AULA 9| 11 e 15 maio 2023

TEMA: REGULAÇÃO DA EXPRESSÃO EM EUCARIOTAS

SUMÁRIO
Regulação da expressão genética em eucariotas: diferentes níveis de
regulação. Diferentes padrões de expressão de genes. Região promotora e
RNA polimerases de eucariotas. Elementos reguladores: activadores e
silenciadores. Metilação Histonas e remodelação da cromatina. Imprinting
genómico. Silenciamento do cromossoma X. Obesidade, herança epigenética e
regulação génica.

COMPETÊNCIAS A ADQUIRIR
. Compreender o complexo padrão de expressão de genes de eucariotas com um
número limitado de proteínas reguladoras.
. Compreender os mecanismos moleculares de regulação génica em eucariotas.
. Perceber o papel da cromatina na regulação de genes de eucariotas.
. Entender o que é a epigenética e de que forma esta influencia a expressão de genes.
. Compreender o conceito de imprinting genómico e o seu papel em doenças genéticas.
. Epigenética e doença: qual a possível relação?
. Compreender de que forma o ambiente determina o padrão da expressão dos genes.

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OVERVIEW OF GENE REGULATION ON PROKARYOTES AND EUKARYOTES

https://www.youtube.com/watch?v=3S3ZOmleAj0

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EUKARYOTIC GENOME ORGANIZATION

ü Complex (number of genes)

ü Transcription and translation in different

compartments

ü Genes with introns (alternative splicing)

ü Each gene has its own promotor

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 WHAT DETERMINES WHICH GENES ARE “ON” AND WHICH ARE “OFF”?

Regulation of gene expression

Is it possible to measure gene expression? YES!

By measuring mRNA levels.

ü Northern analysis
ü RT- PCR (cDNA)
ü Microarrays
ü Transcriptome analysis (connecting the
genome to gene function - when and where?)

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 WHY EUKARYOTIC GENE EXPRESSION IS MORE ELABORATE?

ü Regulation of gene expression occurs in a tissue-specific

manner and is influenced by epigenetic marks

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GENOME SIZES VS COMPLEXITY

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NON-CODING DNA

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MAIN COMPONENTS OF THE HUMAN GENOME

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EUKARYOTES REQUIRE COMPLEX CONTROL OVER GENE EXPRESSION

ü multiple chromosomes
ü different genes are turned on and off in specific cells
ü different cell types are generated through differential gene
regulation.

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 DIFFERENT LEVELS OF REGULATION OF EUKARYOTIC GENE EXPRESSION

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HOW FAST DO MRNA´S DEGRADE: mRNA HALF LIFE

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MAJOR LEVELS OF REGULATION OF EUKARYOTIC GENE EXPRESSION

Major levels of control

Transcription: limits the

amount of mRNA that is
produced from a particular
gene.

Translation of mRNA into

proteins; even after a protein
is made, post-translational
modifications can affect its
activity.

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PROKARYOTIC AND EUKARYOTIC GENE EXPRESSION AND REGULATION

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ENVIRONMENTAL INFLUENCES ON GENE EXPRESSION

Chemicals, drugs, light, temperature, others??

Ex1:

Ex2:

Gene C: controls fur colour in Himalayan rabbits

15 - 25°C: geneC: active Above 30°C: geneC: inactive 14

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 IMPORTANT ELEMENTS REGULATING GENE TRANSCRIPTION

ü Promotor region
ü Transcription factors
ü Activators (enhancers) and silencers.

GC rich region
CCAAT Box
cis (close to the promotor)

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TRANSCRIPTION FACTORS (TF) ARE REGULATORY PROTEINS THAT ACTIVATE GENE
TRANSCRIPTION

TF´s can be classified by their three-dimensional protein

structure and its specificity relies on the consensus
sequences to which they bind.
ü basic helix-turn-helix
ü helix-loop-helix
ü zinc finger proteins.
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THE COMPLEXITY OF EUKARYOTIC GENE EXPRESSION

ü It is combinatorial: it requires the coordinated interactions of

multiple proteins

ü TF´s are activated by signal transmission (ex: cytokines and

growth factors

ü Eukaryotic genes are usually “Off”; “On” state requires

interaction of regulatory proteins 17
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REGULATION OF GENE EXPRESSION IS TISSUE-SPECIFIC

ü No cell type other than red blood cells expresses beta globin.

