10 05 2006 Lifescience 1

阮麗蓉
中研院基因體研究中心助研究員
台大分子醫學研究所兼任助理教授
Functional Genomics
中研院基因體研究中心
Tel: (02) 2789-9930 ext: 253 (O); 2789-8775 (L)
ljjuan@gate.sinica.edu.tw
Gene Expression
Sep 27, 2006

國防生科特論
阮麗蓉實驗室
Functional Genomics
Tel: (02) 2789-9930 ext: 253 (O); 2789-8775 (L)
Virus-Cell Interaction
Sep 27, 2006

國防生科特論
阮麗蓉實驗室
Functional Genomics
Tel: (02) 2789-9930 ext: 253 (O); 2789-8775 (L)
Could chromatin modifiers be the
common targets?
Sep 27, 2006

國防生科特論
Transcriptional Regulation in Eukaryotic Chromatin
Inactive chromatin
Core histones H2A, H2B, H3 and H4
DNA
Linker histone H1
Alberts et al
Nature 1997 389, 251
Cell Dec 2005
ouse embryonic stem cells including decreased

ll for three H1 genes global nucleosome spacing,
croarray reduced
ethylation of specific CpGs local chromatin compaction,
thin the regulatory regions of some
andof
e H1 regulated genes is reduced. decreases in certain core
histone modifications.
Inactive chromatin
DNA
Linker histone H1
Active chromatin
Inactive chromatin
DNA
Linker histone H1
DNase I hypersensitive sites

DNA demethylation
Histone hyperacetylation, Reduction of
H1 content
Incorporation of histone variants
Active chromatin (H2A.Z and H3.3) and/or non-histone
chromosomal proteins (HMG proteins)
These regulatory
DNAs,
enhancers,
silencers and
insulators are
scattered over
distances of
Cis and Trans Elements of
roughly 10 kb in
Transcription fruitflies and
100 kb in
mammals.
Nature
(2003)
424, 147
Inactive chromatin
DNA
Linker histone H1
Active chromatin
Inactive chromatin
DNA
Linker histone H1
Activators
Active chromatin
Inactive chromatin
DNA
Linker histone H1
Activators
Coactivators
Active chromatin
Inactive chromatin
DNA
Linker histone H1
Activators
Coactivators
Scaffold
Histone modifiers
ATP-dependent chromatin remodeling complexes
DNA demethylases
Active chromatin
Inactive chromatin
DNA
Linker histone H1
Activators
Coactivators
Scaffold
Histone modifiers
DNA demethylases
Active chromatin
Inactive chromatin
DNA
Linker histone H1
Activators
Coactivators
Scaffold
Histone modifiers
DNA demethylases
Active chromatin
Inactive chromatin
DNA
Linker histone H1
Activators
Coactivators
Scaffold
Histone modifiers
DNA demethylases ？
Active chromatin
Inactive chromatin
DNA
Linker histone H1
Repressors Activators
Corepressors Coactivators
Scaffold Scaffold
Histone modifiers Histone modifiers
ATP complex ATP-dependent chromatin remodeling complexes
DNA methylases DNA demethylases ？
Active chromatin
Inactive chromatin
DNA
Linker histone H1
Activators
Coactivators
Scaffold
Histone modifiers
DNA demethylases
Active chromatin
Santos-Rosa and Caldas, Euro J. Cancer (20
Amino Acid
Inactive chromatin
DNA
Linker histone H1
Activators
Coactivators
Scaffold
Histone modifiers
DNA demethylasesHistone acetyltransferase (HAT)/
Histone deacetylases (HDAC)
K-Ac
Active chromatin
Inactive chromatin
DNA
Linker histone H1
Activators
Histone Coactivators
methyltransferase Scaffold
(HMT)/ Histone modifiers
demethylase ATP-dependent chromatin remodeling complexes
DNA demethylasesHistone acetyltransferase (HAT)/
K-Ac
Active chromatin
Inactive chromatin
DNA
Linker histone H1
Activators
Me-K DNA demethylasesHistone acetyltransferase (HAT)/
K-Ac
Active chromatin
Inactive chromatin
DNA
Linker histone H1
Activators
Me-K DNA demethylasesHistone acetyltransferase (HAT)/
Me-R Histone deacetylases (HDAC)
K-Ac
Active chromatin
Me-K
Me-R
K-Ac
Me-K
Me-R
K-Ac Histone Code
Me-K
Me-R
K-Ac Histone Code
Recognized by specific factors

