors are encoded by multiple different genes, its a
family of factors, a family that can bind DNA and may or may not activate
transcription. These factors are physiologically very important for all innate
and inflammatory reactions. All inflammatory reactions where you have
pain with inflammation or any kind of inflammatory effect you ALWAYS
activate NF-kB , NF-kB is the reason why you have inflammation!!!!!If
you take Anti-Inflammatory or painkillers, asperin, acetominophin all of
these are inhibiting the activation of one of the NF-kB pathways!!!!!! We
even know what component of the pathway they are inhibiting.

Some of the things that NF-kB does in normal health, in normal physiology
without NF-kB B-Lymphocytes would NOT develop, without NF-kB you
would not have Antibodies, B-cells would not divide, so T-Cells wouldnt
develop either, without NF-kB your whole adaptive immune system will be
dramatically compromised. You cant fight infections without NF-kB!!!!!
These are adaptive immune response that is not inflammation. Inflammation
is a rapid response. Adaptive immune response requires cell develop and
takes a longer period of time, the initial effect is an inflammatory reaction,
so called Innate Response , adaptive response you adapt to a response, but
NF-kB is involved in BOTH. But NF-kB does a lot of other BAD things,
double edge sword. Every single one of these diseases above NF-kB

impacts on. NF-kB becomes inappropriately activated and contributes to

the outcome of disease. It can contribute to the onset of progression of the
disease and the physiological consequences, so its a good cop and a bad cop
at the same time. Even though there are a lot of drugs that inhibit NF-kB
you have to be really careful on how long you want to inhibit it and how
much you want to inhibit it, if you inhibit it too much even though you
might help a disease context , adaptive immunity will be screwed up
eventually. Have to careful, also on this list is cancer as well.

This here when NF-kB is constituvely activated (activated all the time) it
makes cells immortal. NF-kB will induce an inflammatory reaction, it will
induce blood vessel cell growth, it will cause tumor cells to become
malignant and metastasis to other site in the body, its also a very potent
Anti-Apotic signal. it helps cells survive for a longer period of time its a
survival signal , One of the reasons NF-kB makes cells immortal because
one of its direct targets is a gene encoding telomerase , telomerase maintains
the length of telomeres , when cells get really old , telomeres reduce
themselves and if you have really short telomeres that impedes cell division
when chromosomes interact and get cell division. You need to maintain the
original length of the telomere to get proper cell proliferation!!! Old peoples
telomeres are shorter then average. NF-kB induces activity of telomerase
.On the top of the slide are all things that can activate NF-kB sooo
Endotoxins will activate it , Cytokines involve in the Innate Immune
Response will activate it, an infection by a virus will activate it , things that
induce cell death will activate it , things that cause cancer will activate it
,carcinogens and all types of stress will activate it , dramatic change in pH

will activate NF-kB. Hypoxia can activate NF-kB, Hydrogen peroxide

will activate NF-kB basically free radicals. You want to reduce the free
radicals in your food if you start to oxidize a lot of things NF-kB will get
activated. So this means that if you all these different ways of activating
NF-kB, it means you can activate this with AN EXTRACELLULAR
SIGNAL or an INTRINSIC SIGNAL INSIDE THE CELL!!!!!
If the amount of free radicals oxidizing compounds increases inside the cell
you can alter pH inside the cell without any extracellular signal at all that
will affect intracellular NF-kB. Many ways to activate
NF-kB!!!!!

Some of the target genes of NF-kB, how does NF-kB do what it does, in
order to do that you need to know who its direct target genes are, direct
target of the transcription factor should help us Understand how it does all
these different things, ChemoKines and Cytokines are things that make cells
divide or make cells migrate to different places in the body ALMOST all of
them are direct targets of NF-kB, adhesion molecules that make cells
interact with other cells are also direct targets, also things involve in
Hydrogen metabolism ,also proteins that help the cell survive , antiapoptic
proteins many of them are direct targets of NF-kB, things that positively
regulate the cell cycle, things that make a cell divide faster!!!! Are direct
targets of NF-kB , two of them are Cyclin D1 and c-MYC. cMYC is a
transcription factor, if make too much cMYC it encodes for a protooncogene
is can be oncogenic because it cause cells to proliferate too fast!!! Too much
growth the potential beginning of the onsets of cancer. NF-kB can be a
cancer promoter or tumor promoter. NF-kB also induces Tissue Remodeling

it directly activates metalloproteases, it induces transcription factors that turn

on Metalloproteases. Also activate other receptors that are critical for
recognizing damaged tissue and inflammatory reactions. All of these are
direct targets of NF-kB , it means that NF-kB binds to their promoter or
enhancer and is critical for their transcription . Interesting Jmjd3 is a
Histone modifying protein; NF-kB even directly regulates proteins that
alter chromatin. Some of NF-kB targets are its OWN INHIBITORS!!!
Inhibitors of NF-kB are its own direct targets, so thats how the cell
regulates NF-kB , if its too active its doing some bad things it will turn on
its own inhibitors, these inhibitors feedback by feedback regulation and shut
down NF-kB. These are multiple inhibitors not just one!!! Even one NF-kB
subunit is an inhibitor of the pathway. So there is negative feedback control
involved in this process that restrained NF-kB so it doesnt get too active. If
any of these things get compromised, YOU CANT RESTRAIN NF-kB and
that contributes to the growth of some cancers.

NF-kB always functions as a dimer!!!, you need two dimers of the NF-kB
for it to bind to the target site.

Here are some activating members of the NF-kB family , here is Rel A,Rel
B,c-Rel each is encoded by a different gene, there is also p50 and p52 which
are synthesize as a precursor protein, initially there is a p105 precursor that

is processed to become p50 and a p100 that is processed to become p52. So

these five guys mix and match with each other! they can homodimerize or
Heterodimerize. The precursors of these NF-kB can be Inhibitors when they
are precursors only when precursor, because these precursors have this
Carboxyl terminal domain that has muilple repeats, Anchor motifs are motifs
in proteins that are sticky motifs that allow it to bind to other proteins, so
IKa just inhibitor another gene, each of these have an anchor motif. also
amino acids that remodel the activator or inhibitor .

This is the way NF-kB generally designed, in the cytoplasm complexed to

an inhibitor, and it depends on which NF-kB it is and which inhibitor it is,
and then what happened is you get a signal from the outside of the cell and
that signal activates Kinases and those Kinases phosphorylate the inhibitor
which target the Ubiquitination and proteasome destruction of the NF-kB
migrates to the Nucleus .

Marcu Lecture 6:
Transcription factors that bind DNA or activators that either drive cell to a
cancerous state or to repressed Cancerous state,
NF-kB

This again shows you the guys that bind DNA and the inhibitors, two of the
inhibitors are precursors of two of the NF-kB that bind DNA if only the
Carboxy-terminus is cleaved off at the upside down triangle liberated the
DNA binding component, and NF-kBs will bind DNA as dimers!!!!!!!!
homodimers and Heterodimers

Shows some of the names of all NF-kB and all the possible combinations,
shows you the NF-kB consensus sequence for the binding site, then you
have these c-Rel or p65(just ex.) homodimers the binding sites are similar

but there some differences, for instance you that this first residue is not a G
in the site preferred c-Rel or p65 homodimers and so on you get the idea.

