Transcription Regulation and Gene Expression in Eukaryotes FS 08 Co-activators and Co-repressors

Transcription

1 Transcription Regulation and Gene Expression in Eukaryotes FS 08 Co-activators and Co-repressors P. Matthias, April 02, 2008

2 Co-regulators: a separate class of transcription factors Do not bind to DNA by themselves Are not part of the basal machinery (GTFs) Basal Machinery Co-regulators link upstream factors to the basal machinery

3 Another layer of complexity: diverse transcriptional coactivators Direct binding of upstream factors to prok RNA Pol recruitment Indirect binding of upstream factors to euk Pol II

4 Upstream factors interact with a diversity of co-regulators, ie. gene -specific co-activator/co-repressor, TAF s and distinct subunits from the mediator complex

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

6 Co-regulators: single polypeptides OBF-1/OCA-B/Bob-1 (co-activator lymphoid lineages) CBP/p300 and ACT (CREB binding protein /co-activator) CIITA (MHC II) Groucho related - Grg4 (co-repressor) PGC-1 NHR TRAP220 (co-activator PPARg2) β-catenin

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

8 The B cell-specific co-activator OBF-1/OCA-B/Bob1 IN VITRO TRANSCRIPTION Luo et al., Cell 71,

9 The B cell-specific co-activator OBF-1/OCA-B/Bob1 IN VITRO TRANSCRIPTION

10 The B cell-specific co-activator OBF-1/OCA-B/Bob1 IN VITRO TRANSCRIPTION EMSA

11 The B cell-specific co-activator OBF-1/OCA-B/Bob1 YEAST TWO HYBRID Strubin et al., Cell 80,

12 The B cell-specific co-activator OBF-1/OCA-B/Bob1 EMSA EMSA

13 In vivo transcription co-activation by OBF-1 IN VIVO TRANSACTIVATION ASSAY

14 OBF-1-POU domain interaction

15 OBF-1 function: phenotype of OBF-1 -/- mice Early B cell development largely normal; reduction in transitional B WT cells and splenic seeding; MZ B cells reduced in Bl6 strain; overall reduction in splenic B cells OBF-1 -/- IgM transcription and production normal; reduced levels of secondary Ig isotypes (IgG, IgA); subset of Vκ genes affected Severely impaired TD immune response; absence of GC formation Nature, 383, primary follicle B cell intrinsic effect; T cells are developmentally and functionally normal in vivo (GC formation, antigen responses) secondary follicle mantle zone centrocytes follicular dendritic cells centroblasts T H cells

16 OBF-1 : two isoforms with different roles? NH 2 COOH MLWQKPTAPEQAPA.. LSASKRKGNILSQAMLW QKPTAPEQAPA.. OBF-1 p35 p40 p35 p34 OBF-1 p40/p34 B cell Yu et al., Immunity 14, 297

17 BAC modifications BAC sequence: LOC / LOC / K07Rik : putative genes derived by automated computational analysis WT p34 Alain Bordon

18 Mice expressing only 1 isoform of the coactivator Modified BACs are introduced into OBF1- KO mice BAC Spleen extracts OBF-1 β -tubulin Alain Bordon No OBF-1 p34 p35 p34

19 CREB and related proteins

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

21 CBP / p300: a large protein

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

23 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).

24 Rubinstein-Taybi

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26 MLL: 3969 aa

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28 LEF-1/TCF-1

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

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

32 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

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

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

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

36 PGC-1 family PGC-1 1 AD L-motif RS RRM 798 PERC/PGC-1β E E 1 PRC 55% 45% % 46% 1664

37 Mediator: large multi-protein complexes

38 Mediators-like co-activator: metazoan cells

39 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

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41 Comparative chart showing the relationships in polypeptide composition of various Mediator-like complexes: TRAP/SMCC (Refs 18,19); PC2 (Ref. 23); DRIP (Ref. 24); ARC (Ref. 25); CRSP (Ref. 28); murine Mediator (mumed)27; and NAT (Ref. 26). 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 (Refs 19,20).