The Human Genome

~ 32,000 Genes

?? 200,000 Proteinsa) alternative mRNA splicing

b) post-translational modifications

Pourquoi est-ce que l’expression génique est régulée ?

Quel est le principal moyen de régulation de la transcription ?

Comment est-ce que ceci est réalisé ?

Régulation Transcriptionnel

Pourquoi la transcription est-elle le mode primaire de la régulation de

l’expression d’un gène?

Le but principal du control de l’expression des gènes chez les

organismes pluricellulaires est l’exécution des décisions

précises au bon moment et dans les

cellules appropriés au cours du

développement et de la différentiation

cellulaire.

Nucleus

DNA

Primary RNA transcript

Mature mRNA

Pol II transcription

Mature mRNA

ProteinNuclear processingCappingSplicingPolyadenylation

Export

Translation

Degradation

Gene Expression:Multiple, Spatially and

Temporally Distinct StepsCarried out by Distinct

Cellular Machinery

Cytoplasm

Regulation Can Be at Several Different Levels

- Dynamic Protein Association

Post-Translational Modifications

Degradation

GENOME TRANSCRIPTOME PROTEOME

Gene expression is regulated at several levels :

1) Transcriptional – the most common way of controlling levels of a transcript (and therefore also the protein it encodes)

2) Post- transcriptional – regulation of aspects of the RNA after it is transcribed, for example, alternative splicing, polyadenylation, capping, transport out of the nucleus, and half- life

3) Translational – factors that affect the efficiency with which an mRNA is translated, such as mRNA capping, tRNA abundance, and modification of initiation factors (eIF- 2phosphorylation for example)

4) Post- translational – regulation at the protein level, mechanisms include protein stability,Covalent modification of the protein(Phosphorylation, Acetylation, Methylation, Ubiquitination, Sumoylation…), protein localization and protein degradation

Le control de la transcription est le principal moyen de régulation

de l’expression de gènes

DNA Condensation

2 mètres d’ADN par cellule

dans 2 x 10-9 cm3 (ou millilitres)

5 x 1010 kilomètres d’ADN par être humain

Euchromatinopen, transcriptionally active

Heterochromatincondensed transcriptionally silent

HISTONES

form the NUCLEOSOME, which DNA loops around.

EUCHROMATIN – less compact; actively transcribed

HETEROCHROMATIN – more compact; transcriptionally inactive.

Heterochromatin can be either constitutive or facultative.

Levels of Chromatin Assembly

Inactive Solenoid 30 nM structureDnase I insensitive

Active Beads-on-a-string structureDnase I sensitive 10-fold

Open Nucleosome-freeDnase I hypersensitive 100-fold

Chromatin Structure

• Core particle - 146-147 bp

• Wrapped twice around histone octamer

• H2A, H2B, H3, H4

• Linker DNA ~15 - 55 bp

• H1 histone - linker associated

Modèle d’empaquetage de la chromatine

Structure d’un Nucleasome

Histones :

H2A = jaune H2B = rouge

H3 = bleu H4 = vert

ADNvert et marron

Components of the Transcriptional Machinery

• RNA Polymerase

• Basal transcription factors

• Sequence-specific transcription factors

• Co-activators/repressors bridging the basal machinery and the other sequence-specific regulators

• Enzymes that covalently modify core histones and other chromatin components

• Enzymes that alter chromatin structure in an ATP-dependent manner

Basal Gene Expression

Pre-initiation complex assembly

• Consensus cis-acting elementsTATA boxInitiator ElementCCAAT box

• General transcription factorsTATA Box binding Protein (TBP)TBP Associated Factors (TAFs)

• Mode of assemblyHoloenzymeSequential assembly

TBPGene X

-35

Basal Transcription

Basal, Very Low Level mRNA Expressioni) Detectable with RT-PCRii) No protein production

TAFs

Regulated Transcription

1. Cis-acting sequences

2. Transcriptional activators/co-activators

2,000 + transcription factors

Combinatorial complexity

1. Proteins (and RNA’s?) that regulate (positively or negatively) transcription initiation

2. Can act via sequence-specific DNA-protein interactions, or via protein-protein interactions

3. Many transcription factors bind to cis-acting regulatory DNA sequences (regulatory elements in DNA) associated with genes

4. Includes: general transcription factors, upstream activators, enhancer-binding proteins, cell-type-specific factors, co-activators, etc.

Transcription Factors

Les facteurs de transcription, qui stimulent ou répriment la

transcription, se fixent à des éléments régulateurs proches de du promoteur

et des amplificateurs

Les facteurs de transcription, sont des protéines modulaires contenant un domaine de liaison à l’ADN et un ou

quelques domaines d’activation ou de répression

Le rôle principal des activateurs et des répresseurs de la

transcription eucaryotes consiste à se fixer à des complexes à

sous unités multiples que régulent la transcription soit en

modulant la structure de la chromatine (effet indirect) soit en interagissant avec la

polymérase II et les facteurs généraux de la

transcription (effet direct)

TBPGene X

TATA-35

Regulated Transcription

Co-activatorprotein

General transcription

factors

Transcriptional activatorsbinding to promoter region

Les activités de nombreux facteurs de la transcription sont régulées

directement par son interaction avec des

hormones et indirectement par la fixation de protéines et

de peptides extracellulaires à des récepteurs présentes à la surface

des cellules.

