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1、Main Points Transcription process of eukaryotic gene. P-TEFb: functions and its Activity regulation I. Transcription process Steps of class II gene transcription Promotor clearence Initiation PIC assembly Termination Elongation TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH RNA polymerase II (Pol II)
2、The class II pre-initiation complex (PIC) RNA polymerase II (Pol II) 12 subunites: Rpb1- Rpb12 About 500Kda of MW Rpb1: largest subunit, CTD: Y1S2P3T4S5P6S7 Yest: 26 repeats C. elegans: 32 repeats Drosophila 42 repeats Mammals: 52 repeats Pre-initiation Pol II CTD PIC assembly IIF IIH IIE PIC Pol II
3、 CTD IIF Pol II CTD IIF II D II AIIB IIH IIE +1 DNA temp TATA PIC Pol II CTD II D II A IIB IIF IIH IIE Initiation 5 5 5 5 5 Pol II 5 5 5 5 5 +1 DNA temp TATA Promoter clearance 20-30 nt Pol II CTD 5 55 5 5 DNA temp TATA II S DSIFNELF MT Pausing Capping Pol II CTD 5 55 5 5 DNA temp TATA II S RNA DSIF
4、NELF Early elongation 5-triphosphatase guanyltransferase N C N C methyltransferase MT MT N C N C Capping Early elongation DNA temp Productive Elongation CycT1 CDK9 P-TEFb RNA CycT1 CDK9 P-TEFb Pol II CTD 5 55 5 5 II S DSIF 2 2 2 2 CycT1 CDK9 P-TEFb NELF Pol II CTD 5 55 5 5 II S DSIF Capped short mRN
5、A 2 2 2 2 P P CycT1 CDK9 P-TEFb Splicing 5 55 5 5 Pol II 2 2 2 2 2 RNA DNA temp RNA U snRNP Productive Elongation Splicing CycT1 CDK9 P-TEFb FCP1 Pol II Pol II CTD 2 5 5 5 5 2 2 2 Pol II 2 5 5 5 5 2 2 2 +1 5 5 5 5 Pol II 5 cap RNA 5 5 5 5 2 2 2 2 Pol II CTD TFIIH NFkB Sp1 NFAT Pol II Steps: Pre-init
6、iation Initiation Promoter clearance Elongation Termination Initiation Early elongation Promoter clearance Elongation TerminationPre-initiation Transcription cycle Transcription Process P-TEFb, its functions and activity regulation P-TEFb: composition and property Discovered in 1995. (positive trans
7、cription elongation factor b) A heterodimer of Cdk9 and cyclin T, including T1, T2a, T2b and Cyclin K. But 80% P-TEFb is CDK9/Cyclin T1. kinase, phosphorylates CTD of Pol II, DSIF and NELF. CycT1 Cdk9 Physiological functions A global transcriptional elongation factor Essential for more than 80% mRNA
8、 transcription Essential for embryo development CTD Pol II RNA NELF Nuc 失败转录 CycT1 Cdk9 P-TEFb 成功转录 RNA P P P P P P P P CycT1 Cdk9 Adapted after Price, MCB 20:2629 (2000) P-TEFb在基因转录过程中的作用机制模式图 B. Pathological functions Exquisitely required for HIV-1 gene expression Abnormally high P-TEFb activity d
9、irect link to cardiac hypertrophy May also relate to tumor progression TAR RNA CTD Pol II 5 5 5 5 CTD Pol II 5 5 5 5 Abortive Transcription CTD Pol II 5 5 5 5 Short RNA CycT1 CDK9 Tat CycT1 CDK9 Tat HIV LTR +1 Tat Tat CycT1 CDK9 Tat CycT1 CDK9 Tat CycT1 CDK9 Tat 2 2 2 2 HIV P-TEFb is essential for H
