1 Εκπαιδευτικό Πρόγραµµα της Οµάδας Μοριακής Παθολογικής Ανατοµικής της Ενότητας «ΑΡΧΕΣ ΜΟΡΙΑΚΗΣ ΚΑΙ ΚΥΤΤΑΡΙΚΗΣ ΒΙΟΛΟΓΙΑΣ» Αθήνα, 6/2/2010 «Μηχανισµοί µεταγραφής Ρύθµιση γονιδιακής έκφρασης» ρ Α. Πίντζας ιευθυντής Ερευνών Επιστ. Υπεύθυνος Εργαστηρίου Γονιδιακής Ρύθµισης Ινστ. Βιολογικών Ερευνών και Βιοτεχνολογίας Εθνικό Ιδρυµα Ερευνών
2 Key Concepts In any given cell, most genes are expressed in low levels Only a small number of genes, whose products are specialised for the cell type are highly expressed ~10,000 expressed genes maybe common to most cell types of a higher eukaryote
3 Gene Transcription and Translation Transcription generates a single-stranded RNA identical in sequence with one of the strands of the duplex DNA. Translation converts the nucleotide sequence of mrna into the sequence of amino acids comprising a protein. The entire length of an mrna is not translated, but each mrna contains at least one coding region that is related to a protein sequence by the genetic code: each nucleotide triplet (codon) of the coding region represents one amino acid. Only one strand of a DNA duplex is transcribed into a messenger RNA. We distinguish the two strands of DNA as depicted in Figure 5.2: The strand of DNA that directs synthesis of the mrna via complementary base pairing is called the template strand or antisense strand. (Antisense is used as a general term to describe a sequence of DNA or RNA that is complementary to mrna.) The other DNA strand bears the same sequence as the mrna (except for possessing T instead of U), and is called the coding strand or sense strand.
4 Promoter structure and function (A) Organization of a generalized eukaryotic gene, showing the relative position of the transcription unit, basal promoter region (black box with bent arrow), and transcription factor binding sites (vertical bars). The position of transcription factor binding sites differs enormously between loci; (B) Idealized promoter in operation. Initiating transcription requires several dozen different proteins which interact with each other in specific ways. These include the RNA polymerase II holoenzyme complex ( 15 proteins); TATA-binding protein (TBP; 1 protein); TAFs (TBP-associated factors, also known as general transcription factors; 8 proteins); transcription factors; Transcription cofactors; and chromatin remodeling complexes
5 Introduction Initiation of transcription: a key mechanism to regulate gene expression -Initiation of transcription is a KEY step to the regulation of gene expression. -It requires the recognition of a particular gene promoter -This induces a multi-step recruitment of the pre-initiation complex. - Promoter recognition and positioning of the TFIID complex (transcription factor complex II D). The TATA box is involved. - The binding of TFIID induces the ordered recruitment of the other general transcription factors and RNA polymeraseii Promoter recognition is the most important step to initiate the transcription. TFIID plays a major role in this process. TFIID is composed of the TATA-Binding Protein (TBP) and several TBP-Associated Factors (TAFs) Pre-initiation complex: PIC In vitro, TBP is sufficient for promoter recognition and PIC assembly. What is the role of the TAFs? - Initiation of transcription
6 Introduction Transcription initiation: the in vivo situation and the role of TFIID In vitro situation/naked DNA chromatin In vivo situation/chromatin structure/other cis-and trans acting factors An entire TFIID is necessary for REGULATED transcription initiation in vivo: It controls the correct promoter recognition captured in chromatin structures and mediates bridging between activators and the basal transcription machinery. In vivo, TFIID is necessary to the control of transcription initiation TFIID, TBP and TAFs. -TFIID is a basal transcription factor, composed of TBP+TAFs (numbered 1,2 etc depending on their MW) -TAFs organized between them and around TBP in a particular order and stochiometry - Core TAFs necessary for stability of the complex. If one of these TAFs is missing, TFIID is destabilized and individual TAFs are degraded. Gangloff et al., Trends Biochem Sci., 2001 Muller and Tora, EMBO J., 2004