BioEssays (2005) 27, 164
BROMO
CHROMO
SANT
Tutor
WD40
SWIRM
Me-K
Me-R
K-Ac Histone Code
Different cellular event

Histone code hypothesis
Me-K
Me-R
K-Ac Histone Code

Curr Opin Cell Biol (2002) 14, 286
Me-K
Me-R
K-Ac Histone Code

Gene activation
Me-K
Me-R
K-Ac Histone Code

Curr Opin Genet Dev (2005) 15,

163
Gene repression
HP1
Me-K
Me-R
K-Ac Histone Code

Histone modification associates with
cancer formation
Me-K
Me-R
K-Ac Histone Code

Active chromatin
cancer formation
M HMT
Shi Huang (2002) Nat Rev

Cancer
Me-K
Me-R
K-Ac Histone Code

cancer formation
An, Kim and Roeder (2004) Cell 117, 735
Me-K
Me-R
K-Ac Histone Code

cancer formation
An, Kim and Roeder (2004) Cell 117, 735
Loss of acetylation at Lys16 and trimethylation at Lys20

of histone H4 is a common hallmark of human cancer.
Fraga et al Esteller (2005) Nat Genet 37, 391
Me-K
Me-R
K-Ac Histone Code

Inactive chromatin
DNA
Linker histone H1
Activators
Coactivators
Scaffold (HAT/HDAC)
Histone modifiers ex: histone acetylase/deacetylase
DNA demethylases
Active chromatin
Inactive chromatin
DNA
Linker histone H1
p53 (JBC 2000) Activators

Coactivators
HDV small δ Ag
(Virology 2004)
Scaffold (HAT/HDAC)
Steroidogenic factor 1 ATP-dependent chromatin remodeling complexes
(MCB 2005)
DNA demethylases
Active chromatin
Inactive chromatin
DNA
Linker histone H1

Coactivators
HDV small δ Ag
(Virology 2004)
Scaffold (HAT/HDAC)
(MCB 2005)
DNA demethylases
Active chromatin
Inactive chromatin
DNA
Linker histone H1

Coactivators
HDV small δ Ag
(Virology 2004)
Scaffold (HAT/HDAC)
(MCB 2005)
DNA demethylases
Active chromatin
Inactive chromatin
DNA
Linker histone H1

Coactivators
HDV small δ Ag
(Virology 2004)
Scaffold (HAT/HDAC)
(MCB 2005)
DNA demethylases
Active chromatin
Inactive chromatin
DNA
Linker histone H1
HCMV IE2 Activators

(EMBO 2004 23, 2269) Coactivators
Scaffold (HAT/HDAC)
DNA demethylases
Active chromatin
許志宏
Viral proteins
A common theme
Chromatin regulators
Viral proteins which regulate histone modifier activity
DNA Viruses
Adenovirus E1A △ HAT Ait-Si-Ali et al. Harel-Bellan, Nature (1998)

▼ HAT Chakravariti et al. Evans, Cell (1999)
Perissi et al. Rosenfeld, PNAS (1999)
Hamamori et al. Kedes, Cell (1999)
E1B ▼ HAT (PCAF/p53) Liu et al. Liao, MCB (2000)
EBV Zta △ HAT Chen et al. Liberman, MCB (2001)
HCMV IE2 ▼ HAT Hsu et al. Juan, EMBO (2004)
HPV E6 ▼ HAT Thomas and Chiang, Mol Cell (2005)

▼△ HMT Juan (Unpublished)
E7 ▼△ HMT Juan (Unpublished)
Viral proteins which regulate histone modifier activity
RNA Viruses
HCV Core △ HAT Gomez-Gonzalo et al. Lopez-Cabrera, Virology (2004)

▼ HAT Virology (2004)
▼△ HMT Juan (Unpublished)
HIV-1 Tat ▼ HAT

(TAFII250/histones/MHC class I genes)
Weissman et al. Singer, PNAS (1998)
Wong et al. Harrod, JBC (2005)
▼ HAT
(Tip60/Mn-dep superoxide dismutase)
Creaven et al. Khochbin, Biochemistry (1999)
▼ HAT
(GCN5/Tip60/CBP/histones)
Col et al. Khochbin, JBC (2002)
△ HAT
(p300/NF-kB) Furia et al. Kashanchi, JBC (2002)
▼ HAT
(p300/PCAF/p53) Harrod et al. Franchin, JBC (2003)
Viral proteins modified by histone modifiers
DNA Viruses
RNA Viruses
HAT HDV Delta Ag Mu et al. Chen, Virology (2004)