10、IV-1 replication and gene expression Tat, a HIV-1 encoded protein. Trans-cyclin T1 gene causes cardiac hypertrophy Trans-vector Trans-cyclin T1 CycT1 Cdk9 7SK snRNA Inactive P-TEFb-7SK snRNP HEXIM1 HEXIM2 LARP7 MePCE HEXIM1 LARP7 Methyl donor CycT1 CDK9 HEXIM1 CycT1 CDK9 HEXIM1 MePCE MePCELARP7 CycT
11、1 CDK9 HEXIM1 MePCE 7SK LARP7 5-Cap 3-tail MePCE 3-tail capped 7SK 7SK 5-Cap 3-tail Trinity compled 7SK snRNP 3-tail Nascent 7SK 5-termini 5-Cap CycT1 CDK9 HEXIM1 7SK snRNP assemble Active P-TEFb for general transcription CycT1 Cdk9 Brd4 P P P P P P P P CycT1 Cdk9 Brd4 CTD Pol II mRNA mRNA Pol II Cy
12、cT1 Cdk9 HEXIM1 7SK RNA CycT1 Cdk9 Brd4 Stress/hypertrophic signals/estrogen/ HIV infection inactive P-TEFb CycT1 Cdk9 Brd4 active P-TEFb Recruitment of P-TEFb for stimulation of transcriptional elongation by bromodomain protein Brd4 (Submitted to: Mol Cell) CycT1 Cdk9Brd4 CycT1 Cdk9 HEXIM1 7SK snRN
13、A Inactive P-TEFbActive P-TEFb CycT1 Cdk9 P P P 3/4 1/4 ? How is 7SK snRNP disrupted? CycT1 Cdk9HEXIM1 7SK snRNA Inactive P-TEFb P P P P P P P P P P P P P P P P Phosphorylated P-TEFb is tagged for inhibition through association with 7SK snRNA PP1 treatment Or Stress: Act D, DRB, UV Dephosphorylation
14、 Active P-TEFb CycT1 Cdk9 HEXIM1 + P P P P P P P Phosphorylation ATP How does cell signaling control the disruption of the inactive complex? Control P-TEFb activity by calcium signaling pathway UV and HMBA can trigger Ca+2 influx F1C2 cell Signal pathway inhibitors CycT1 Cdk9 HEXIM1 7SK snRNP Inacti
15、ve P-TEFb P P P PPP P P P P P PPP P P Stress treatment CycT1 Cdk9 Active P-TEFb P P P P P P P HEXIM1 + A calcium-dependent signaling pathway controls P-TEFb transcriptional activity (in writing) Inhibitor: UV: HEXIM1 CDK9-f 1 2 3 4 5 Calcium signaling pathways Ca+ InfluxCycT1 Cdk9 HEXIM1 7SK snRNP I
16、nactive P-TEFb P P P PPP P P P P P PPP P P CycT1 Cdk9 Active P-TEFb P P P P P P P HEXIM1 + Cell membrane L-type channel F1C2 cell Calcium CycT1 Cdk9 HEXIM1 7SK snRNP Inactive P-TEFb P P P PPP P P P P P PPP P P Stress treatment CycT1 Cdk9 Active P-TEFb P P P P P P P HEXIM1 + F1C2 cell Ca+ Influx Calc
17、ium signaling pathways F1C2 cell Ca+ Influx CaM-dependent protein kinases CaM CaM-dependent protein phosphatases CaMKK, CaMKI, CaMKII, CaMKIV Calcineurin (CaN, PP2B) W7 CsA, FK506 F1C2 cell W7, CsA, FK506 CycT1 Cdk9 HEXIM1 7SK snRNP Inactive P-TEFb P P P PPP P P P P P PPP P P Stress treatment CycT1
18、Cdk9 Active P-TEFb P P P P P P P HEXIM1 + A calcium-dependent signaling pathway controls P-TEFb transcriptional activity (in writing) F1C2 cell Ca+ InfluxCaMPP2B Hiding and seeking . Who is the second one? isoform of PP1 catalytic subunit involves in mediating the disruption of 7SK snRNP F1C2 cell C
