Download DNA METHYLATION

The Human Genome
~ 32,000 Genes
?? 200,000 Proteins
a) alternative mRNA splicing
b) post-translational modifications
Régulation Transcriptionnel
Pourquoi est-ce que l’expression
génique est régulée ?
Pourquoi la transcription est-elle le
mode primaire de la régulation de
l’expression d’un gène?
Quel est le principal moyen de
régulation de la transcription ?
Comment est-ce que ceci
est réalisé ?
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.
Regulation Can Be at Several Different Levels
GENOME
TRANSCRIPTOME
Nucleus
Gene Expression:
Multiple, Spatially and
Temporally Distinct Steps
Carried out by Distinct
Cellular Machinery
DNA
Pol II transcription
PROTEOME
Cytoplasm
Primary RNA transcript
Protein
Nuclear processing
Capping
Splicing
Polyadenylation
Translation
Mature
mRNA
Mature mRNA
Export
Degradation
Post-Translational
Modifications
Degradation
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
Euchromatin
open, transcriptionally active
Heterochromatin
condensed 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 structure
Dnase I insensitive
Active
Beads-on-a-string structure
Dnase I sensitive 10-fold
Open
Nucleosome-free
Dnase 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
ADN
vert et marron
Histones :
H2A = jaune
H2B = rouge
H3 = bleu
H4 = vert
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 elements
TATA box
Initiator Element
CCAAT box
• General transcription factors
TATA Box binding Protein (TBP)
TBP Associated Factors (TAFs)
• Mode of assembly
Holoenzyme
Sequential assembly
Basal, Very Low Level
mRNA Expression
i)
Detectable with RT-PCR
ii) No protein production
TAFs
TBP
Gene X
-35
Basal Transcription
Regulated Transcription
1. Cis-acting sequences
2. Transcriptional activators/co-activators
2,000 + transcription factors
Combinatorial complexity
Transcription Factors
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.
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)
Co-activator
protein
General
transcription
factors
TBP
Gene X
Transcriptional activators
binding to promoter region
TATA
-35
Regulated Transcription
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/CBP
PCAF
TAFII 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
Histone
acetyltransferase
Histone
deacetylase
Hypoacetylation
Strong
internucleosomal
interactions:
Acetylation has two
functions:
Neutralize the positive
charge on the lysine
residues
Destabilize interactions
between histone tails
and structural proteins
Hyperacetylation (Yellow)
Weak internucleosomal interactions:
histone tails do not constrain DNA,
which is accessible to transcription factors
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 sequences
irreversibly repressed state
Mitosis erases methylation only temporarily
Cytosine methyl-transferases
of vertebrates
have preference
for hemi-methylated targets
Because of that newly synthetized DNA strand
will receive same methylation pattern
as parental strand
DNMT1 enzyme
= maintain methylation
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 CpG
binding 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.
Recent Added Complexity
+ ubiquitination
+ sumoylation
+ methylation
+ phosphorylation
+ histone substitution
Modifications courants des acides amines des Histones
Histone Substitutions
H2AZ
H2AX
H3.3
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 - XH2
Alpha Thalassemia + Mental Retardation
Chromatin Modification
Chromatin Remodeling
SNF/SWI
Transcription Factor
Modification
Acetylation
Phosphorylation
DNA Methylation
CpG dinucleotides
MeCP2
Histone Substitution
H2AZ
H2Ax
H3.3
Histone Modification
Acetylation
Ubiquitination
Sumoylation
Methylation
Phosphorylation
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
DNMT-I
HDAC-I
Epigenetic Therapy:
Restoration of Gene Function
Silenced
Gene
Function
Evidence of restoration of function
by epigenetic therapy
ER
Estrogen
Receptor
Restored expression from estrogen
sensitive genes
RARB2
Retinoic acid
receptor
Restored growth suppression by
retinoic acid
MLH1
Mismatch
repair
Correction of mismatch repair
defect
P16/INK4a cell-cycle
regulation
Restoration of RB checkpoint
P14/ARF
Restored normal cellular
distribution of MDM2
MDM2
inhibition
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.
Régulation Transcriptionnel
Pourquoi est-ce que l ’expression
génique est régulée ?
Pour faire les protéines
appropriées dans les bonnes
cellules au bon moment.
Pourquoi la transcription est-elle le
mode primaire de la régulation de
l’expression d’un gène?
Pour obtenir un meilleur control
avec une efficacité maximale.
Quel est le principal moyen de
régulation de la transcription ?
« Turning on-and-off » l’amorçage
de la transcription.
Comment est-ce que ceci
est réalisé ?
Par des protéines que se lient à l'ADN et
qui augmentent ou répriment la liaison
de la ARN polymérase au promoteur
Par des modifications de la chromatine