Ces protéines constituent donc des cibles moléculaires pour des

médicaments agissant comme agonistes ou antagonistes afin de

réguler l’activité cellulaire à travers

l’expression génique.

Les récepteurs nucléaires constituent

une superfamille de facteurs transcriptionnels dimériques à doigt à zinc C4 qui fixent des

hormones liposolubles et interagissent avec des éléments

spécifiques de réponses dans l’ADN.

La liaison des hormones aux récepteurs nucléaires induit des

changements conformationnels qui modifient leur interaction avec d’autres

protéines

Les récepteurs nucléaires hétérodimériques (récepteurs de l’acide rétinoïque, de la vitamine D ou de l’hormone thyroïdienne) sont présents uniquement dans

le noyau.

En l’absence d’hormone ils répriment

la transcription de gènes cibles, une fois leur ligands fixés ils

activent la transcription.

General Scheme for Activation of Gene Transcription by Nuclear Hormone Receptors

Les récepteurs des hormones stéroïdes sont des récepteurs nucléaires homodimériques.

En l’absence d’hormone, ils sont piégés dans le cytoplasme par

des protéines inhibitrices.

Une fois associés a leur ligands, ils peuvent subir une

translocation vers le noyau et activer la transcription des gènes

cibles.

Epigenetics

  “Any heritable changes in gene expression (influencing on gene function)

that occur without a change in DNA sequence”

Such changes cannot be attributed to changes in DNA sequence (mutations)

but they can be as permanents as mutations (difficult to reverse)

Major mechanisms of epigenetic changes

Three important factors that play clear roles in transcriptional regulation are known:

– DNA METHYLATION –

A subset of cytosine (C) residues could be modified by methylation.

– HISTONE ACETYLATION

Histones can be modified by acetylation.

- IMPRINTING (non-coding RNAs involved)

Histone acetylation and gene expression

• HISTONES in transcriptionally active genes are often ACETYLATED

Acetylation of lysine residues in histones :– Reduces positive charge, weakens the interaction

with DNA.– Makes DNA more accessible to RNA polymerase II

• Enzymes that ACETYLATE HISTONES are recruited to actively transcribed genes.

• Enzymes that remove acetyl groups from histones are recruited to methylated DNA.

There are additional types of histone modification as well, such as methylation of the histones.

Histone Acetyltransferase (HAT) Complexes

P300/CBPPCAFTAFII 250

• Conserved lysines in the N-terminal tails of histones

• Post-translational - Reversible

• Localized - ~2 nucleosomes

• Can also acetylate other proteins involved in transcription

Histone Deacetylation Complexes (HDAC)

Histone acetylation²

HypoacetylationStrong

internucleosomal interactions:

Hyperacetylation (Yellow) Weak internucleosomal interactions: histone tails do not constrain DNA,

which is accessible to transcription factors

Histone acetyltransferase

Histone deacetylase

Acetylation has two functions:

Neutralize the positive charge on the lysine residues

Destabilize interactions between histone tails and structural proteins

Mitosis does not erase acetylation, but merely distributes histones, between the daughter

chromosomes.

Specific acetyltransferases (red) end up distributed between the daughter chromosomes, too. Once segregated, an acetyltransferase would acetylate the adjacent nucleosomes (yellow) and thereby spread over the entire chromatin domain.

DNA Methylation

• Genes that are transcriptionally inactive are often METHYLATED.– In eukaryotes, cytosine residues are modified by methylation.

• Typically, the sites of methylation are CpG dinucleotides (vertebrates); CpG islands in front of genes are mostly unmethylated

DNA Methylation

• 5-methylcytosine 5’-CpG-3’

• CpG only 10% of predicted frequency in eukaryotic genomes

• Deaminated methylcytosine - to - thymine

• ~70% of remaining CpGs are methylated

often in repeat sequencesirreversibly repressed state

Mitosis erases methylation only temporarily

Cytosine methyl-transferases of vertebrates

have preference for hemi-methylated targets

DNMT1 enzyme = maintain methylation

Because of that newly synthetized DNA strand will receive same methylation pattern

as parental strand

DNMTs coordinate transcriptional repression

with histone deacetylases (HDACs)

and methyl-CpG binding proteins (MBDs)

DNMT-mediated gene silencing

Transcriptional Repression by Methylation

Binding to methylated CpGs by Methyl CpGbinding proteins (MeCP2/MBD1,2,3)

Mutations of MeCP2 in humans - Rett Syndrome(similar neurologic phenotype in KO mice)

Changes in DNA methylation during mammalian development

DNA methylation and histone acetylation can be maintained through replication

This allows the packing of chromatin to be passed on - just like a gene sequence.

– However, differences in chromatin packing are not as stable as gene sequences.