This slide shows you p52 , p52 loves to Heteridimerize with Rel B, and it
likes to Homedimerize with itself when Rel B is not around, p52 and Rel B
Heterodimers bind to a certain consensus sequence , The left side of the
slide shows you p50 and p65 . Notice p52 had this extension on its Carboxyterminus because whats illustrated here is the precursor, p100 is the
precursor for p52, RelB is complexes to p52 in the Cytoplasm as the
precursor to p52, not as the monomer or cleaved form because its the
precursor this is the inhibitory domain and then Rel B, p52 bound to the
precursor in that dimer stays in the Cytoplasm wont go into the nucleus sort
of in a dormant state or repressed state in the cytoplasm, you need a signal
generally coming from the outside of the cell and what that signal does is it
activates a Kinase cascade one of the ones it recruits is NIK(NF-kB
INDUCING KINASES) , then IKKa/1 this Kinase is activated by NIK .
IKKa/1 is the one that phosphorylates a specific serine rite at the junction of
this amino terminus and carboxytermnius and when that Serine gets
phosrylated that makes that protein a target for mono-Ubiquitination for
Ubiquitin ligase then additional Ubiquitination for Ubiquitin ligase that
polyubiquitnate things and now proteasome comes in to play(Cell bio can
blow me) And what the Proteosome is going to do cleave and destroy this
Carboxyl Terminus but it spares this Amino Terminus so then you are left

with a Mature p52, and now mature p52 and Rel B can go into the Nucleus
as that Heterodimer that they were in the beginning and now they might bind
their target sites and activate transcription. Very similar thing happens on
the left side of the slide but the players are different here you have p50 and
p65(CreI) these are Heterodimers and a signal comes from the outside of the
cell activates a Kinase called IKKBeta/2 this phosphorylates IkBalpha
inhibitor which is keeping this Heterodimer in the Cytoplasm!!!!!!!!!!,
when you phosphorylate IkBalpha two specific amino terminus Serines that
same thing happens MonoUbiquitination, PolyUbiquitination destruction by
the Proteasome, the only thing that gets destroyed is the Inhibitor the one
thing that is Ubiquinated thats the nice thing about the Proteosome its
ONLY
GOING
TO
DESTOY
SOMETHING
THAT
IS
UBUIQINATED!!!!! If you dont have that specific Ubquitnation its a very
targeted degradation process. Proteasome vey important because you need to
keep proteins down to a certain level and now after this inhibitor is
destroyed, now p50 and p65 as a Heterodimer can go into the Nucleus and
now maybe it binds its target site and maybe activates transcription.
Carnonical: Means generally that this is the pathway that is generally
happening ALL THE TIME!!!, most of the time is a situation where NF-kB
is involved in regulating genes that cause stress/inflammation then when you
get a signal coming from the outside of the cell , independent of any new
protein synthesis very rapidly IKKbeta gets activated and this happens
VERY RAPIDLY!!! So this Heterodimer would bind DNA become in a
active state and that was the only factor out of the Enhancer and promoter
that you were missing and then you get transcription, youll get initiation
and also release from the start site, both things can then happen and that will
be very fast.
The pathway on the right has a Lag time off approximately 2 hours and this
requires New Protein Synthesis, so that is NOT TYPICAL of the family
therefore we called it Non-Canonical, NIK is constantly being destroyed by
the Proteasome you always have very low levels of NIK so what you have to
do is, is stop the Proteasome from doing that, in order to stop the
Proteosome from doing that you need to synthesize a new protein that is an
Inhibitor of Ubiquitin Ligases, suffice to say that you dont have enough
NIK and it takes two hours to solve that problem and that requires new
protein synthesis, so in the slide the NonCanonical pathway is protein
synthesis dependent.
Canonical Pathway: Is protein synthesis INDEPENDENT
When you are infected by a Virus or Bacteria, the very first reaction you get
is a Canonical reaction and thats the first pathway, but then eventually if

you have some Immunity to the Virus or bacteria B-Lymphocytes and TLymphoctyes become involved, and they will help the B-Lymphocytes make
antibodies that will attack the invader, but that has an extremely lag period.
NF-kB very critical regulator of the development of B-Lymphocytes and
T-Lymphocytes. One of the major reasons why Adaptive Immunity is
delayed is because the Non-carnonical pathway is delayed, you dont make
Antibodies rite away, you still need this pathway and is still protein
synthesis dependent. And just remember that the Innate Inflammatory
Responses is quicker.

This just shows you all the possibilities of NF-kB , Homo and Hetero
Dimerizing , and it also shows the Target genes , also shows you the DNA
Binding sites of these targets genes, sites of their promoters and enhancers
and these sites are highly conserved in Evolution , evolutionary conserved
regions that are found in mice and humans, and you can see that the binding
sites are highly conserved . If you look at the fruit fly there are things that
look like NF-kB in the fruit flies it has different names. He says that yeast
dont have this.

Here is part of the Conadral activation mechanism, what we knew before

1997 , was the Conadral pathway ,we didnt know shit for the
NonConadral Pathway. We knew that in the Conadral Pathway there was
an Inhibitor, IcappaB alpha and we knew it was complexes to these guys in
the cytoplasm , we knew it was phosphorylated and Ubuiquitinated and we
then knew this guy goes into the Nucleus as this p50,p65 Heterodimer,
(p65 is sometimes called RelA). Labs give it different names!!!! Rel is the
name of a DNA binding domain discovered in a chicken(dont know why ) .
So the NF-kB binding domain was originally discovered in chicken. They
knew that there has to be some kind of Kinase that is doing this , because the
initial signal is to phosphorylate , because these are Serines , we knew it had
to be a Serine/Threonine kinase . There is two MAJOR classes of Kinases,
Serine/Threonine and Tyrosine Kinases!!!!!! So we knew that it had to be
a Serine.Threonine kinase and we knew that it wasnt active all the time and
some signal is activating it!!!!! We knew some of the signals like bacterial
Viral Infection, Cytokines we really didnt know what IcappB kinase was.

So as of the summer of 1997

People discovered was the following, there was in the cytoplasm

bimolecular complexes of Kinases, two different Kinases IKK alpha and
IKK Beta!!! And this complex has a certain archechiture, its locked in this
conformation by an Adaptor protein IKK GAMMA
IKK: Inhibitor of NF-kB Kinase !!!!!! OK SO IKK alpha and Beta is a
Kinase BUTTT IKK GAMMA IS NOT A KINASE!!!! OMGG someone
decided to name it NEMO like finding Nemo(NF-kB essential Modulator)
it modulates the activity of the Kinase complex sort of like an adaptor
protein or scaffolding protein and the purpose of this protein is very
important in this pathway , what it does is that it senses all of these different
signals , different kinases cascades , all these cascades dropping in on this
one complex. All of these things are protein where things coming from
outside of the cell are impacting on their receptors when they bind, and
when they bind they set up a signal transduction pathway and each pathway
has its own unique attributes, that have adaptors proteins and Kinases in
them, but the ONE THING THEY ALL HAVE IN COMMON IS they all
communicate at the end with NEMO!!!!!!!!!!!(IKK GAMMA(nOT THE
CLOWN FISH) OK!! When all these guys are communicating with IKK
Gamma what happens is that there is some specific Ubiquitnatins that
happen , specifically Ubiquitnations in Nemo, and those Ubiquitnations alter
the conformation of the complex , and now what starts to Happen is these
Kinases become Activated , and in order for them to become active they
require phosphorylation have so to speak a Kinase activation domain and

that domain gets phosphorylated to become active and the kinase that do this
activation one of them is MEKK3 etc and these guy have to communicate
with Nemo to be able to activate one of these Kinases. OK if you activated
(IKK Beta you drive the Conadral pathway), if you activate (IKK alpha
you drive the NonConadral Pathway.) Some of the things that activate IKK
alpha will also activate IKK beta , BUT MANY MANY things that activate
IKK Beta CANT Activate IKK alpha . Making IKK Alpha more restricted
and unique!!! Because you have to activate in almost all cases a NIK if you
want to activate IKK Alpha !!!!! There are some cells that have abnormal
degree of regulation and there is another Kinase beside NIK but its not in a
normal cell

Shows you the proteasome, some activators of the Canonical pathway when
they set up their receptors they set up a signal transduction that activates
NIK, and then NIK can activate IKKalpha and it could phosphorylate the
p53 precursor , as a consequence you get these things happen, on the left
side where you activate IKKBeta all these other things he told us like the
stress responses, the inflammatory response , its also a cell survival signal
and also in addition to survival if you activate it too much ,cells will start to
prematurely age. So NF-kB will do many things to the cell, inflammation,
stress, premature aging if its too active and cell survival. And because of the
survival aspect this can contribute to cell proliferation and NF-kB finds
itself being a tumor promoter!!! Many tumor cells will have Hyperactive

NF-kB , cancer cells can overcome Natural aging because they are
immortal. In order to do that they lost Tumor Suppressor genes and are
required for synapses responses. Cancer cells have mutations in a lot of other
genes lost normal control, you need to use negative regulators, so called
suppressors, so in cancer cells you have overactive cell growth, normally
NF-kB is tightly regulated remember some of the targets of NF-kB are its
own inhibitors Icappa Balpha is the major one , p100 the precursor of p52 is
also a direct target of NF-kB , these direct targets of NF-kB are direct
targets of the Canonical pathway NOT THE NONCANONICAL
PATHWAY!!!!!
Icappa B alpha is a target of the first pathway, p100 is a direct target of the
IKKbeta dependent p52 /p65 pathway these are NOT targets of the
NonCanonical pathway
IMPORTANT Concept: There is cross talk between the two pathways and
this cross talk is set up in which the first pathway induce Inhibitors of both
pathways , that because p100 is also a target of the first pathway and p100 is
the primary Inhibitor of the NonCanonical pathway, p100 again does
double duty when you cleave p100 you get p52. So p100 has two different
functional roles in the NonCanonical pathway its an Inhibitor as a
precursor and after you cleave it p52 is needed with RelB TO ACTIVATE
GENE EXPRESIION!!!!!!! BECAUSE IT HAS TO FUNCTION AS A
DIMER!!!!!

