Initiation de la transcription et Facteurs de transcription

Transcription

1 Initiation de la transcription et Facteurs de transcription Aurélio Balsalobre Institut de Recherches Cliniques de Montréal Laboratoire de génétique moléculaire Jeudi 18 octobre 2012

2 Plan du cours Introduction The actors How is RNA synthesis initiated? Focus on the transcription factors Illustration through the POMC promoter study

3 Introduction 1- 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

4 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

5 Introduction 3- 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

6 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

7 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

8 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

9 The actors 2- General transcription factors (GTF) Benoit Coulombe, IRCM

10 Les acteurs transcriptionnels 2- Other general transcription factors There are other actors that are present for general purpose: # 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. transcriptional activity 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

11 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

12 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

13 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

14 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 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

15 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

16 How is RNA synthesis initiated? 2- The sequential model Coulombe B & al. (1999) Microbiol Mol Biol Rev. 63, 457

17 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

18 How is RNA synthesis initiated? 3- The holoenzyme discovery Ossipow et al., Cell 1995

19 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

20 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

21 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

22 b- Mechanisms of actions Destruction of the nucleosomal structure Recrutement of coactivators/corepressors Recrutement of the general transcription factors

23 HAT, HMT, Kinase ATP-dependent remodeling (SWI/SNF) GTF

24 Adapted from Cosma MP, Mol Cell 2002 Aug;10(2):

25 c- TF as targets of many regulations Transcriptional control: abondance and specificity post-transcriptional control: splicing Translational control: pause post-translational control : phosphorylation Interactions: dimerization Cellular distribution

26 Transcriptional control: abondance and specificity * TF found in almost all cellular * TF specific to some conditions cellular specificity developmental regulation circadian control etc...

27 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

28 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

29 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

30 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

31 The Hypothalamo-Pituitary-Adrenal axis secretion transcription Hypothalamus CRH secretion transcription Glucocorticoids secretion transcription synthesis Pituitary anterior POMC ACTH The corticotroph cell Adrenals

32 Regulation (simplified!) of the POMC gene LIF (activation) Tissue-specifique synergy CRH (activation) Gluco (repression) STAT3 NeuroD1/Pan1 Nur77 Tpit Pitx1 Pan1/Pan1 GR TATA -480 Distal Central Proximal Minimal +63

33 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 2 nd level of recrutement (coactivators and corepressors)

34 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 Rapporteur : Luciferase, CAT, GFP 5 region (promoter) Enhancers Response elements multimerized

35 Importance of the DECE region in the POMC promoter (level of expression) Therrien et Drouin, 1991

36 Specificity of the combined DE + CE regions 3 cellular cell types cortico somato fibroblaste Therrien et Drouin, 1993

37 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 Rapporter 5' region Response elements multimerized

38 Synergy between Tpit and Nur77 on the Tpit response element Lipofection in GH3 cells 2159 % Activité Ctl Nur77 Tpit Nur77+Tpit CE3 CE3 CE3 Luciferase

39 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

40 ChIPseq data over POMC locus Novel enhancer Tpit Pitx1 IgG Pol2

41 Pax7 and melanotrope identity corticotropes Tpit POMC Pax7+ melanotropes POMC ACTH POMC αmsh PC2 AVP AVPR1b CRH CRHR1 DA DRD2 Gc Pax7? GR POMC GR POMC

42 ChIPseq data over Pcsk2 locus AtT20-Pax7 cells are a good in vitro model Comparaison du recrutement de Tpit en fonction de la présence de Pax7 Quels sont les facteurs qui l influencent, quels sont les mécanismes qui l expliquent?

43 Pax7 re-programming of Tpit-dependent differentiation Chromatin marks associated with Pax7-dependent changes in Tpit binding «pioneer» transcription factor in AtT-20 Pax7 cells: new Tpit peaks 3655 peaks conserved Tpit peaks 6989 peaks lost Tpit peaks 5711 peaks Pax7 peaks Tpit peaks in neo cells Tpit peaks in Pax7 cells FAIRE peaks in neo cells FAIRE peaks in Pax7 cells H3K4me peaks in neo cells H3K4me peaks in Pax7 cells kb from Tpit peak center

44 A classical TpitREpal under Tpit peaks A new composite DNA binding motif for Pax7 under Pax7 peaks TpitREpal 70% of sequences HD 55% of sequences Paired Brachyury DBS CGTGACTAATTAA Pax7

45 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 2 nd level of recrutement (coactivators and corepressors) Whole genome analysis Chromatin analysis : epigenetic

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