7 Introduction Other TAF-containing complexes are involved in transcriptional regulation HAT activity Muller and Tora, EMBO J., 2004
8 TFIID not so basal after all! TBP and TAFs are regulated Transcriptional regulation: rate, tissue specificity, isoforms Post-translational regulation: modifications, stability, cellular localization Alternative complexes: several TBP-containing and TBP-free TAF- containing complexes to control transcriptional initiation Target gene specificity, induced by cell signals and acting via specific interactions with TF Ex: TBP is up-regulated by the RAS signalling pathway; contribute to mechanisms of cellular transformation in colon carcinoma cell lines. (Johnson et al., Mol. Cell Biol.) a specialized isoform of TAF6 is induced and cleaved via apoptotic signaling pathways, to selectively alter transcription. (Bell et al., Mol. Cell.Biol.) Inactivation of TAF4 induces serum-independent autocrine growth and activation of the transforming growth factor beta signaling pathway. (Mengus et al., EMBO J.) Cell-type-selective induction of c-jun by TAF4b directs ovarian-specific transcription networks. (Geles et al., Proc Natl Acad Sci U S A
9 Οι µεταγραφικοί παράγοντες ρυθµιζονται απο κυτταρικά σήµατα Στο κλασικό µονοπάτι ΜΑΡ κινασών, επιτυγχάνεται µε την µετακίνηση της ίδιας της κινάσης ΜΑΡ στον πυρήνα, όπου φωσφορυλιώνει µεταγραφικούς παράγοντες- στόχους Ένα εναλλακτικό µονοπάτι είναι η φωσφορυλίωση ενός κυτταροπλασµατικού παράγοντα. Αυτός µπορεί να είναι ένας µεταγραφικός παράγοντας που στη συνέχεια µετακινείται στον πυρήνα ή µια πρωτεΐνη που ρυθµίζει έναν µεταγραφικό παράγοντα (π.χ. τον απελευθερώνει για να πάει στον πυρήνα)
10 Generalized signalling pathway
11 Ternary complexes of eukaryotic transcription factors and promoter DNA
12 οµές µεταγραφικών παραγόντων στο DNA ΙΙ
13 WNT signals through β-catenin
14 NF-κB activation
15 The interactions of Jun and Fos proteins components of AP-1 transcription factor The transcription factor AP-1 is formed by dimerization of Jun proteins (c-jun, JunB and JunD) or heterodimerization of a Jun protein with a Fos protein (c-fos, FosB, Fra-1 and Fra-2). AP-1 binds primarily to the 12-O-tetradecanoylphorbol-13- acetate (TPA) response element. Jun and Fos proteins contain a basic-region leucine zipper (bzip) domain and can interact with other bzip proteins, such as MAF and ATF, which allows them to target the camp response element (CRE), and with the p65 subunit of nuclear factor B (NF- B). A dominant-negative mutant form of c-jun (TAM67) interacts with any of the three Jun or four Fos family proteins and/or p65, inhibiting the transactivational activity of AP-1 and NF- B.
16 Regulation and function of genes coding for AP-1 factors Abundance of AP-1 proteins Activity of AP-1 factors Transcriptional regulation of genes Phosphorylation Modulation of protein stability JNKs (ubiquitination) c-jun ser 63, 73 Interaction with transcriptional coactivators e.g. CBP e.g. c-jun, c-fos
17 AP-1 and signal transduction c-fos c-jun Transcriptional activation of immediate early genes: c-fos, c-jun Cell surface stimuli P AAA AAA Translation P Heterodimer and homodimer formation Jun Fos Fra Jun Jun Jun JunJunB Fos Jun TGAGTCA DNA binding and regulation of target gene expression Fra JunB Jun Fos
18 Structure of the members of Ets family proteins Ets, DNA-binding (Ets) domain; HLH, helix loop helix domain (Pointed domain); AD, activation domain; ID, auto-inhibitory domain; RD, repression domain.
19 Functional cooperation of Ets family proteins with other transcription factors and co-activators on various cellular promoters and enhancers (a) Ets-1 or PEA-3 activates the MMP (matrix metalloproteinase) promoters in cooperation with AP1 (c-fos/c-jun) on the RRE (Ras-response element). (b) TCF (Elk-1, Sap-1) activates the c-fos promoter in cooperation with SRF (serum response factor). (c) PU.1 activates myeloid-specific promoters in cooperation with AML1 and C/EBP. (d) Fli-1 activates megakaryocyte-specific promoters in cooperation with GATA-1. (e) PU.1 activates the immunoglobulin (Ig) heavy (H) chain enhancer in cooperation with E12, Ets-1, TFE3 and Oct-2. (f) Ets-1 activates the T cell receptor (TCR) enhancer in cooperation with CREB/ATF, TCF-1, AML1 and GATA-3.