What this slide is telling you is that they are some genes that are regulated
by NF-kB that are activated rite away and others that are activated slow, so
early activation and late activation, you can guess what are the NF-kB that
are doing this, for instance the guys that are activated late, which are the NFkBs? NF-kB are the transcription factors!!!!! P50, p65, remember the
NonCanonical pathway takes time, so it makes kind of sense for genes that

take a while to be activated ARE PROBALY TARGETS OF THAT

PATHWAY. At top left corner thats what a gene looks like that is involved
in early activation next to it promoter involved in late activation, in the early
activation it shows a RelA Homodimer : these love to bind to their binding
sites, rite at the center is a A/T base pair that says that this is maybe an NFkB site is a Rel A homodimer site. Now the gene in the RIGHT its a G/C
and p52 homodimers LOVE to bind to that , you see this guy BcL3 and
Tip60 they come along with p52 homodimers and that may activate
transcription!!!! Why does p52 homodimer require certain things and the
other RelA COMES ALONG AND RITE AWAY brings CBP/p300 (BIG
COACTIVOR!!!!!) Key is that BCL3 is bringing along the Histone Acetly
transferase called Tip60
But what the HELL is BCL 3?

So when he say that you cleave this p105 and it liberates this amino
terminus, notice that the protein that is liberated whether it be p50 or p52 is
smaller in size to the other NF-kBs , it missing something that the other NFkBsn have , the other NF-kB have a transcriptional activation domain, thats
what TD stands for in the slide , these guys p50 and p52 are missing it .

Now if p52 bound to that site in slide above ^ as a Homodimer and BCl3
wasnt available that gene will NOT TURN ON. Because p52 doesnt have
an Activation domain to recruit anybody!!! Its missing ability to recruit
anybody!!! Chromatin remodelers cant be recruited, Histone Acetylases cant
be recruited, So sometimes p52 brings along with it BCl3 , BCl3 is a coactivator protein that binds that binds to p52 , BCl3 as a Co-Activator will
still bring the other guys , but p52 needs to be in a complex with BCl3 to do
that, these other guys RelA doesnt have to do that , as long as cREL,RELB are there ( these have activation domains and one activation
domain is enough!!!!) . At the bottom of the slide it shows genes that
persistently active (doesnt stop). So early in the pathway what happens to
persistent genes they have two of these sites not one!! Will have ability to
bind different members of the NF-kB family in different locations in their
enhancer or promoter or both!!!! So early you get activation of transcription,
but the reason why activation is persistent, its because late the complex
binds to the G/C center, on which in early activation it was empty!!! It was
empty early because it takes time, and now in both complexes, these are all
working. Ok but now look at bottom left corner, WORKING DOESNT
NESSACRILY MEAN POSTIVE, WORKING CAN BE NEGATIVE!!!!
Both are p52s, you see that these p52s late in activation have two different
A/T and G/C , p52 will bind to A/T ,prefers G/C but can still bind A/T. If
you have a gene that has a composite promoter like this , you can have G/C
and A/T homodimers in both places , but look you still recruit Bcl3 but now
Bcl3 is going to do something totally different , ITS RECRUITING AN
HDACs!!!! Omggg not an AcetylTransferase, shit the same protein near is
recruiting the Co-Activator and other protein near is recruiting an HDAC!!!

This is an Allosteric Effect, and the Allosteric effect is cause by the binding
site of DNA being different, it means that p52 has different conformations!!!
And the p52 Bcl3 complex has different conformations. In the Co-Repressor
conformation-binding site is where an HDACs binds to, in the p52 CoActivator conformation a binding site appears where a Co-Activator binds
to. Transcriptional control is Cool, you dont want over expression or under
expression YOGURT(YOU) WANTs HOMESTATIC EXPRESSION!!!!

This shows you all the pathways that activate NF-kB , different aspect of the
Canoical pathway, NonCanocial pathway. Ok he told us that p100 is
complexed to RelB and p100 it has p52 , does that mean thats the only thing
p100 complexes to? FUCK NO ,p100 will also complex to p65 and p50 if
they are not complexed to Icappa Balpha , so some of the p50 , p65 in the
cytoplasm some of it is not with I cappa Balpha ,some of it is complexes to
p100, but its still repress and is still in the Cytoplasm because then p100
provides amino terminal domain, so in this context what p100 is doing , in
order to liberate p50 and p65 , p100 is only functioning as an Inhibitor!!! If
you get rid of p100 if you get rid of the Carboxy terminus, p52 then
dissociates with the complex, AND NOW p50 and p65 will go into the
nucleus, THIS IS VERY IMPORTANT, sometimes inflammatory reactions
are not acute they are chronic(dont go away) , evidence that this mechanism
has evolved in Chronic Inflammatory response, WHY? Well this is activated
late but its persistent!!! Also the guy that is introduce early you induce the
expression of IcappaB alpha(this will send the NF-kBs BACK TO THE

CYTOPLASM) So the first pathway has a way to suppress all the time
reduce its activity by targeting the expression of its own Inhibitor!!! In the
other guy you need this DELAYED RESPONSE these guys p50 and p65 the
genes they induce are the same genes they will induce in the other pathway
but they will just keep them on. MOST of the p50 and p65 is in this module
2 of the diagram; there is JUST a fraction of it that is in this p100
complex!!!! That means there are a total of four NF-kB modules,
heterodimer module p50 and p65 (also p50 and p30) etc We called these
four different NF-kB signaling modules that are all in the Cytoplasm ALL
end up inducing expression of NF-kB target genes.
He told us that IKKalpha is a Kinase, IKKalpha has a NLS and IKK beta
doesnt, and when you activate IKKalpha some of it goes directly into the
Nucleus and IKKalpha has other protein targets which are NOT NF-kB .
IKKalpha has protein targets which are regulating the Nucleosome
structure and if you phosphorylate those targets by IKKalpha has an effect
that relaxes the Nucleosomes!!! This means that IKKalpha is not just
inhibitor of NF-kB Kinase alpha, remember HMGB1 as an architchitrual
protein and it has another life if its outside the cell in which it binds a
receptor and activates stress and Inflammation responses and damaged tissue
responses. IKKalpha ALSO has at least another life, in one life its the
activator of a Non-Canonoical pathway and it has another role in the
Nucleus that has nothing to do with that. This means that IKKalpha can up
regulate the expression of many, many genes that are NOT EVEN
TARGETS OF NF-kB., it also a positive effector of chromatin relaxation
by having targets in the Nucleus. In addition to BCl3 theres another protein
similar to BCl3 called IkBzeta( this will also do what BCl3 does to p50
sometimes its activating and sometimes its repressing(if its brings in a
HDAC) NF-kB targets thousand of genes and these are some of the
ways!!!!!

This just shows you that

NF-kB are also targets of Kinases
themselves!!!!So DNA binding domain, IkBa dimerization domain,

Transcriptional activation domains (See RelA has two of them) these

different regions are targets for acetylation and phosphorylations by
acetlyase and kinases. And what these modifications do to NF-kB is some of
them enhance its ability to bind DNA!!! And some of them enhance NFkB ability to recruit p300. And some of them target NF-kB for destruction
by the proteasome!!!! A lot of these modifications happen mostly in the
Cytoplasm and to some degree in the Nucleus!!! And these are posttranslational modifications of NF-kB in the DNA binding domain, most of
these you find in Rel A. , and these modifications modulate the binding
activity of transcription. This means you might have a p50/Rel A(p65) that
binds DNA and gets into the Nucleus because its liberated by IkBa so you
activated IKKbeta its bound to DNA but just because its bound to DNA
doesnt mean it will be able to activate transcription , reason its a maybe is
because it has to be properly phosphorylated and acetylated and then it
will activate transcription. So additional control involved. The acetylation is
particularly important as a mechanism, why do you have two,
phosphorylation and acetylation?
The phosphorylation will enhance the ability to activate transcription. Guess
what the acetylation does? If Rel A is Hyper Acetylated it cant bind IkBa
anymore!!!! So when you active IkBa expression to make the protein, you
cant thread it back out to the cytoplasm because the Rel A that is bound to
DNA says IM acetylated IkBa I CANT BIND TO YOU NOW SRRY
IMMA BE TURNED ON ALL THE TIME ; ) maybe im a cancer cell. So
this is one way you can DE-regulate NF-kB if you acetylate Rel A too
much!!! Because it cant bind to its Inhibitor anymore.

