Transcrip)on Regula)on And Gene Expression in Eukaryotes Cycle G2 (lecture 13709) FS 2014 P MaFhias & RG Clerc

Transcription

1 Transcrip)on Regula)on And Gene Expression in Eukaryotes Cycle G2 (lecture 13709) FS 2014 P MaFhias & RG Clerc P. MaFhias, March 26th, 2014 Co- ac&vators / co- repressors Cell- specific / factor- specific co- ac&vators and co- repressors Mul&- component co- ac&vators and co- repressors

2 The Players in Transcription Regulation Chromatin regulators DNA-binding transcription factors (upstream factors) Chromatin regulators Coactivators and corepressors: Mediator, etc.. Basal Machinery: RNA PolII, GTFs Coactivator Mediator" RNAPII" TFIIH" TFIIF! TFIIE" TFIIA" TFIIB" TFIID" Basal Machinery DNA-binding TFs Gene

3 Co- regulators: a separate class of transcrip&on regulators Do not bind to DNA by themselves (i.e. are not TFs) Are not part of the basal machinery (i.e. are not GTFs) Basal Machinery B. Lewin, Genes VIII Co- regulators link upstream factors to the basal machinery

4 Another layer of complexity: diverse transcrip&onal co- ac&vators Direct binding of upstream factors to prok RNA Pol recruitment Indirect binding of upstream factors to euk Pol II

5 Co- regulators according to R. Tjian Lemon and Tjian, Genes & Dev. 14:

6 Mediators-like co-activator: structural and funtional aspects TRX activity termed Mediator is necessary to support TRX activation in vitro, but distinct from TAF s and USA co-activators 2 groups of proteins involved: SRB s (suppressors of RNA Pol B) - suppressors of partial truncations in the CTD of the largest subunit of Pol II and RGR1-MED s, GAL11, SIN4, etc) Mediator activity is a large polypeptidic complex often associated with Pol II and GTF s (>1MDa, ~25 polypeptides) In metazoa, mediator complexes (eg. SMCC/TRAP, DRIP) contain homologs of yeast SRB s and MED; also wide range of co-activators eg. TRAP s (depending on physiological conditions) and Pol II subunits Different Mediator complexes are modular and contain various peptide subunit composition

7 Mediator: large multi-protein complexes

8 Mediators-like co-activator: metazoan cells

9

10 Comparative chart showing the relationships in polypeptide composition of various Mediator-like complexes: TRAP/SMCC; PC2; DRIP; ARC; CRSP (Ref. 28); murine Mediator (mumed)27; and NAT. Each column depicts the components of a single complex, whereas each row indicates the presence (blue box) or clearly established absence (yellow box) of a given subunit within the different complexes. Boxed numbers indicate the molecular weights (in kda) of subunits whose identities have been firmly established from reported amino acid sequences or immunoblot analyses; unboxed numbers indicate the molecular weights (in kda) of polypeptides whose sequences have not yet been reported. Horizontal lines linking the boxes indicate that the subunits are identical. Other designations for selected subunits are given to the left. The +/- signs in PC2 and CRSP indicate subunits that are variably associated. Asterisks mark subunits with homology to yeast Mediator subunits. For p36 in TRAP/SMCC and PC2, and for p34 in mumed, corresponding degradation products are also indicated. For TRAP/SMCC, 150a and 150b refer to two polypetides that are both present in the band previously referred to as TRAP150.

11 GENES & DEVELOPMENT 20:

12 GENES & DEVELOPMENT 20:

13 GENES & DEVELOPMENT 20:

14

15

16 Co- regulators: single polypep&des OBF- 1/OCA- B/Bob- 1: co- ac&vator of Oct factors in lymphoid lineages CBP/p300 and ACT: CREB binding protein /co- ac&vator CIITA: co- ac&vator for MHC II genes Grg4: co- repressor, Groucho- related PGC- 1: co- ac&vator for NHRs FOG1: co- ac&vator for GATA factors TRAP220: co- ac&vator of PPARg2 beta- catenin: co- ac&vator in the Wnt pathway Notch intracellular domain: co- ac&vator of CSL/Rbp- Jk Aire: co- ac&vator (?) media&ng the promiscuous expression of many genes