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 EPIGENETIC MECHANISMS

ü Changes to the genome that affect gene expression without changing the
DNA sequence

https://www.youtube.com/watch?v=i9a-ru2ES6Y
https://www.youtube.com/watch?v=kp1bZEUgqVI&t=4s 19
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 EPIGENETIC MECHANISMS

Can we reprogram genes?

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 EPIGENETIC MECHANISMS: PROOF OF CONCEPT

Epigenetics | Moshe Szyf | TEDxBratislava

https://www.youtube.com/watch?v=SrqmuYvk3iQ

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EPIGENETIC MECHANISMS: PROOF OF CONCEPT

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GENE EXPRESSION CAN BE REGULATED BEFORE THE INITIATION OF TRANSCRIPTION
BY EPIGENETIC MARKS

ü DNA methylation: chromatin more tightly bound resulting in

downregulation or inhibition of transcription

ü Acetylation of histones: loosens bindings and promote transcription 23

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EX: EPIGENETIC REGULATION OF GENE TRANSCRIPTION

ü Not all genes are active at all times.

ü Methylation might play a role in the regulation of gene expression

Ex: Methylation status of the beta-

globin locus

Unmethylated in cells that

expressed beta-globin

Methylated in other cell types

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THE IMPORTANCE OF DNA METHYLATION IN DEVELOPMENT AND IN DISEASE

ü DNA methylation is an epigenetic signaling tool that cells use to lock

genes in the "off" position and thus it is somehow involved in gene
expression
§ embryonic development
§ genomic imprinting
§ X-chromosome inactivation
§ preservation of chromosome stability
§ human diseases

Transcription initiation sites

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INTERPLAY BETWEEN THE CANCER GENOME AND EPIGENOME

“…the evidence linking epigenetic processes with cancer is

becoming “extremely compelling”…”

ü Protooncogenes: normal genes

that code for proteins that
stimulate cell cycle. Ex: ras

ü Oncogenes: cancer-causing
genes; can result from a
mutation in a protooncogene:
activate TF´s that promote
undifferentiated cell growth,
resulting in cancer.

ü Tumor supressor genes:

promote synthesis of cell-cycle
inhibiting proteins (“guardian
angel of the genome”). Ex:p53 26
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METHYLATION AND GENE SILENCING

ü Methylation of CpG islands may lead to inappropriate gene

silencing
e.g.: tumour supressor genes, ex: p53

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METHYLATION AND GENE SILENCING

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DNA METHYLATION: DIAGNOSIS (DNA MARKERS)

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DNA METHYLATION IN HUMAN DISEASE

Imprinting: one of the genes within a pair is somehow switched

"off”, leading to monoallelic expression 30
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DNA METHYLATION & IMPRINTED GENES
Mendel found out (about peas!!) that:

ü The two parents contribute equally to the character

ü The effect of an allele is independent of whether it comes from

the mother or the father

This is not true for some mammalian genes, the imprinted

genes!!!

Dominance is meaningless!!! 31
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IMPRINTED GENES

Silencing usually happens

through the addition of methyl
groups during egg or sperm
formation

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DNA METHYLATION & IMPRINTED GENES

ü Imprinted alleles are silenced: genes are either expressed only from
the non-imprinted allele inherited from the mother or the father.

Imprinted = silenced

Maternal imprinting: allele is expressed if it is inherited by the father

Paternal imprinting: allele is expressed if it is inherited by the mother

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IMPRINTING-RELATED DISORDERS

The consequence of parental imprinting:

the imprinted genes are expressed as if there was only one
copy of the gene (even though there are two)

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 GENOMIC IMPRINTING AND DISEASE
A mutation in one allele (either genetic or epigenetic) can result in the
absence of one or more gene products, thereby leading to a number of
well-known imprinting disorders.

E.g. Prader-Willi syndrome (PW) and Angelman syndrome (AS): epigenetic, sex-specific gene
silencing

Imprinted
Paternally
inherited= ON
Maternally
inherited= ON
Imprinted

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https://www.youtube.com/watch?v=VYbL_CQx2Dk&t=9s
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DNA METHYLATION AND INACTIVATION OF CHROMOSOME

ü An example of low transcribed genes due to heavy methylation

ü Most of the genes are silenced, and the chromosome has epigenetic
marks (methylation and deacetylation of histones and hypermethylation of its DNA)
ü Genes on the inactivated chromosome remain inactive in all the
descendents of these cells.