This shows you which Serines are targets for phosphorylation, which
Lysines are targets for Acetylation, it tells you whether it affects DNA
binding, activation, transcription or decrease in activation and so on so
fourth.

Another property of the pathway, analogous to Steroid receptors)remember

for Steroid Receptors that in the absence of their Hormone , steroid receptor
is already bound to DNA but its recruiting HDACs!!!! , Then when it binds
its hormone that goes into the nucleus , changes the conformation and now
the HDACs dont get recruited instead you recruit Co-Activators having
them turn independent of new protein synthesis estrogen response genes
expression. NF-kBs has a similar mechanism, conceptually speaking but the
details are different, So in the absence of a signal that activates NF-kBs. In
the slide p65 Homoodimers are already in the Nucleus and some p50 dimers
are in the nucleus these guys got in the Nuclues because there wasnt enough
Inhibitor to go around in the Cytoplasm and there held in check by IkBa and
the precursor of p50 which is p105, DONT ASSUME THAT THERE IS
ENOUGH INHIBITOR TO KEEP ALL OF THEM IN THE

CYTOPLASM!!!!! Something arent with the Inhibitor and then they get
into the Nucleus even though there is NO signal but you still dont activate
genes, because p 65 and p50 homodimers if they bind to their Target
sequence they recruit HDACs and so they keep those targets Repressed,
actively repressed, its active repression just like in Steroid Hormone
receptors, in the absence of the Hormone!!! Active Repression because they
are recruiting something thats keeping the DNA on the Nucleosomes
(constrained state) . Ok so now you get a signal, you activate IKKbeta , a
lot more p50 and p65 gets into the Nucleus and p65 is appropriately
phosphorylated so you can enhance DNA binding and activate transcription ,
look what happens these guys that were bound to DNA in the absence of the
signal , they come off and get replaced by these guys (p50 and p65 guys can
then recruit a Co-Activator!!!! In order for this to exist there has to be rapid
dynamic exchange (it means that the Homodimer guys arent on the DNA all
the time , but if the other guys are not on these genes they stay repressed . So
DYNAMIC EXHANGE IS CRITICAL for repression in the absence of a
signal and rapid activation in the presence of a signal, AGAIN this rapid
activation is protein synthesis independent, its IKK beta driven like in stress
and inflammation.
You can use a modified version of pFRET to actually look at this dynamic
exchange, can prove by experimental technique that things are on DNA and
things that come off the DNA!!!
Marcu Lecture 7:

Again this concept of that NF-kBs. Cells control it very tightly because of
all of the things it can do, also under constant negative feedback control , if
you activate the NF-kBs. Inducing Kinases, which activate NF-kBs. You do
a time course, so you have degree of activation and this over time, if you just

have one activating signal here, you see very rapidly you induce NF-kBs.
But then you see in minutes it again drops off because of the negative
feedback control, if you induce NF-kBs. Again it will go up but it wont go
up as high as it did before, then if you wait longer it will come up again, so
there is more then one negative feedback modulator!!!!! IkBalpha is one
of them, but on the slide of negative feedback there was also p100, which is
a negative feedback inhibitor of the NonConical pathway!!! And the Conical
pathway has another target called A20, A 20 is a deUbiquitnase, what is
does is it removes Ubiquitnations from proteins, in order to activate the
IKKs especially the IKK beta, remember that IKKalpha are in a complex
with this guy called NEMO!!!!! And Nemo interprets signals coming from
multiple pathways Upstream!!!! And Nemo is required for those signals to
activate IKK Beta!!!!! NOT IKK alpha. IKK alpha functions independent
of NEMO!!!!!!!!!!! You can use NEMO knockout cells and the IKK alpha
is perfectly fine!!!! But the IKK Beta is totally compromised!!!!! Because
IKK beta cannot be phosphorylated in the absence of NEMO, but there are
also Ubiquitnations that happen, NEMO gets Ubiquitnated , there are two
types of Ubiquitnations , one results in Proteosome destruction because it
has to do with the specific Lysine that is being Ubuiqunated , there other
Ubuiquitanase that dont Ubiquitnate the target Lysine that is recognize by
the proteasome , other lysines will be Ubiquitnated these other specialized
Ubiquitnases and generally what happens when you Ubiquitnate on other
Lysines , this allows proteins to make complexes with each other or alter
protein conformation that might put protein in an active state or inactive
state!!!!! In this context NEMO needs a Ubuiqutination to put it in a active
conformation in order for the signal to get to IKK beta!!!!!! A20 is a
deUbiquitnase and A20 will remove those Ubuiquitnations!!!!!!! So NFkBs. is driving the expression of a deUbiquitnase that will shut the signal
down , even before getting to point to phosphorylate IkBalpha, more then
one way this being inhibited and more then one step in the signaling
pathway!! Intermediate step Ubuiquitnation and final step of
phosphorylation of IkBalpha because it accumulates more and you make
more of it!!! And thats what causes these cycles, which is illustrated to
some degree in the cell.

A technique that lets you look at Dynamic exchange as a way to regulate

NF-kBs target genes . Dynamic exchange of NF-kBs. ON AND OFF THE
DNA!!!! to modulate the level of expressions of their target genes . So NFkBs. gets to the cell, nuclear entry it goes to Chromatin!, allosteric
effects,covalent modifications all of these things are happening that are gene
specific depends on the NF-kBs. Target gene, remember you have to make
the Chromatin accessible, you have to recruit a Co-Activator, you have to
recruit the mediator, you have to release from the Start site!!!!!! And it
depends what that genes needs that is a NF-kBs. Target gene, some of them
will turn on rite away because the only thing you need is NF-kBs. And if
turns on that FAST NF-kBs is probably facilitating release from the
start site!!!! And the Mediator and Pol 2 will probably be there already , and
if it takes longer, and if it takes longer you will probably need to move
around Nucleosomes. Relax Chromatin so on and so fourth. Basically the
text box tells you that in order to modulate these targets you also have
exchange of dimers on and off the sites NF-kBs. dimers, remember THEY
ONLY BIND AS DIMERS!!!!!!!!!!!!!!!!!

FRET recovery after photo bleaching, where you have two different proteins
and each has a different Fluoroform, one fluoroform takes the signal from
the other that allows the signal of the first one to stay longer in
time!!!!Binding As a way to see if they are nearby each other in DNA. There
is another version of this called FRAP, and the flip side of FRAP called
Flip.
FRAT (fluorescence recovery after photo bleaching), simpler because it
only requires One fluorophore , only need to make a fusion protein of any
fluorphore protein generally GFP!!!! By recombinant DNA techniques GFP
was put in Translational reading frame with NF-kBs. p65 , but was
attached to p65 in such a way that p65 remains totally functional because
GFP as a flurophopre is a modular domain and NF-kBs has it own modular
domains one that binds DNA and one that is an activation domain all of
these domains will be functional. You put these into cells by Transpection
techniques or viral infection techniques and because its a plasmid vector
you are overexpressing this protein, tremendous amount made because you
drive the expression with promoter-enhancer in vector!! So there is so much
expression of it that there is no way that there is enough IkBa to keep it in
the cytoplasm!!! And it goes into the nucleus because it has a NLS which is
functional even though, and remember if IkBa is not bound to p65 the NLS
is VISIBLE and it goes to the Nucleus.