17 Versa&le Transcrip&onal Coac&vators Spiegelman & Heinrich Cell, 119,

18 The B cell- specific co- ac&vator OBF- 1/OCA- B/Bob1 Namalwa: B cells EMSA Luo et al., Cell 71, IN VITRO TRANSCRIPTION

19 The B cell- specific co- ac&vator OBF- 1/OCA- B/Bob1 IN VITRO TRANSCRIPTION Luo et al., Cell 71,

20 The B cell- specific co- ac&vator OBF- 1/OCA- B/Bob1 Luo et al., Cell 71, IN VITRO TRANSCRIPTION

21 The B cell- specific co- ac&vator OBF- 1/OCA- B/Bob1 IN VITRO TRANSCRIPTION EMSA Luo et al., Cell 71,

22 The B cell- specific co- ac&vator OBF- 1/OCA- B/Bob1 YEAST TWO HYBRID Strubin et al., Cell 80,

23 The B cell- specific co- ac&vator OBF- 1/OCA- B/Bob1 EMSA EMSA Strubin et al., Cell 80,

24 In vivo txn ac&va&on by OBF- 1/OCA- B/Bob1 Strubin et al., Cell 80, IN VIVO TRANSACTIVATION ASSAY

25 OBF- 1- POU domain interac&on

26 Nat. Immunol. 2, 69-74

27 The B cell- specific co- ac&vator OBF- 1/OCA- B/Bob1 Molecular Cell 42,

28 The B cell- specific co- ac&vator OBF- 1/OCA- B/Bob1

29 The B cell- specific co- ac&vator OBF- 1/OCA- B/Bob1

30 CREB and related proteins

31 CREB Structure of the CREB basic region/leucine zipper domain (amino acids ) (bzip) bound to the somatostatin CRE (TGACGTCA)

32 CREB and CREM use different co-activators CBP/p300 (CREB binding protein) ACT (activator of CREM in testis)

33 CBP / p300: a large protein

34 Co-activation by CBP/p300 (and others?)

35 Versa&le Transcrip&onal Coac&vators Spiegelman & Heinrich Cell, 119,

36 PCAF (p300/cbp-associated factor), a factor that competes with E1A for binding to p300/cbp. A: Schematic representation of PCAF and yeast GCN5. The amino terminal domain of PCAF is required for its interaction with p300/ CBP. B: The competition between E1A and PCAF for binding to p300/cbp is depicted. The interaction of E1A with p300/cbp is much stronger than that of PCAF (indicated by the relative thickness of the arrows). Therefore E1A is able to selectively displace PCAF from p300/cbp (indicated by the red X for the PCAF interaction).

37 CBP pathologies Rubinstein-Taybi

38 CBP fusions

39 MLL: 3969 aa

40 LEF-1/TCF-1

41 The life of β catenin within the cell. Valenta T et al. EMBO J. 2012;31: by European Molecular Biology Organization

42 The Wnt beta-catenin pathway

43 The Wnt beta-catenin pathway

44 The Notch pathway

45 PGC-1 is a potent coactivator in mammalian cells GR! PGC-1! GRE! TATA! Luciferase! vector + PGC-1 Luciferase units PGC-1 (µg) no hormone corticosterone 50 nm dex 50 nm

46 PGC-1 is not a general coactivator of transcription Luciferase units (x10 4 ) p50/p Gal4 - VP Gal4 - GR LBD 2 50 PGC

47 Fold-enhancement by coactivator PGC-1 enhances the activity of many nuclear receptors Fold-enhancement by coactivator ER AR MR GR 0 PGC SRC-1e

48 PGC-1 is expressed in a tissue-specific manner brain heart skeletal muscle colon thymus spleen kidney liver small intestine placenta lung peripheral blood leukocytes 7.5 kb

49 NR PGC-1 function in energy homeostasis Cold exposure Fasting Physical exercise PGC-1 PGC-1 txn TR/ other NRs PPARγ? GR /HNF4 PPARα gluconeogenesis fatty acid oxidation Hormones mitochondrial biogenesis respiration

50 PGC-1 domains and functions Leu-motif RS domains 1 acidic basic txn activation receptor interaction regulation 798 RNA-binding motif oligomerization subnuclear localization protein stability

51 PGC-1 family PGC-1 1 AD L-motif RS RRM 798 PERC/PGC-1β E E % 45% PRC 1 48% 46% 1664