HMT HDV Delta Ag Li et al. Lai, J. Virol. (2004)
HMT HIV-1 Tat Boulanger et al. Richard, J. Virol (2005)
HAT HIV-1 Integrase Cereseto et al. EMBO (2005)
HMT HCV Core Juan (Unpublished)
Viral proteins which utilize histone modifiers
DNA Viruses
HPV E7 displace HDAC Longworth, Wilson and Laimins, EMBO (2005)
EBV OrPi remodeling Zhou et al. Liberman, EMBO (2005)

EBNA3C Subramanian et al. Robertson, J Virol. (2002)
LMP-1 DNA methyltransferases
Tsai et al. Chang, PNAS (2002)
HCMV IE2 HDAC/HMT/Mi-2/DNA methylation

Juan (unpublished)
RNA Viruses
HIV-1 Tat displace HDAC Kumar et al. Galande, MCB (2005)

Lancet 366, 549, (2005)
Virology 235 (1997) 302-310
Virology 332 (2005) 329-336

Welcome to Genomics Research Center,
Academia Sinica, Taipei !
Cancer and pandemic diseases

Stem cell research
Welcome to Genomics Research Center,
Academia Sinica, Taipei !
Chi-Huey Wong
Cancer and pandemic diseases

Stem cell research
阮麗蓉實驗室
Functional Genomics
Tel: (02) 2789-9930 ext: 253 (O); 2789-8775 (L)
Could chromatin modifiers be the
common targets?
Sep 27, 2006

國防生科特論
Eukaryotic RNAs and RNA polymerases
Inactive chromatin
RNA polymerases
Active chromatin
RNA
Alberts et al
Robert Roeder
Rockefeller University
Nature (1969) 224, 234
Multiple forms of DNA-

dependent RNA polymerase in
eukaryotic organisms
Roeder and Rutter

resistant low h
10
igh
hnRNA
snoRNAmicroRNA
resistant low h
10
igh
hnRNA
microRNA
Sequence of human CTD
Eur. J. Biochem. (2003) 270, 3859

Things happened during gene activation
Inactive chromatin
RNA polymerases
Basal transcription machinery

DNA demethylation
Histone hyperacetylation,
Active chromatin methylation…
Reduction of H1 content
Cis and Trans Elements of Transcription
Initiator, Py2CAPy5
TFIID: TBP+TAFs -3 to +5
Alberts et al
TFIIA
activates TFIID
TBP by TAFII230
relieving the binds to TBP
repression at its DNA-
caused by binding
TAFII230 surface in
solution
TFIIF
contains TFIIB
RAP74 which provides
is an ATP- surface
dependent recognized
DNA helicase by RNAP
and can melt
TFIIE:allow
the DNA, and
RNAP to
RAP 38 which
move on
binds to
RNAP
Allow RNAP F20.11,
to bind to Gene VII
DNA
TFIIH
contains
ATPase,
helicase and
kinase
activities
Can
phosphorylate
CTD of RNAP
And relieve
RNAP from
promoter
F20.11,
Gene VII
Orphanides and Reinberg, 2002, Cell 108, 439
CTD
Orphanides and Reinberg, 2002, Cell 108, 439

Inactive chromatin
Key factors regulate gene-specific

transcription in eukaryotic chromatin?

DNA demethylation
Histone hyperacetylation, Reduction of
TBP/RNAPII H1 content
(Ubiquitous)
Active chromatin (H2A.Z and H3.3) and/or non-histone
Gene-specific transcriptional activation in eukaryotic chromatin
Inactive chromatin
DNA binding domain

Activators (Tissue, Cell, developmental stage-specific)
Coactivators
Scaffold As a signal or code
Histone modifiers ex: acetyltransferases (HAT)
DNA demethylases?