The point of the technique is the following, there is a lot of p65 that went
into the nucleus, some of it is going to bind DNA and some it is NOT , what
this technique allows you to do is that it allows you to look at the life time
NF-kBs. that is bound to DNA to prove that something is bound to DNA,
the way you prove that you are looking at something bound to DNA is you
make another fusion protein , same kind of strategy he explained with
HMGB1 , you make a site directed mutation in NF-kBs. p65 subunit DNA
binding domain, the site directed mutation drops its binding activity by
three order of magnitudes so it cant bind DNA but it goes into the nucleus
just like before , its NLS signal remains Intact !!!!! So you compare these
two proteins the wild type protein attached to p65 and the site-directed
mutant fused to p65 and you do a time course and look at the fluorescence.
So the kind of result you get is the following: You first photobleach and then
you look at fluorescent recovery, the mutant p65 recovers FASTER THEN
THE WILD TYPE PROTEIN. The Mutant recovers faster, what is
happening is the following, the wild type protein can bind DNA, the
Mutant protein CANT bind DNA, so whats happening is this guy is
slowed down a little bit , the difference in the two curves is that one is bound
to DNA and the other one is not. The guy that is bound to DNA his
exchange in real time is just a little bit slower and the guy that is not bound
is constantly being hit by the laser basically he has much greater mobility!!!
So ask yourself how was this experiment done? Was this experiment done
by shining the whole laser on the whole nucleus? Or was the laser shined on
just a little part of the nucleus. You could put a filter on the nucleus and
what you do is except one little spot that the laser goes through! Really what

you are looking at in this experiment is you are looking at only what
happens in that little box, the rest of the nucleus is not hit by the laser so no
bleaching or fluorescence .So in the little box if you cant bind DNA it moves
fast in the little box you could move a little slower if you bind DNA!!!!!!
The reason why that a guy that can bind DNA is slower is because it stays
on the DNA it does have a resonance time thats less then a minute and then
this guy is bleached, and then goes off the DNA and then another guy
replaces him coming from a part of the nucleus where the laser didnt hit
anything he binds DNA and now the laser sees him and you get protection
from bleaching, the protection is caused by a combined DNA!!!!! The
reason its an exchange is because first the laser hits it, its bleached, after
DNA binding is done, after a matter of some seconds , another molecule
p65-GFP replaces it coming from a part of the Nucleus where the Laser
didnt hit anything!!!!! And anything that is not bound to DNA is moving
extremely rapidly the only thing that is going to slow it down is if its bound
to DNA and you can prove it because the site directed mutant is slightly
faster!!!! That difference in the curves is all you get with regards to binding
DNA!!!!

In order to prove that its NF-kBs Binding to its DNA Sequence and not
some other one.. DONT FORGET that at the beginning of this experiment
you overexpress this protein you are making a shitload of this protein, it
might start to bind to things that it shouldnt be binding to. You have to do
one more control experiment beside the site directed mutant to prove what
you are looking at in your experiment NF-kBs.p65 BINDING TO NF-kBs.

SEQEUNCES!!! So now you Co-transpect(which means you mix two DNA

together) you take NF-kBs.p65 DNA and you mix it with another plasmid
and this other plasmid has an exact NF-kBs. DNA binding site, (384 NFkBs. DNA BINDING SITES) it integrates in chromosome depending on the
technique you use and now do the laser trick, and when you hit a part of the
nucleus with a Laser, there is one spot that is incredibly bright, its so bright
green you only see this when you Co-Transpect this has to be somewhere
the plasmid integrated in some chromosome , since you 384 NF-kBs. p65
YOU HAVE THE POTENTAIL TO BIND 384 GFP p64 to bind to this
one thing!!! So its incredibly bright green, the bright green starts getting
bleached!!! At t=0 its totally bleached but then light comes back again it
comes back again because those guys are p65 GFPs that came from
somewhere else in the Nucleus that you didnt shine the laser on, if you do
this with a Mutant p65 YOU dont see the bright thing at all!

You can do the technique one more way, you can flip it, so what FLIP is if
you can put a filter on the whole nucleus, you can put a filter on a little spot
and then the REST OF THE NUCLEUS IS HIT BY THE LASER!!!!!! So
its the flip side situation, with regard to what the laser hit. If you just hit a
little piece of the Nucleus, what you see that the bleach goes away , it takes a
little bit longer for the cluster to be bleached ,whats happening is that
something thats green leaving the DNA and being replaced by someone
thats not DNA because he came from someplace else in the Nucleus. You
end up proving your point that Dynamic exchange, even if you overexpress
something its really fast. Can you imagine what happens if you dont

overexpress something, its probably even faster, very difficult to measure, it

means that these guys that are binding to DNA, making enhancers function
,recruiting co-activators they are really coming on and off the DNA really
fast!!!!! Remember he said that in the absence of a signal NF-kBs. are
repressing their target sites because the dimers that are bound are going to
recruit HDACs and after you give a signal extremely fast , these guys
because of Dynamic exchange they are coming on and off the DNA all the
time and now there is an active NF-kBs. Sending it to the nucleus in an
active state and now replaces the repressor and it actives transcription. If you
didnt have Dynamic exchange you would of not made this observation

The point of this slide is to show how NF-kBs. is regulating gene expression
, not just by binding to DNA and recruiting co-activators but ALSO
influencing the Histone Code!!!!!!!! And also affecting transcription from
the start site, it will modulate the structures of the Nucleosomes, by
recruiting appropriate proteins that are chromatin modifying proteins, will
also help genes initiate transcription, also help genes with polymerase
release from the start site. Primary response genes activated in less then
two hours, they generally have CpG islands, in their promoter they have
more then one CpG dinucleotide a property of these very rapidly active
genes, if you look at the gene before its activated, but Sp1 has already
recruited a Histone Acetyltransferase , its helping NF-kBs. before it even got
there, its already helped relax the chromatin , even Polymerase is there with
the Mediator , so some Nucleosomes already got out of the way, now you

bind NF-kBs. look what NF-kBs. is recruiting , its P-TEFb (RNA Pol 2
Transcription elongation factor) this has kinase activity , specialized kinase
that phosphorylates the CTD of RNA Polymerase and thats what the
polymerase needed to release from the start site, this gene that is activated
very fast, NF-kBs. doing that independently of initiation of transcription
, its by release from the start site because it can release P-TEFb and that was
needed, thats why it can activate so fast!!!!!
A secondary Response gene: You need more then two hours, maybe this
gene was in Heterochromatin to begin with, or maybe it was in a state where
it was primed for transcription, maybe a pioneer factor is bound and maybe
not, NF-kBs. is recruited but thats not enough , nucleosomes get modified
specific acetylation and methylations required , NF-kBs. again recruits PTEFb but more towards the end. This does not mean its not releasing from
the start site, but its doing two things now, its facilitating formation of the
pre-Intiation complex , its facilitating Initiation and releasing from the start
site NF-kBs.IS HELPING EVERTHING Because it was needed very
early .

Some NF-kBs target genes are being actively repressed!!!!!!!!!( The NFkBs. repression mechanism is in the absence of a signal if the gene has
p50 homodimers,p65 homodimers and they recruited a HDAC facilitating
repression in the absence of a signal some genes do it this way) Other genes
have a much more potent way of doing it, they have another DNA binding
site for another protein in the diagram it shows cJUN. The cJUN

transcription factor binds to AP1 sites, if its a JUN homodimer it will

recruit this protein NCoR and NCoR is like a co-repressor that will recruit
HDACs so that DEAcetlyates the Histones, you see this repression complex
puts this bar across the start site , you dont get transcription initiation
because it cancels transcription from happening.
So now you activate NF-kBs. LPS (polysaccharide on the surface of the cell
set up a signal transduction pathway, which is one rapid way of activating
NF-kBs. p65 brings along a chromatin modifier ph12, p65 is binding as
Heterodimer as it should , what pH12 is doing is that it is removing triple
methylations from the K20 Lysine from Histone H4 , in addition to
recruiting an HDAC before it also recruits a methylase that makes sure you
keep the DNA on the Nucleosome , by putting a triple methylation on K20 ,
THEN YOU HAVE THE phf 12HISTONE demethylase which is
removing the methylations and now you get acetylations because you
recruited your co-activator , now you will get initiation and maybe activate
these target genes of NF-kBs. and release from the start site.
So NF-kBs has the ability to recruit Co-Activators, Histone demethylases
and kinases that phosphorylate the tails of RNA Polymerase and chromatin
remodeling complexes. All of these things, it depends what the gene needs,
to be activated also depends on the binding sites of other transcription
factors that are over the enhancer and promoter that NF-kBs. is in
associating with.

This shows you about genes and whether if its constituve or late.

Shows the multiple ways NF-kBs. can activate transcription , depending on

what the gene needs. Showing how NF-kBs are playing different functional
roles.

Above is another kind of signal transduction pathway; it involves a

transcription factor called STAT!!! STAT needs to be phosphorylated to
dimerize , so you have a Cytokine (facilitates two receptor molecules
making a dimer, when that dimer forms it has a Kinase associated with them
called JAK and JAK kinase becomes activated because of receptor
dimerization, that kinase phosphorylates STAT , STAT dimerizes goes into
the nucleus binds DNA and maybe activates transcription!!!!! So this is a
situation where you are not dealing with an Inhibitor , that in order for this
protein to dimerize in an active state , it needs to be phosphorylated, that
wasnt the NF-kBs mechanism. NF-kBs does get phosphorylated but that
enhance its ability to activate transcription!!!!! It doesnt facilitate its
ability to enter the nucleus or bind DNA. In this case this STAT CANT enter
the Nucleus and bind DNA unless its phosphorylated in the cytoplasm!!!!

