Initiation de la transcription
et
Facteurs de transcription
Aurélio Balsalobre
Institut de Recherches Cliniques de Montréal
Laboratoire de génétique moléculaire
Jeudi 1 octobre 2015
● Introduction
● The actors
● How is RNA synthesis initiated ?
● Focus
on the transcription factors
● Illustration
through the POMC promoter study
Introduction
1A- Where does transcription stand in the cell’s life ?
The central role of RNA is critical in the genetic information flux
Cellular diversity reflects the transcriptome diversity
DNA
mRNA
Proteins
Introduction
1B- Basics about DNA, RNA and proteins : Gene structure
1
Exon 1
Enhancer
2
4
2
3
mRNA variant 1
4
Promoter
1
5’ UTR
2
3
CDS
4 mRNA variant 2
3’ UTR
Protein
Introduction
2- Transcription steps
1- Preinitiation
Recrutment of factors on the DNA and PIC formation
2- Initiation
DNA opens and RNA synthesis
After 9 nt, most factors dissociate: Promoter Escape
3- Elongation
RNA synthesis (processivity)
4- Termination
the RNA PolII dissociates and the DNA closes
5-Maturation
The mRNA is the target of many modifications
(capping, splicing and polyadenylation)
kvhs.nbed.nb.ca/gallant/biology/transcription.jpg
Introduction
3A- The regulation of transcription
The regulation of transcription can be done at several steps (PIC, elongation, termination,
maturation, without mentionning post-transcriptional, translational and post-translational ).
However, the transcriptional regulation is critical during the preinitiation step by the
antagonistic action of the chromatine structure itself on one side and on the other side the
combined action of transcription factors and coregulators.
Repression by the condensed structure of the chromatine
Targetting of the promoter region
Regulation by activators/repressors
Regulation by coactivators/corepressors
3B. Condensed structure of the chromatin
http://www.daviddarling.info/
The actors
1- RNA polymerase II
the enzyme which catalyzes the transcription reaction
2- General transcription factors
proteins involved in the PIC formation
3- Transcription factors
regulatory proteins which bind DNA
4- Coregulators
regulatory proteins which do not bind DNA
Basal
transcription
Regulated
transcription
The actors
1- RNA polymerase II
The RNA polymerase II is a complex of 12 units (± 600 kDa) with a particular domain (CTD for
C-Terminal Domain) which consists into 7 aa repetitions (26 to 52 times, according to the species)
Properties of each RNA polymerases (I, II and III)
Uses ribonucleotides
Enzyme dependant of a DNA template
No primer required
Gnatt & al. (2001) Science 292, 1876
The actors
2- General transcription factors (GTF)
The purified RNA PolII is not able to initiate in
vitro transcription by itself. What are the others
required factors ?
TFIIA, TFIIB, TFIID, TFIIE, TFIIF et TFIIH
Almost all GTF are composed of several subunits.
This is the case for TFIID which is composed of
TBP (TATA-Binding Proteins) and several TAF
(TBP-Associated Factors).
Only TBP is able to bind DNA (TBP is also
involved in the transcriptions depending on RNA
PolI and III)
Matsuit T & al.(1980) J Biol Chem. 255, 11992
The actors
2- General transcription factors (GTF)
Benoit Coulombe, IRCM
Les acteurs transcriptionnels
2- Other general transcription factors
There are other actors that are present for general purpose:
transcriptional activity
# Mediators: They are only required for the regulated transcription (not for the basal
transcription); it is a link between the general trancription machinery and the transcription factors.
Its existence has been suggested through the squelching effect.
Squelching: the excess of free activators in
solution binds and titrates out an essential
component for the transcritional (i.e. mediator),
thus inhibiting instead of activating the
transcription because the mediator becomes a
limiting factor.
transcriptional activator amount
# Chromatin remodeling factors (like the Swi/Snf complex): they act on the nucleosomal
structure and on the DNA helicity
The actors
2- General transcription factors (GTF)
Despite their name, most general transcription factors are not essential for the majority of genes.
Through loss-of-function experiments it has been shown that they are only required for a variable
subset of genes
Holstege FCP & al. (1998) Cell 95, 717
The actors
3- The transcription factors
Trancription factors bind, by definition, a specific target sequence on DNA, promoter or
enhancer region. They are mainly composed of a DNA binding domain and an activation domain.
They are critical actors for the regulation (activation but also repression) of transcription.
Most often they act as a group in a synergistic way, through protein-protein interaction with
themselves and with coregulators and GTF.
Specific transcriptional machinery
General transcriptional machinery
PIC
GTFs + RNA PolII
Enhancer
Promoteur
The actors
3- The coregulators
The coregulators, by definition, do not bind DNA. They act only through protein-protein
interaction by recrutment of multiprotein complexes. They can act
on the DNA structure (this requires ATP hydrolysis) like the Swi/Snf complex
on the DNA or proteins composition (this is independant of ATP). It could be acetylation,
methylation or phosphorylation
How is RNA synthesis initiated ?