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 INACTIVATION OF X CHROMOSOME
https://www.youtube.com/watch?v=Y9vXhmI5FXM

ü In cats, the fur pigmentation gene is X-linked; depending on which copy of the X
chromosome each cell chooses to leave active, either an orange or black coat
color results.

ü X inactivation only occurs in cells with multiple X chromosomes, which explains

why almost all calico/tortoiseshell cats are female.

ü A recessive mutation carried in an active X, can have profound adverse

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phenotypic effects
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EXPRESSION OF COAT COLOR: MOSAIC EXPRESSION

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 OBESITY, EPIGENETICS, AND GENE REGULATION

ü Genetically identical mice

ü Reared under identical

conditions

ü Why are they so different?

ü Epigenome makes the difference!: agouti

genes are “off” in normal healthy mice.

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WHAT TRIGGERS THE EXPRESSION OF THOSE GENES?
ARE ENVIRONMENTAL FACTORS INVOLVED?

DNA methylation of agouti (other genes)

was decreased by 31%.

ü E.g.: Bisphenol A alters the action of organisms' epigenomes by

removing methyl groups from DNA.
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 41
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 ROUTES OF DIRECT EXPOSURE TO BPA

ü ~97% test + for BPA

Ø continuously exposed to
low doses of BPA
BFA Polymers break down by saliva enzymes

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THE REGRETTABLE SUBSTITUTION – FROM ONE BISPHENOL TO ANOTHER

The majority of these chemicals have been found to

disrupt the sensitive endocrine (hormone) system that
plays a crucial role in the development and functioning
of our bodies and those of wildlife.

These endocrine disrupting chemicals (EDCs) have

been shown to cause or being associated with:

i) feminization of fish
ii) neurodevelopmental problems
iii) impaired fertility
iv) certain cancers (breast cancer)
v) diabetes
vi) obesity
vii)coronary heart disease.
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EPIGENETICS (EPI=ABOVE): ABOVE GENETICS

Study of inherited traits caused by mechanisms other than changes

in the underlying DNA sequence.

ü Epigenetic changes can switch genes on or off and determine which proteins
are transcribed; “Genetic Marks” 44
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 EPIGENETIC REPROGRAMMING

ü The course of methylation and demethylation during

development

Passive demethylation of
maternal genome Remethylation; Maintenance of
somatic epigenetic marks
Rapid demethylation of
paternall genome

https://www.youtube.com/watch?v=QHuF6RS10nk
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 EPIGENETIC REPROGRAMMING

ü The course of methylation and demethylation during

development

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EPIGENETIC REPROGRAMMING IN PRIMORDIAL GERM CELL DEVELOPMENT

Derived from somatic cell

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EPIGENETICS AND ENVIRONMENT: ANY RELATIONSHIP?

ü Contaminant-induced epigenetic dysregulation: transmitted as chemical

signals (through the blood stream to the developing fetus) that are laid
down as epigenetic marks that affect the fetus genes

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https://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-019-0659-4
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HOW DO EPIGENETIC CHANGES AFFECT GENES?
https://www.youtube.com/watch?v=IYJ_nd9glvw

Methyl donating substances

ü Epigenetic marks can be stable, inherited, yet also reversible; only genes in the
germ line cells will be passed down;
ü Factors like diet, stress, nutrition can make an imprint on genes passed from
one generation to the next. Thus can be under genetic control, but also
influenced by environmental factors. 49
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 HOW DO EPIGENETIC CHANGES AFFECT GENES?

Agouti gene: a pleiotropic gene: associated to a number of diseases (e.g.: obesity)

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 HOW DO EPIGENETIC CHANGES AFFECT GENES?

Stressed rats

GR: Glucocorticoid receptor- helps to handle stress situations

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HOW DO EPIGENETIC CHANGES AFFECT GENES?

How can epigenetics affect YOU?