Here it shows you that STAT is a family and you can see here, STATs and
NF-kBs collaborate, that would be a gene that has NF-kBs binding site and
STAT binding site in its promoter and enhancer, this gene would need those
two signaling pathways.
How cancer cells are different from Normal Cells, where transcription
factors fit in the altered cell physiology that is characteristic of a cancer cell.
Need to ask questions if you are doing this as a Normal protein or mutated
protein.

Here you take primary cells from the mouse and the famous primary cell
would be skin cells. These cells shouldnt divide indefinitely!!!! Every round
of cell division their telomeres get progressively shorter and shorter!!!!!!!
Cells can go through crises and make a ball of cells (rapidly dividing)
Transformed cells are dividing to fast and they dont respect the boundaries
of their neighbors and they are not contact inhibited!!! You can have more
cells in a smaller amount of space!!!! Sometimes one of these cells escape
and can form another ball equivalent to Metasis of the tumor !!!!!!But still
the cells may not be immortal. Unless they became immortal in the crisis
phase!!! You need to avoid fast growth. If you want to become a cancer cell
you need another protective mechanism cells have , thats Apoptoiss
(programmed cell death) , If a cell divides too fast , Apoptosis is a form a
programmed cell death in which the cell TOTALLY disintegrates
Necrosis: A dead cell but not Chopped up.
Cancers cell also have an altered Cytoskeleton

Cancer cells can change their morphology, something about their

cytoskeleton where they cant detach well. Similar type of thing we they are
contact
inhibited,
Normal
cells
spread
out

What causes this at the level of genes? Mutations of course damage to DNA
Xrays,gamma rays, radiation, all kinds of shit!!
Mutations can also be cause by cells that go to S phase to quickly, the
polymerase that replicated DNA might have error if you go through S-phase
too fast!!!!! So you start putting in the wrong nucleotides and thats then
transmitted to a daughter cell and the daughter cell has a mutation. There
could be something wrong with DNA Repair not being able to keep up with
fast dividing cells. One mutation can mutate a class of genes that are positive
regulators of cell growth control these are genes that are facilitating
progression of the cell cycle, helping cells go from G0 to G1 S to G2, one

you mutate one of these lets say you make it better then it was before , a
mutation can make a protein more active , or mutation makes the protein
have a longer half life so it stays around longer to make to much cell
division, if this happens you might get Cell transformation.
Oncoproteins or proteins that if you mutate them, have a positive effect on
cell growth and could facilitate hyperplastic growth, too much cell division
and maybe lead to cancer if other things happen. These classes of proteins

have many different functions in the cell, some of them are growth factors
that bind to receptors, the receptors themselves and some of them are
transcription factors etc You dont have to be a Transcription factor to
oncoprotein
He talks about Oncoproteins that are transcription factors, and other
transcription factors that repress these proteins ability to withdraw from the
cell cycle.

We are diploid organisms!!!!! Here you have a mutation of one gene and the
other one is good, but if anything happens to the good one , here good gene
is silence and this is abnormal activity , at the top you have a situation where
LOH(Loss of Heterozygosity) that would mean when chromosomes split
and daughter cells sometimes you dont get separation of two chromosomes
and you only get one chromosome and its replicated, you can lose the good
allele and instead use the mutated allele, so you would have mutation on
both daughter cells. Sometime in some cancers you only need to mutate one
allele and that is enough to compromise the function of the Locus, this
would be Haploinsufficient tumor suppressors.
For you to get cancer YOU HAVE TO MUTATE SEVERAL NEGATIVE
MODULATORS THAT MAKE SURE THE CELL CYLCE GOES AT
THE RITE RATE!!!!!!!!
That make sure you dont go to S phase to fast, also things that make sure
that cells apoptosis themselves when they are bad. All of these are so-called
Tumor Suppressors two different types of tumor suppressors one that
regulates the cell cycle and its a co-repressor of transcription called Retino
blastoma protein critical regulator of the start of the cell cycle co repressor
of trandscription by other transcription factors!!!!!and then another one p53.
p53 IS MUTATED IN 50% of all Human cancers, because p53 is one of
the only tumor suppressors working as a transcriptional Activator binding
DNA that activates the transcription of genes and the targets of p53 cause
synesisent or apoptosis(these are the things you are losing) but its a
transcriptional activator!!!!

One proto-oncogene that is positive for transcription! Remember that they

are multiple mutations happing in these cells at the same time. Cancer is an
accumulation of mutations in the end it gives you a cell that divides too fast,
survive and be immortal!!! The slide shows you stepwise progression in
Colon cancer

pRb is the gatekeeper in the G1 phase of the Cell Cycle, functions as a

transcriptional Co-Repressor!!!!
It represses the activity of E2F
transcription factor

MYC is a transcriptional activator that belongs to a class of proteins that

have this Carboxyl terminal domains, that is sort of like a Leucine Zipper
(Helix-Loop-Helix) but not exactly, but it allows MYC to Homodimerize or
dimerize with a target protein and that creates DNA binding domain like
CREB . Slide shows you that there is c-MYC which has this Helix-loopHelix domain that needs to interact with target protein, has a very complex
activation domain that is subjected to different phosphorylations it has this
MYC binding site CACGTG , MYC will only bind if there is twelve base
pairs the three base on each side of this core binding site can vary a lot , the
core is important because this is where the protein is making contact with the
DNA cant bind if you have at least a twelve base pair sequence and it binds
as a dimer!! Max are Myc like genes on Homologous chromosomes, there
is N-Myc and L-Myc and have different activation domains, all bind the
same sequence but their activation domain is different!!!
MAX IS THE ONLY THING THAT WILL DIMERIZE WITH THESE
GUYS INSIDE THE CELL AND that will bind DNA and maybe activate
transcription, these Myc could cross dimerize with each other but the bond
they form is extremely weak, its so weak they are basically incompatible
with each other. IF c-MYC goes with MAX then you can bind DNA and
activate transcription

Marcu Lecture 8:
You see that the entire Nucleus was fluorescent, the reason the whole
nucleus is fluorescent you are looking GFP but you are not looking it at with
a high power laser.

In this FLIP experiment, you use a high power laser, and you bleached the
Whole nucleus, you protecting a region from the high power laser.
In the FRAP experiment trying to measure the dynamic exchange, p65
bound to DNA and at certain rate that is what you are trying to measure, but
then when it gets replaced it gets replaced by a GFP that is outside where the
laser is, Its a P65 GFP coming from the outside where the laser would be.
Coming somewhere outside the nucleus because things are rapidly moving
around all the time, the p65 GFP that was where the laser was dissociates
and is replaced by a green GFP.
If something bound to DNA it will stay there for a little while but guy that
cant bind to DNA wont bind.
What is the difference between the pbFret with HMGB1? And the Frap?
The laser is just being used to bleach, what was in the HMGB1 experiment
to look at the proximity of two molecules in DNA to look at if they were
nearby each other, in the FRAP experiment you are just looking

p65 coming on and off the DNA.