• Heritable but potentially reversible changes in gene expression are called EPIGENETIC phenomena

– Vertebrates use these differences in chromatin packing to IMPRINT certain patterns of gene regulation.

– Some genes show MATERNAL IMPRINTING while other show PATERNAL IMPRINTING.

• The alleles of some genes that are inherited from the relevant parent are methylated, and therefore are not expressed.

+ ubiquitination+ sumoylation+ methylation+ phosphorylation+ histone substitution

Recent Added Complexity

Modifications courants des acides amines des Histones

Histone Substitutions

H2AZH2AXH3.3

Variations of nucleosome stability

The Increasingly Complex Code

Location specific

Quantitative and Qualitative

Epigenetic Modification

Chromatin Remodeling Complexes

SWI/SNF Family

• Facilitates gene activation by assisting transcription machinery

to gain access to targets in chromatin.

• Multi-subunit complex - ~10 units

• Very low abundance ~150 complexes/cell

• Molecular weight - 2 MDa

• Destabilize nucleosomes/ disrupt DNA histone contacts

• ATP- dependent activity

Chromatin Remodeling Complexes

SWI/SNF Family

Related to a Helicase superfamily of proteins

RAD16/RAD54/ERCC6 - DNA Recombination/Repair

SWI2/SNF2/brahma - Transcription

ATR-X Syndrome

Mutations of Helicase - XH2Alpha Thalassemia + Mental Retardation

Chromatin RemodelingSNF/SWI

Histone ModificationAcetylation

UbiquitinationSumoylationMethylation

Phosphorylation

DNA MethylationCpG dinucleotides

MeCP2

Histone SubstitutionH2AZH2AxH3.3

Transcription FactorModification

AcetylationPhosphorylation

Chromatin Modification

Solid evidence demonstrates that in

addition to genetic alteration, aberrant

epigenetic regulation, such as silencing

of tumor suppressors, is used by

cancer cells to escape growth and

death control mechanisms

EPIGENETIC MODIFICATIONS AND CANCER

Epigenetic modifications and particularly the methylation of cytosines 5’ of guanine residues (CpGs) in gene promoter regions is an essential regulatory mechanism for

normal cell development.

DNA methylation can inactivate tumor suppressor genes by inducing C>T transitions in somatic and germline cells

and by altering gene transcription. On the other hand, hypomethylation of specific sequences

may reactivate the expression of potential oncogenes.

Thus, aberrant hyper- and hypomethylation are considered crucial steps leading to cancer development.

Thus, compounds able to influence the epigenetic status of a cell have promise for cancer treatment.

DNA metyl transferase inhibitors (DNMT-Is) as Decitabineand Azacitadine have been already largely

tested in cancers. (One of the most recent results obtained 30–60% response rates in leukemias)

Histone deacetylase inhibitors (HDAC-Is) as SAHA, valproic acid, MS-275, Depsipeptide and phenylbutyrate,

exhibit impressive anti-tumor activity potentiated by little toxicity in vitro, ex vivo and in vivo models

HDAC-I

DNMT-I

Epigenetic Therapy:Restoration of Gene Function

Restoration of RB checkpointcell-cycleregulation

P16/INK4a

Restored normal cellulardistribution of MDM2

MDM2inhibition

P14/ARF

Correction of mismatch repairdefect

Mismatchrepair

MLH1

Restored growth suppression byretinoic acid

Retinoic acidreceptor

RARB2

Restored expression from estrogensensitive genes

EstrogenReceptor

ER

Evidence of restoration of functionby epigenetic therapy

FunctionSilencedGene

Hypomethylating Cytosine Analogues

An important component of the actions spectrum of HDAC-Is is the induction of the

cyclin-dependent kinase inhibitor p21WAF1/CIP1,

Very recent data point to an exciting potential of HDAC-Is that may explain the selective anti-cancer activity of these compounds in

AML models : the induction of TRAIL/Apo2L and its death receptors.

(activation of the TRAIL death pathway is well known to be more toxic for tumor cells than for normal cells.)

HDAC Inhibitors Clinical Trials

• Butyrate, Phenylbutyrate

• Depsipeptide

• SAHA

• MS-275

• Valproic acid

Studies on the molecular basis that

modulate epigenetic events during

tumorigenesis, and their effect on

differentiation and apoptosis

pathways, alone or in combination

with other drugs, will provide new

tools to fight cancer.

Pour faire les protéines appropriées dans les bonnes cellules au bon moment.

Pour obtenir un meilleur control avec une efficacité maximale.

« Turning on-and-off » l’amorçage de la transcription.

Par des protéines que se lient à l'ADN et qui augmentent ou répriment la liaisonde la ARN polymérase au promoteurPar des modifications de la chromatine

Régulation Transcriptionnel

Pourquoi est-ce que l ’expression génique est régulée ?

Quel est le principal moyen de régulation de la transcription ?

Comment est-ce que ceci est réalisé ?

Pourquoi la transcription est-elle le mode primaire de la régulation de l’expression d’un gène?