Change DNA demethylation
in Histone hyperacetylation, Reduction of
histone – TBP/RNAPII H1 content
DNA (Specific genes)
interacti
on Active chromatin (H2A.Z and H3.3) and/or non-histone
Gene-specific transcriptional repression in eukaryotic chromatin
Inactive chromatin
Repressors
Corepressors
Scaffold
Histone deacetylases RNAPII
ATP-CRC
DNA methylases

histone – (Specific genes) H1 content
DNA
interacti
Robert Roeder
Rockefeller University
1. Discovery of three RNA
polymerases in 1969
(Location, purification,
subunit, function)
2. Promoter recognition and
accurate initiation
(nuclear extract preparation
and fractionation)
3. General transcription factors
and preinitiation
complex formation
Nature (1969) 224,4.234
Gene-specific transcription
factors
Multiple forms of DNA-dependent RNAat 1980 and
(TFIIIA, 1st found
polymerase in eukaryotic
clonedorganisms
Euk txn factor 1984)
5. Transcription coactivators
Roeder and Rutter
Preparation of the nuclear extract
–
one of the most highly cited paper
in the field
Kinkakuji Temple, Kyoto, Japan, 2006
IGBMC, Strasbourg, France
Laszlo Tora at IGBMC
Insect cells
HeLa
Antibody Core
Dish Wash Core
Gene-specific transcriptional activation in eukaryotic chromatin
Inactive chromatin
Exchangeable subunits in
coactivator/corepressor complexes
Factor modifications: SUMO …
Repressors Activators (Tissue, Cell, developmental stage-specific)
Corepressors Coactivators
Scaffold Scaffold As a signal or code
Histone deacetylases Histone modifiers ex: acetyltransferases (HAT)
ATP-CRC ATP-dependent chromatin remodeling complexes
DNA methylases DNA demethylases?

histone – TBP/RNAPII H1 content
DNA (Ubiquitous)
interacti

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阮麗蓉

Sep 27, 2006

Sep 27, 2006

Sep 27, 2006

ouse embryonic stem cells including decreased

DNase I hypersensitive sites

Recognized by specific factors

Recognized by specific factors

Different cellular event

Recognized by specific factors

Different cellular event

Recognized by specific factors

Different cellular event

Recognized by specific factors

Different cellular event

Curr Opin Genet Dev (2005) 15,

Recognized by specific factors

Different cellular event

Recognized by specific factors

Shi Huang (2002) Nat Rev

Recognized by specific factors

Different cellular event

An, Kim and Roeder (2004) Cell 117, 735

Recognized by specific factors

Different cellular event

An, Kim and Roeder (2004) Cell 117, 735

Loss of acetylation at Lys16 and trimethylation at Lys20

Recognized by specific factors

Different cellular event

p53 (JBC 2000) Activators

p53 (JBC 2000) Activators

p53 (JBC 2000) Activators

p53 (JBC 2000) Activators

HCMV IE2 Activators

Adenovirus E1A △ HAT Ait-Si-Ali et al. Harel-Bellan, Nature (1998)

EBV Zta △ HAT Chen et al. Liberman, MCB (2001)

HCMV IE2 ▼ HAT Hsu et al. Juan, EMBO (2004)

HPV E6 ▼ HAT Thomas and Chiang, Mol Cell (2005)

HCV Core △ HAT Gomez-Gonzalo et al. Lopez-Cabrera, Virology (2004)

HIV-1 Tat ▼ HAT

HAT HDV Delta Ag Mu et al. Chen, Virology (2004)

HPV E7 displace HDAC Longworth, Wilson and Laimins, EMBO (2005)

EBV OrPi remodeling Zhou et al. Liberman, EMBO (2005)

HCMV IE2 HDAC/HMT/Mi-2/DNA methylation

HIV-1 Tat displace HDAC Kumar et al. Galande, MCB (2005)

Virology 332 (2005) 329-336

Cancer and pandemic diseases

Cancer and pandemic diseases

Sep 27, 2006

Nature (1969) 224, 234

Multiple forms of DNA-

Roeder and Rutter

Eur. J. Biochem. (2003) 270, 3859

Basal transcription machinery

DNase I hypersensitive sites

Orphanides and Reinberg, 2002, Cell 108, 439

Key factors regulate gene-specific

DNase I hypersensitive sites

DNA binding domain

DNase I hypersensitive sites

DNase I hypersensitive sites

DNase I hypersensitive sites

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