19、ycT1 Cdk9 HEXIM1 7SK snRNP Inactive P-TEFb P P P PPP P P P P P PPP P P CycT1 Cdk9 Active P-TEFb P P P P P P P HEXIM1 + A calcium-dependent signaling pathway controls P-TEFb transcriptional activity (in writing) F1C2 cell Ca+ InfluxCaMPP2B PP1 PP2B + PP1 ? In vivo: PP2B and PP1 work cooperatively to
20、disrupt the inactive complex In vitro: PP2B and PP1 directly disrupt inactive complex PP2B: let me work first. F1C2 cell Calcium CycT1 Cdk9 HEXIM1 7SK snRNP Inactive P-TEFb P P P PPP P P P P P PPP P P Stress treatment CycT1 Cdk9 Active P-TEFb P P P P P P P HEXIM1 + F1C2 cell Ca+ InfluxCaMPP2B PP1 Ca
21、+2/PP2B and PP1 pathways cooperatively mediate UV and HMBA induced disruption of inactive complex How do PP2B and PP1 cooperatively disrupt inactive P-TFb complex? In vitro. Only PP1 can dephosphorylate pT186 of CDK9 In vitro. PP2B and PP1 work cooperatively to dephosphorylate pT186 of CDK9 CycT1 Cd
22、k9 Inactive P-TEFb P PHEXIM1 7SK snRNA PP2BPP1 Two steps dephosphorylation fro dissociating 7SK snRNP CycT1 Cdk9 plasma membrane LTCC Ca2+ PP2B nucleus PP1 CaM PP2B PP1 inactive P-TEFb CycT1 Cdk9 UV or HMBA P P HEXIM1 A calcium signaling pathway dependent two-step dephosphorylation to release P-TEFb
23、 CycT1 Cdk9 ? PHEXIM1 pT186 Brd4 How is liberated P-TFFb Recruited to chromatin Active P-TEFb for general transcription CycT1 Cdk9 Brd4 P P P P P P P P CycT1 Cdk9 Brd4 CTD Pol II mRNA mRNA Pol II CycT1 Cdk9 HEXIM1 7SK RNA CycT1 Cdk9 Brd4 Stress/hypertrophic signals/estrogen/ HIV infection inactive P
24、-TEFb Brd4 CycT1 Cdk9Brd4 ? Dynamic shift between different complex. CycT1 Cdk9 active P-TEFb HEXIM1 CycT1 Cdk9 active P-TEFb 1/4 Modified Nuclear Fractionation Chromatin-free fraction (LSF) Nucleus Low-salt buffer extraction High-salt buffer extraction Chromatin-bound fraction (HSF) Nuclear lysis f
25、raction (NLF) Nucleus Nucleus Almost all of Brd4 associate with chromatin at basal state Stimuation induces Brd4 release from chromatin The release of Brd4 from chromatin is essential from P-TEFb recruitment The release of Brd4 from chromatin is HDAC-dependent The release of Brd4 from chromatin is e
26、ssential from elongation CycT1 Cdk9 Productive elongation HEXIM1+ 2 2 2 2 PP2B+PP1Kinase? T186 CycT1 Cdk9 P Brd4 mRNA Pol II 5 5 5 5 Ac Ac HDAC Recruit P-TEFb to promoter mRNA Pol II H4 tail 5 5 5 5 H4K5/8 deacetylated Signal Deacetylate H4K5/8Ac mRNA Pol II Ac AcBrd4 Acetyl- nucleosome 5 5 5 5 CTD Release Brd4 from chromatin 7SK T186 7SK CycT1 Cdk9P HEXIM1 7SK snRNP Ai_Fig7 CycT1 Cdk9 P Adaptor Brd4Brd4 HDAC Signal ? CycT1 Cdk9 Kinase T186 CycT1 Cdk9 P ? Thank you!