1- The promoteur recognition
There are several sequences on the DNA that allow precise binding at the +1. However, TBP is the
only GTF that can bind DNA. All others interactions among GTF are thus protein-protein
interactions.
The TATA sequence, situated at about 25-30 upstream of the +1, is well known but it is not the
only one. There is also the INR, for which the consensus sequence is 5’-PyPyCAPyPyPyPyPy-3’.
There is the DPE (Downstream Promoter Element) and other poorly caracterized sequences. It is
noteworthy that many promoters contain one consensus sequence but many promoters contain a
combination of 2 or more sequences.
Burke TW & al. (1996) Genes Dev. 10, 711
How is RNA synthesis initiated ?
2- The sequential model
TFIID binds DNA through TBP
TFIIA enhances DNA binding (replaced by TFIII on the Inr)
TFIIB acts as an adapter between TFIID and RNA PolII
TFIIF + RNA PolII are recruted. TFIIF (not shown here)
avoids RNA PolII binding on DNA (outside promoter regions)
TFIIE helps to recrut TFIIH
TFIIH (not shown here) is a critical player because it contains
two important activities : helicase and kinase
Buratowski S & al. (1989) Cell 56, 549
How is RNA synthesis initiated ?
2- The sequential model
Coulombe B & al. (1999) Microbiol Mol Biol Rev. 63, 457
How is RNA synthesis initiated ?
3- The holoenzyme discovery
The holoenzyme has been first identified in the yeast. It is a very
large complex composed by the RNA PolII and several GTF in
solution (not bound on the DNA). Its composition is variable
depending on the laboratory but it is capable to activate an in vitro
transcription.
The holoenzyme has been then purified in mammalian cells (coIP
with a monoclonal antibody against cdk7, a TFIIH subunit).
The holoenzyme is very variable, reflecting the different techniques
that are used to purify it and the large diversity of interactions that are
part of its structure.
Ossipow et al., Cell 1995
How is RNA synthesis initiated ?
3- The holoenzyme discovery
Ossipow et al., Cell 1995
How is RNA synthesis initiated ?
4- The « promoteur escape »
Transcription starts with rounds of abortive initiations leading to the synthesis of short
transcripts, 2 - 10 nt (Abortive Initiation). Eventually, the RNA PolII goes on and separates from
most GTF (except TFIIF).
It is noteworthy that the RNA PolII exists in two forms: the non-phosphorylated form (PolIIA)
and the phosphorylated form (PolIIO). The phosporylation is mainly done by TFIIH.
The non-phosphorylated RNA PolII is present in the PIC and in the holoenzyme. It corresponds
to the RNA PolII that is not engaged in the elongation. However, the phosphorylated RNA PolII is
only found during elongation and it is never found in the holoenzyne nor at the PIC.
fragment de dégradation
protéolytique
Ossipow et al., Cell 1995
Focus sur les
Les facteurs de transcription (FT)
a- Modular structure
Different families of transcription factors
b- Mechanisms of action
c- TF: targets of many regulations
a- Modular structure of transcription factors
 DNA binding domain (helps to classify the TF)
 basic domaine (bZIP and



bHLH)
zinc fingers (nuclear receptors)
Helix-turn-Helix (homeodomain)
β sheets (NF-kB, p53)
 Protein-protein interaction module (activation or
repression domain)
 with the general transcription factors
 homo and heterodimerization with other TF
 with coactivators-corepressors
 Ligand binding domain
b- Mechanisms of actions
 Destruction of the nucleosomal structure
 Recrutement of coactivators/corepressors
 Recrutement of the general transcription factors
HAT, HMT, Kinase
ATP-dependent remodeling (SWI/SNF)
GTF
Adapted from Cosma MP, Mol Cell 2002 Aug;10(2):227-236
c- TF as targets of many regulations
 Transcriptional
control: abondance and specificity
 post-transcriptional
 Translational
control: pause
 post-translational
 Interactions:
 Cellular
control: splicing
control : phosphorylation
dimerization
distribution
 Transcriptional
control: abondance and specificity
* TF found in almost all cellular
* TF specific to some conditions
cellular specificity
developmental regulation
circadian control
etc ...
 post-translational
control
* The TF is modified in order to regulate its activity, its DNA
binding affinity, its interaction with other proteins, its half-life, ...
* Common modification:
phosphorylation, acetylation, ubiquitination, sumoylation
* these modifications allow a very fast control of the genetic
program in response to signaling pathways
 Interactions:
dimerization
* Homodimerization
* Heterodimerization (among the same family)
One of the partner can modify the DNA binding
specificity or the recrutement of a specific coactivator
* Heterodimerization (two different families)
The DNA sequence can be a new composite site
 Cellular
distribution
* The
TF can be sequestred in the cytoplasm (NFκB)
or at the cellular membrane (STAT).