In the HMGB1 experiment, what the experiment was is you are trying to see
when HMG1 is bound to DNA and Glucocorticoid receptor is bound to
DNA are they binding nearby each other in any of the sites they bind are
they in association with each other, one is a transcription factor and the other
is DNA architurel protein, you need two fluroforms, one fluroform fused to
one protein and the other fused to other protein , one flouroform takes away
energy from the first one in the pbFRET experiment when using the High
power laser , because in this experiment the only thing you are using is a
high power laser and you are relying on the second fluorform to take some
of the energy away from the first one flourphore so you can see the color.

cMYC is a Helix-loop-Helix type transcription factor has activation

domain,DNA binding domain , you need the Helix-loop-Helix for an
indirect reason ,once this complex forms you get a active DNA Binding
Domain , it binds to a 12 base pair sequence , see other members of the Myc
family they also have activation domains and their activation domains
determine their differential function because they bind to the exactly same
sequence. If you mix these guys together like you Myc-Myc for some reason
their Helix-loop-Helix are not compatible with each other, cant dimerize
they dissociate to rapidly, EVERY one of these in order for them to bind
DNA they need a partner protein but not one of themselves, ONLY one
protein in the cell can do this and thats the Max protein, Max makes
Heterodimers with all of these Myc proteins and now they can bind this

target sequence, and activate different genes some genes similar, some
different, because of difference in their activation domains!!!! Depending
on what the gene needs to transcribe!!! Max does not have a activation
domain, the only thing it is a DNA-Binding domain, just a very small
protein where its only 150 amino acids, the Myc protein is 439 amino acids.
Max doesnt have a repression domain, it just has a DNA binding domain,
the only purpose that Max has is to allow Mycs to bind to their target
sequence , if you have a MAX knockout cell , no MYCs will bind anything ,
MAX IS ESSENTIAL FOR MYC TO BIND TO DNA AND ACTIVATE
TRANSCRIPTION!!!!!!!!! Because Max is required for Myc to form that
DNA-Binding domain as a Heterodimer!!!!1

There are other proteins kind of like MAX but not exactly!!!! E-BOX is the
CACGTG binding site, ,Myc helps a lot of proliferation and self renewal of
cells ,also drives Heterochromatin to the EuChromaic state so it an
activating transcription factor , without its Activation domain of course it
cant do that , Myc facilitates Cell growth, cell cycle entry, proliferation
also a tumor promoter if its overexpressed , but it also directly induces
Cell death . The only way MYC will induce Apoptosis, it has direct target
genes is if Myc is OVEREXPRESSED , then some target genes that require
more Myc , some of them are inducing Apoptosis and those same genes that
induce apoptosis usually also require another transcription factor and thats

the p53 tumor suppressor , so these are special genes that need a lot of
Myc and most of the time active p53 in order for them to turn on. What this
slide also shows you is that the only thing MAX will bind to , if there is too
much expression of these other proteins that MAX likes to bind to , there
may not be enough MAX for Myc to bind to. Another thing is that they
could be facilitating transcription repression!!!! The key here is that when
Max binds to these other guys, can they still bind to Myc? The only way
these guys will take Max out of the equation, is if Max interaction with them
is mutually exclusive with regard to its interaction with MYC, its NOT
mutually exclusive, Max will interact with Myc and the other guys at the
SAME TIME. These other guys point at E-box and that arrow is then goes to
repression, this means Max interacting with these other guys are repressing
transcription of MYC-Max , all of these other things are blocked off , if you
go the MYC-MAX way everything is fine , thats because these other guys
are not with Max.

The core-binding site is in the center of the slide, around this are all other
Myc heterodimers that are binding to this site, MYC-MAX of course binds,
cell cycle entry, myc/max activation of transcription, growth cell cycle
progression. LOOK max/mad sin3 and some of these other ones you hit
he binding site and now what happens is, is you recruit a Histone
Deactelyase complex and this transcriptional co-repressor Sin3 , only the
co-repressor will bring along the HDAC , the HDAC has to be recruited by
a Co-Repressor . MAX CANT BIND SIN, BUT MAX/MAD can bind Sin,

so if Max is in a dimer with one of these guys and it binds this core-binding
site THE RESULT IS ACTIVE TRANSCRIPTIONAL REPRESSION!!!!!!
At the level of Epigenetic change in the Nucleosomes, you are deacetlyating
the Nucleosomes, so that is an epigenetic mechanism, its an active
mechanism because an enzyme is involved, however if MAX/MAX
homodimers bind AND max can homodimerize to itself MAX/MAX can
also bind, it breaks the transcriptional circuit because it doesnt have an
activation domain but its not a mechanism of active repression sort of you
dont have an activation domain and you cant activate transcription because
your missing something because it only comes from one of the Mycs. The
HDACs are NOT INVOLVED IN THIS MAX/MAX only can be recruited
by the co-repressor, which is the other guy that interacts with Max. Maybe
these guys one way they are working is because you take Max away from
Myc!!!! Its very clear from the diagram that the only thing MAX needs to
repress transcription is one of these other guys but its not interacting with
Myc instead of interacting with Myc , its interacting with one of these other
guys. However there is still enough Mad to interact with Myc at the same
time. THERE IS A TREMONDOUS AMOUNT OF MAD IN THE CELL
AT ANY GIVEN TIME VERY LITTLE MYC.
Myc under normal steady state conditions has a half life of Ten to fifteen
minutes, the protein auto destructs by the proteasome within ten to fifteen
minutes, it has the half life of a bacterial protein even though its a protein in
mammalian cells. MAX HAS A HALF LIFE OF MORE THEN TWELVES
HOURS!!!!!! Tremendous difference between Myc and MAX stability. Myc
turns over very fast, Max barely turns over at all, so always a tremendous
amount of MAX and very little Myc!!!! Myx/Max once it binds DNA it
drives Cell Cycle progression ,its going to make cells grow!!! And if you
dont want abnormal cell growth, you really have to have LOW Myc/Max
all the time, it has to be mostly MAX/MAX or Max with one of the other
guys shown in the slide, if you jack up the amount of Myc/Max, there is
mutations in Myc that have been discovered in some cancers and then the
HALF life of the MYC protein goes up and finds itself more often with Max
and Max less often with itself, or just activate the transcription of Myc and
because the mRNA goes up and even if the protein half life is still low , you
still have more protein because there is more MYC mRNA to make it . Too
much Myc/Max binding to this core binding site will give you
Hyperplastic growth , cells go through S phase very rapidly , its at least a
doubling of cell growth rate or more , its really fast which could lead
potentially to cancer , the only reason that it doesnt contribute to cancer on
its own, to much Myc will also give you apoptosis , now if you lose the

p53 tumor suppressor , if its mutant or you dont have it anymore , then you
might go to cancer and if you lose this the apoptosis is also loss , you need
p53 for high levels of Myc to cause apoptosis. There are some survival
factors that are up regulated in cancer BCl2 is one of them, it part of a family
of proteins that are Antiapoptic proteins, BCl2 and other proteins are direct
targets of canonical NF-kBs signaling, they are direct NF-kBs targets So if
NF-kBs is to active , and at the same time you have too much active Myc
then you have an Antiapoptic protein that is accumulated and the cell may
not be able to commit suicide because Anti apoptosis and apoptosis they
antagonize each other , BUT IF p53 is still there you still may not have
cancer all you may have is an Immortal cell , immortalization but not
transformation , not transformation that the cell still respects its neighbors
,the cell cant metastize to another site ,its not a malignant cancer. If you
want a malignant cancer most of the time you have to get rid of p53. Also
to much Myc will also repress cell differentiation, if you operate too much
Myc , the cell keeps going through cycle of cell division and that inhibits
from cells to differentiation , but many times when a cell differentiates it
must be post-mitotic , it CANT BE DIVIDING AND THEN IT
DIFFERTAITES .So Myc antagonizes differentiation of cell types .

How does Myc do all of theses things? The Slide shows Myc/Max binding
to its core sequence, one of Myc activation domain recruits its own big CoActivator complex called TRRAP (which is just a BIG as CBP/p300) .
Myc PREFERS TRRAP!!!!! And TRRAP brings along GCN5 .
This is a case of terminal differentiation many times instead of having
Myc/Max you have Max with MAD these reason is you need to down
modulate the genes that Myc is activating that drive the cell cycle, many
cells that differentiate are not in cell cycle anymore and what Myc is doing,

that is antagonizing that if its with Max, but if Myc is not in the Equation,
Max can pair with one of these other proteins and can recruit SIN3 and
HDAC and is binding the same target sites and same cell cycle progression
gene , at the bottom of the slide MAX /MAD will be recruiting CoRepressors and puts it in inactive state , thats why one of the main reasons
Myc is antagonistic to differentiation, Max/MAD are required for cells to
differentiate!!! But if Myc and Mad thats antagonistic to cell differentiation.