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 IT´S ALL EPIGENETIC´S FAULT?

https://www.youtube.com/watch?v=81rFpRsF80c 53
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 ALL BECAUSE OF EPIGENETICS!

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 Example 1: NATURE VS NURTURE AND EPIGENETICS

Darwin and Freud walk into a bar. Two alcoholic mice — a mother and her son —
sit on two bar stools, lapping gin from two thimbles.
The mother mouse looks up and says, “Hey, geniuses, tell me how my son got into
this sorry state.”
“Bad inheritance,” says Darwin.
“Bad mothering,” says Freud. 55
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Example 2: EPIGENETICS OR WEIRD INHERITANCE?
-EXPERIMENTAL EVIDENCE (TRANSGENERATIONAL INHERITANCE)

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Example 3: COCAINE ADDICTION IN MONKEYS: REPROGRAMMING THE GENES

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THE EPIGENOME REARRANGES!

Is there a future of epigenetics drugs?

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 YOU CAN POSITIVELY INFLUENCE YOUR EPIGENOME!

YOU CAN START BY…

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 60
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SOME EPIGENETIC DISEASES

ü Causes and simptoms

Other diseases that might be related to epigenetic marks: Asthma, Lupus, Diabetes,
Huntington, Alzheimer …. Other?

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MORE ON GENE REGULATION

ü Alternative splicing, a type of gene regulation

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MORE ON GENE REGULATION

ü Alternative splicing, a type of gene regulation

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ALTERNATIVE SPLICING: ANTIBODY DIVERSITY EXPLAINED

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 COMPETÊNCIAS ADQUIRIDAS

ü ser capaz de reconhecer a complexidade da regulação da expressão

génica em eucariotas;
ü reconhecer diferenças na regulação de genes em procariotas e em
eucariotas;
ü reconhecer o papel dos diversos elementos implicados na regulação
de genes de eucariotas (factores transcricionais, activadores,
silenciadores);
ü reconhecer a influência de diversos factores na expressão de genes
(ambiente, temperatura, radiação, químicos, etc.);
ü entender o papel da metilação na regulação da expressão de genes,
no silenciamento de genes e a sua implicação em doenças genéticas
e no cancro;
ü reconhecer o papel da metilação no imprinting genómico e na
inactivação do cromossoma X;
ü entender o conceito de “epigenética”;
ü reconhecer o splicing alternativo como processo de regulação da
expressão de genes.
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 COMPETENCES ACQUIRED

ü be able to recognize the complexity of the regulation of gene expression in

eukaryotes;
ü recognize differences in gene regulation in prokaryotes and eukaryotes;
ü recognize the role of the various elements involved in the regulation of eukaryotic
genes (transcriptional factors, activators, silencers);
ü recognize the influence of several factors on gene expression (environment,
temperature, radiation, chemicals, etc.);
ü understand the role of methylation in the regulation of gene expression, gene
silencing and its implication in genetic diseases and cancer;
ü recognize the role of methylation in genomic imprinting and inactivation of the X
chromosome;
ü understand the concept of “epigenetics”;
ü recognize alternative splicing as a process for regulating gene expression.

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 APOIO AO ESTUDO- revisão de conceitos

1) Em eucariotas o processo de regulação da expressão de genes é mais

fina. Qual é a grande diferença entre a regulação de genes de
procariotas e eucariotas?
2) Indique os elementos essenciais da regulação da transcrição de genes
de eucariotas.
3) De que forma a metilação influencia a expressão dos genes.
4) O que entende por “imprinting” genético? Qual é o possível impacto que
este pode ter na informação genética de um indivíduo.
5) O ambiente pode ter algum papel determinante na regulação da
expressão de genes? Dê um exemplo.
6) O que é a epigenética?
7) De que forma as alterações epigenéticas podem afectar os genes?

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STUDY FOLLOW-UP

1. In eukaryotes the process of regulating gene expression is finer. What is

the big difference between the regulation of prokaryotic and eukaryotic
genes?
2. List the essential elements of the regulation of eukaryotic gene
transcription.
3. How methylation influences gene expression.
4. What is genetic imprinting? What are the possible impacts of genetic
imprinting on an individual's genetic information.
5. Can the environment play a determining role in the regulation of gene
expression? Give an example.
6. What is epigenetics?
7. How can epigenetic changes affect genes?

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