* In
all cases, translocation of the TF to the nucleus is
induced by a signal (signaling pathway). This regulation is
very fast (seconde to minutes) because the TF is already
present in large amount in the cell
Illustration du rôle et de l’importance des FT
à travers l’historique de l’étude
du promoteur du gène POMC
dans le laboratoire du Dr Jacques Drouin
The Hypothalamo-Pituitary-Adrenal axis
Hypothalamus
Negative feedback
secretion
secretion
transcription
transcription
Negative feedback
secretion
transcription
CRH
Pituitary
POMC
gene
Glucocorticoids
synthesis
ACTH
Adrenals
Corticotroph cell
AtT20 cell as a model
Regulation (simplified !) of the POMC gene
LIF (activation)
Tissue-specifique synergy Gluco (repression)
CRH (activation)
NeuroD1/Pan1
Nur77
Pan1/Pan1
Tpit Pitx1
GR
STAT3
TATA
-480
+63
Distal
Central
Proximal
Minimal
Cloning of the required promoter region
Analysis of this region (sequence, mutations, deletions,…)
Positioning of the response elements (specific DNA sequences)
Cloning of the transcription factors that bind these sequences
Analysis of these TF
Expression, synergy, mechanisms
2nd level of recrutement
(coactivators and corepressors)
Transfection in cells (which ones ?)
This experiment is used to determine the required sequences which allow an
efficient and specific level of transcription in vivo
Promoteur natif ou artificiel
5’ region (promoter)
Enhancers
Response elements multimerized
Rapporteur : Luciferase, CAT, GFP
Importance of the DECE region in the
POMC promoter (level of expression)
Therrien et Drouin, 1991
Specificity of the combined DE + CE regions
3 cellular cell types
cortico
somato
fibroblaste
Therrien et Drouin, 1993
Cotransfection in cells (which ones ?)
In this experiment we cotransfect an expression vector for our TF under
investigation with a rapporter to determine the effects of the TF on a specific
promoter. The cell type is very important because the cell may produce other TF
that can interfere with your experiment.
Endogenous proteins ?
Strong Promoter
Expression vector
(TF or other protein)
Promoteur natif
ou artificiel
5' region
Response elements multimerized
Rapporter
Synergy between Tpit and Nur77 on the Tpit response element
Lipofection in GH3 cells
2500
2159
% Activité
2000
1573
1500
1000
500
100
70
Ctl
Nur77
0
CE3 CE3 CE3
Tpit
Luciferase
Nur77+Tpit
Chromatine ImmunoPrecipitation (ChIP)
Crosslink with formaldehyde
Sonication
Immunoprecipitation with a specific antibody (or Ig as
control) and agarose or magnetic beads
Decrosslink, DNA purification and quantification
In vivo analysis of whole-genome recruitment on DNA
ChIP experiment allows the quantification of the binding of a
protein on the chromatin in vivo. It can be a direct binding (a
transcription factor, histone) or indirect binding through another
protein that binds directly to the chromatin.
The quantification can be done by PCR, by hybridization with an
array or by massive sequencing (ChIPseq)
Advantage : quantitative and qualitative informations about the
recruitment of your protein of interest in the genome, replicate is
not essential
Inconvenient : Price, Analysis
Limitations : antibody, knowledge of the genome
ChIPseq data over POMC locus
Novel enhancer
Pol2
Tpit
Pitx1
IgG
Pax7 and melanotrope identity
Pax7+
Tpit
corticotropes
POMC
POMC → ACTH
melanotropes
POMC → αMSH
PC2
AVP
AVPR1b
CRH
CRHR1
Gc
GR
DA
DRD2
Pax7 ?
POMC
GR
POMC
ChIPseq data over Pcsk2 locus
AtT20-Pax7 cells are a good
in vitro model
Pax7 re-programming of Tpit-dependent differentiation
Chromatin marks associated with Pax7-dependent changes in Tpit binding
« pioneer »
transcription factor
conserved Tpit peaks
6989 peaks
new Tpit peaks
3655 peaks
Pax7 peaks
Tpit peaks in neo cells
Tpit peaks in Pax7 cells
FAIRE peaks in neo cells
FAIRE peaks in Pax7 cells
DIFFERENT PROFILES
H3K4me peaks in neo cells
H3K4me peaks in Pax7 cells
-2 -1 0 1
2
-2 -1 0 1
2
kb from Tpit peak center
Heatmap representations of data
Conserved
Tpit peaks
New
Tpit peaks
FAIRE signal
Profile
AtTNeo
FAIRE signal
Heatmap
ATAC signal
Heatmap
AtTPax7
AtTNeo
AtTPax7
The best peaks were selected.
The corresponding sequences are analyzed for a common motif (with windows of
different sizes) with different softwares
A classical TpitREpal under Tpit peaks
A new composite DNA binding motif
for Pax7 under Pax7 peaks
TpitREpal
70% of sequences
55% of sequences
Brachyury
DBS
CGTGACTAATTAA
HD
Paired
Pax7
Cloning of the required promoter region
Analysis of this region (sequence, mutations, deletions,…)
Positioning of the response elements (specific DNA sequences)
Cloning of the transcription factors that bind these sequences
Analysis of these TF
Expression, synergy, mechanisms
2nd level of recrutement
(coactivators and corepressors)
Whole genome analysis
Chromatin analysis : epigenetic