20 Schematics of transcriptional regulation of hematopoietic cell differentiation Schematics of transcriptional regulation of hematopoietic cell differentiation. HAB, hemangioblast; AB, angioblast; EC, Endothelial cell; HSC, hematopoietic stem cell; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; MEP,myeloid-erythroid progenitor; GMP, granulocytic-monocytic progenitor; ProB, pro-b cell; ProT, pro-t cell; Ery, erythrocyte; Meg, megakaryocyte; Neu, neutrophil;m, macrophage; B, B cell; NK, natural killer cell; T, T cell. Note the levels of PU.1, GATA-1 and other lineage-specific transcription factors are critical to determine the subsequent cell fates of hematopoietic progenitors.
21 Signalling specificity in the TGF-ß superfamily
22 Οι 9 πρωτεΐνες Smad χωρίζονται λειτουργικά σε τρεις κατηγορίες: Οι εξειδικευµένοι ενεργοποιητές µονοπατιών είναι οι Smad 2 και 3 (που µεσολαβούν στην σηµατοδότηση TGFβ/ ακτιβίνης) και οι Smad 1 και 5 (που ενεργοποιούν τη σηµατοδότηση BMP). Η Smad 4 είναι συνπαράγοντας που µπορεί να διµερίζεται µε όλες τις εξειδικευµένες Smad. Οι ανασταλτικές Smad δρουν ως ανταγωνιστικοί αναστολείς των ενεργοποιητών Smad, προσδίδοντας ακόµη ένα επίπεδο πολυπλοκότητας στο µονοπάτι. Στην υπεροικογένεια TGFβ, κάθε προσδέτης ενεργοποιεί ένα συγκεκριµένο υποδοχέα που σηµατοδοτεί µέσω ενός χαρακτηριστικού συνδυασµού Smad πρωτεϊνών. ιάφορες άλλες πρωτεΐνες προσδένονται στα διµερή των Smad και επηρεάζουν την ικανότητά τους να επιδρούν στη µεταγραφή
23 The Smad family: Diagrammatic representation of the three subfamilies of Smads
24 Smad oligomerisation Pictorial representation of the plasma membrane receptor kinases that phosphorylate the C-termini of R-Smads (light colour), leading to homo-oligomerisation (a dimer shown for simplicity)
25 Smad nucleocytoplasmic shuttling The five pathways shown are: Smad2 nuclear import after release from SARA (pathway 1); Smad3 nuclear import mediated by importin-ß1 and Ran (pathway 2); Smad4 shuttling mediated by the exportin Crm1 (pathway 3); putative Smad2 (4) and Smad3 (5) export pathways marked with question marks
26 Transcriptional regulation by Smads Two examples, one for gene induction and one for gene repression are shown. Chromatin in nucleosomal configuration is depicted by an arrow indicating promoter activation and a vertical line depicting promoter silencing. Smads are shown as heterodimers of phosphorylated (small black circle) R-Smad Smad4
27 Chromatin changes involved transcriptional regulation Chromatin changes involved in epigenetic transcriptional repression. In unmethylated nucleosomal DNA, acetylation of core histones and binding of transcription factors to DNA leads to active transcription of genes by RNA polymerase II (`transcription on'). Methylation of CpG islands in DNA near to gene promoters is associated with chromatin changes such as deacetylation and methylation of core histones, and this leads to exclusion of transcription factors. This results in loss of gene transcription (`transcription off').
28 Various aspects of the transcription process and its regulation by histone modification (A) Schematic representation of a nucleosome. Yellow represents the histones. Dark red depicts the histone tail that can be modified to loosen DNA (purple) winding. The dark red circle represents a tail without an acetyl (Ac) group. The dark red 'banana shape' represents a histone tail with an acetyl group, relieving the tight packaging of the DNA. (B) Transcriptional repression and activation in chromatin. Yellow circles represent core histone octamers; in the upper panel, acetylated histone tails (dark red) are depicted emerging from the octamer. DNA is purple, and the solid black arrow represents complex movement. Both histone acetyltransferase (HAT; activation) and HDAC (repression) require several cofactors (for DNA binding, for recruitment of the complex, for remodelling of the DNA helix to reduce the accessibility of transcription factors) for their activity Biochemical Journal (2003) Volume 370
29 Chromatin structure regulates transcriptional activity Nucleosomes consist of DNA (black line) wrapped around histone octomers (purple). Post-translational modification of histone tails by methylation (Me), phosphorylation (P) or acetylation (Ac) can alter the higher-order nucleosome structure. Nucleosome structure can be regulated by ATP-dependent chromatin remodellers (yellow cylinders), and the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Methyl-binding proteins, such as the methyl-cpg-binding protein (MECP2), target methylated DNA (yellow) and recruit HDACs. a DNA methylation and histone deacetylation induce a closed-chromatin configuration and transcriptional repression. b Histone acetylation and demethylation of DNA relaxes chromatin, and allows transcriptional activation.