Myc regulates a lot of different things of cell physiology, MYC IS A DNA

BINDING TRANSCRIPTIONAL ACTIVATOR!!!!!! It interacts with
Max as a dimer to bind DNA.
RNA pol 1 that drives the transcription of ribosomal RNA
RNA pol 3 that drives the transcription of 5SrRNA and tRNA .OMG MYC
regulates these tooo in a positive way. But not by Binding DNA, MYC
interacts with other Co-Factors that then get recruited to these other
polymerases, Myc interacts with TF11B , Myc also interacts with PTFFB
(Release factor)
SO MYC IS REGULATING TRANSCRIPTION ALMOST EVERY
SINGLE thing in the FREAKING CELL AT THE SAME TIME Key is that
if there is ENOUGH MYC!!!!!
But remember there is not a lot of Myc , normally Cell must keep Myc
levels really low , because if you drive the transcription of almost every
single thing in the whole cell , the cell will basically go rite through into S
phase .
Remember that Steroid receptor have a Hormone Binding domain and that
domain functions in a Modular way, the way an activation domain and a

DNA binding domain does, and steroid receptors respond to Hormones

because they have the Hormone binding domain and when the Hormone
binds it alters their conformation so they can bind to the Co-Activator.
One more thing about the Hormone Ligand Binding domain of steroid
receptors, it doesnt care if its attached to a steroid transcription factor, you
can actually clone the domain that just binds the Hormone and you can fuse
it to MYC!!!! In translational reading frame put it in a C-terminal , so Myc
has full functionality , so it can still interact with Max and still in activate
transcriptional . But now at its C-termnius it has the ligand binding domain
of the estrogen receptor, just made an estrogen response Myc protein, now
Myc wont work unless Estrogen is there to alter its conformation.
What regulates Endogenous Myc are growth factors, growth factors that
bind their receptors set up a Signal transduction pathway that activates Myc
transcription, Myc needs growth factors in order for it to turn on. Other wise
Myc levels are even lower if the c-MYC gene is not being transcribed that
much.
But now if you got this Myc protein that has an estrogen receptor binding
domain attached to it , now you give the cell estrogen it will go all the way
to S-phase !!!!However Myc will let the cell grow and go to S phase, you
will literally get DNA synthesis and the cell is growing, Well Myc drives
Pol 1, Pol2.Pol 3, MYC HAS NO PROBLEM TO UPREGULATE
EVERYTHING TO LET THE CELL GROW to the rite size and go to
DNA synthesis except cell division is not to good!!! Its freaking amazing
how much Myc will do!!!!!!! To cell growth control THATS WHY
UPREGULATION OF MYC IS A REALLY BAD THING!!!! To happen to
the cell.
If you have a p53 wild type cell and Myc is at high levels, the cell will
commit suicide!!!!
If p53 is Mutant then cell CANT suicide probably CANT then maybe these
things can happen in respond to let say the estrogen hormone.
The only thing that Myc will interact that will directly interact with CTD is
PFTEFFB that kinase that phosphorylates it.

In addition Myc will drive the transcription of Pol 2 targets genes, in

different ways, one Myc/Max in E box gives you transcription, if its Max
WITHOUT Myc its HDACs YOU DONT GET TRANSCRIPTION1!!1!!
Look at third case, there is MYC/MAX but its not binding to DNA instead
theres another protein in between and thats the guy that is binding to DNA,
the name of that protein is MIZ1 and interacts with Myc/Max but when this
happens you dont get transcription!!!!!! POL 2 targets of Miz1 are
suppressed when in a complex with Myc/Max , these are just other things
Myc is doing . You also see Myc regulating Pol 3 targets, here Myc is
interacting with TF11B and facilitates its recruitment to the promoter.

Ok so you have MYC/MAX driving some of the expression of its direct

targets, some of its targets are Cylins and Kinases that drive the Cell Cycle.
Here it shows MYC/MAX binding Miz-1, Max is just coming along for the
ride look at the bar it means you block transcription, and you are you
blocking the transcription of Cyclin-dependent kinase inhibitors!!!!! ,
Kinases are activated by growth factors that then put cyclins in a active state,
some of Mycs direct targets are cylin kinases, but there is another protein
that is saying slow down Myc/Max it says Im Miz 1 when I bind DNA I am
going to activate the transcription of Cyclin-depedndent kinase inhibitors!!!
That will inhibit the activity of the kinases that induce transcription!!!!! Myc
then says FUCk you Miz1, Myc says go and try to activate those targets and
I will complex with you and turn off your targets. Myc gets its way and
THATS WHY YOU GET CELL CYCLE PROGRESSION. This sort of a
Balancing act going on. So if there is too much MYC it says I have a lot of
me to inactivate Miz1 but also drive by own genes at the same time.

Just shows what are some of the MIZ- 1 target genes are. P15 cyclin
dependdent kinase inhibitor and p21 these are direct targets of Miz1 but
when Miz 1 is interacting with MYC the circuit is broken and then these
genes p15 and p21 are not actiavated!!! And one of these genes p21 is a
direct target of p53 tumor suppresor , so one way p53 regualates cell cylce in
a NEGATIVE WAY to induce some of the same genes Miz-1 is inducing .

This tells you Myc has a complicated activation domain!!!!!!!! Myc can
recruit Co-Activators ,Mediator ,Chromatin remodoling proteins , its really a
multi-functional activation domain

Some of the things Myc recruits and how it recruits things because
activation domain has different parts to it.

This also shows how c-Myc will interact with pTEFb and will recruit it to
MYC/MAX binding sites, even if Max is not there and it gets to
transcription complex and TEFb is going to phosphorylate the tail of
polymerase and release from the start site.

He ask himself what are the genes that turn on if he Up regulates Myc? And
the genes that turn off, Used DNA microarray technology, he sequence
thousands of messenger RNAs in the cell at the same time. He found that if
you up regulate Myc you up regulate the transcription of thousand of
genes, then you look at their promoters and enhancers, they DONT have
Myc/Max binding sites there, so how the hell does Myc do that, its up
regulating those genes but its not binding to their DNA, it turns out since
you can recruit pTEFB , Myc is sort of like a transcription factor that is a
transcriptional amplifier , it amplifies the expression of many genes that
it doesnt even bind to its promoter or enhancer!!! See how if you
upregulate Myc thousands of genes get up regulated because the polymerase
is rapidly releasing from the start site, since Myc can recruit pTEFB. When
Myc is amplifying so many targets at the same time its not because its
binding to DNA ITS BECAUSE ITS RECRUITING pTEFB .

So what happens in Cancer, as you learn the cell keeps TIGHT CONTROL
of Myc , low level of transcription, low stability of the protein , Most
dramatic example in how you can up regulate Myc is in lymphoid cells ,
Burkers Lymphomas is a type of B-Lymphoid cancer , it always presents
the same chromosome abnormality , most of the time a fusion between
chromosomes 8 and 14 , always chromosome 8 involved as a fusion partner
and you are breaking chromosome 8 every single time in the same place at
the 8 q- position and replacing it with another chromosome , chromosome
break and the rejoining happens between two different chromosomes, near
the telomere of chromosome 8 thats where the Myc Locus is, you break
rite nearby the Myc locus , that encodes the c-Myc gene , you are fusing
the locus that encodes antibody genes to the Myc Locus , so what Myc is
getting is its getting an extremely strong transcriptional enhancer that
determines high level transcription of immunoglobins heavy chain genes and
the reason why Immunoglobins heavy chains are up regulated is because
they have these cell identity type enhancers that dramatically up regulates
their transcription but only in lymphoid cells and you are not making
antibodies in other cells because this enhancer is cell type specific. Burketts
Lymphoma is a B-Cell its the rite cell type , have tremendous expression

but one of the Immunoglobin chromosomes is fused to Myc , so the

enhancer of the Immunoglobin locus thinks Myc is an Immunoglobin
gene and up regulates it!!!!!!!!! Marcus lab was one of the labs that
discovered this!!!! They first didnt know what the heavy chain locus was
fused to and then they found it that it was fused to Myc.
Myc can also Up regulate microRNAs which will alter transcription post
translation ally!!!

Another way you can deregulate Myc in colon cancer, its by a different
mechanism, you dont have a chromosome abnormality, you have a problem
with making to much B-Catenin.( Transcription co-activator) and usually
B-Catenin is constantly being degraded so the cell usually doesnt make a lot
of this Co-Activator but in the signaling process that activates B-Catenin is
damaged or B-Catenin enhances its stability and that can happen if you get
some mutation in some cell plasma proteins that regulate its turnover!!!,
mutations common in colon cancer , if too much B-Catenin it goes into the
nucleus and guess what B-Catenin does as a Co-Activator OMG IT
ACTIVATES THE TRANSCIPTION OF MYC!!!B-Catenin is the coactivator for Tcf-Lef transcription factor, which is one of the transcription
factors you need to drive Myc transcription, so you get too much MYC
transcription!!!!! Because you are not properly regulating this co-activator,
thats the way you do it in Colon cancer. pRB is mostly a transcriptional corepressor , and how it negatively regulates E2F transcription factors drive
their target genes , pRB is a negative regulator of the cell cycle and E2Fs
are positive regulators of the cell cycle , pRB is a special co-repressor
only interacting with DNA bound E2F and how p53 feeds into this.