HIV-1 Tat complexes reveal subunit composition of active P-TEFb and stable association with 7SKsnRNP

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1 HIV-1 Tat complexes reveal subunit composition of active P-TEFb and stable association with 7SKsnRNP Bijan Sobhian and Monsef Benkirane (Institute of human genetics, Montpellier, France)

2 Many ways to activate but a common requirement: P-TEFb Establishment of a latent provirus is a multifactorial process Multiple drugs targeting various blocks to transcription (HDAC,HMT,DNMT) or inducing activating pathways (NFkB, STAT5, NFAT) have been shown to reactivate a latent virus. However, the HIV-1 LTR is a stalled promoter, thus all will require the action of PTEFb for pause release. HMBA activates the LTR by increasing P-TEFb activity (Contreras et al 2007) Drugs:TSA, Prostratin, Tar-RNA PS-5 CTD P-TEFb PS-2 PS-5 CTD Pol-II NTEFs Transactivator Pol-II NTEFs Latent provirus = stalled LTR Activated provirus

3 Tat mediated HIV-1 transcriptional activation Tar-RNA PS-5 CTD Pol-II NTEFs Abortive transcription Latency P-TEFb Tat PS-2 PS-5 CTD Pol-II NTEFs Processive elongation Virus production

4 P-TEFb: Current view HEXIM1 HEXIM1 MEPCE 7SK-RNA LARP7 Brd4 Active P-TEFb Inactive P-TEFb complex Transcription elongation (Bensaude O, Zhou Q, Kiss T, Price DH, Coulombe B, Fischer U)

5 Regulation of P-TEFb by Tat: Current view Tat HEXIM1 MEPCE 7SK-RNA LARP7 Inactive P-TEFb complex Tat Active P-TEFb Transcription elongation (Barboric et al 2007, Sedore et al 2007)

6 P-TEFb: Current view - BRD4 complex purification: BRD4/P-TEFb/Mediator (Ozato K.) -ChIP: BRD4 is found at promoter regions upon activation while P-TEFb and other elongation factors () associate throughout the coding region(byun et al) -In vitro transcription: BRD4 associates with PIC and dissociates upon elongation (Brady JN) T29 Brd4 Active P-TEFb?? (Meisheng et al 2009) BRD4 recruits P-TEFb to promoters BRD4 associated P-TEFb is not the elongating P-TEFb complex

7 What is the subunit composition of active or elongating P-TEFb? Tat forms stoichiometric complexes with P-TEFb Tat recruits P-TEFb to elongating RNAPII Active P-TEFb should co-purify with Tat

8 Purification of Tat associated proteins Strategy: Immunoaffinity purification of mammalian protein complexes (Ogryzko V. and Nakatani Y., Methods in Enzymology 2003) Tandem affinity chromatography from HeLa S3 cells stably expressing FLAG and HA tagged TAT-101 (etat) or mock cells 1) Growing 4L of suspension culture 2) Preparing Dignam nuclear extract 3) FLAG-IP followed by HA-IP 4) Visualization of etat and associated proteins by silver staining 5) Mass spectrometry????? etat FLAG HA Purification of active P-TEFb?

9 Tat forms stoichiometric complexe(s) with PTEFb FLAG/HA-IP MW S3 S3Tat etat 14 6

10 Stoichiometric interactions with different classes of elongation factors (PTEFb, s, ) and common MLL fusion proteins (AFF1,,, AFF4) involved in Leukemia FLAG/HA-IP MW S3 S3Tat AFF1 AFF4,2,MEPCE, LARP7,, 37 31, EAF1 21 etat 14 6

11 Stoichiometric interactions with the 7SKsnRNP FLAG/HA-IP MW S3 S3Tat AFF1 AFF4,2,MEPCE, LARP7,,, EAF1 etat FLAG/HA-IP S3 % Protein coverage RNAse protection assay with full length 7SK S3Tat 7SK

12 TAT associated complexes S3Tat nuclear extract FLAG-IP Glycerol gradient sedimentation IP against:,, and LARP7

13 Tat forms two biochemically and functionally distinct complexes S3Tat nuclear extract FLAG-IP Glycerol gradient sedimentation Glycerol gradient: Immunoblot 10% 40% (Fr) AFF1 AFF4 Fraction 7 Tatcom1 Tat EAF1 AFF1 AFF4 HA(eTat) LARP7 Fraction 11 Tatcom2 MEPCE MEPCE Tat 7SK 7SK-RT-QPCR Fold increase LARP7 Tat (Fr) CTD-Kinase assay 32 P-GST-CTD

14 Tat associated complexes form in a Jurkat T-cell line FLAG-IP Jurkat Jurkat_Tat AFF1 AFF4 LARP7 HA(eTat)

15 Expression levels and interactions in PBMC Cell extract -IP IgG-IP PHA/IL CD3/CD Time (hrs) AFF4 HEXIM ERK(1/2) GST pull down on activated PBMC AFF1 AFF4 MEPCE GST-Tat GST Active P-TEFb and 7SKsnRNP subunits are induced upon T cell activation. Both, active and 7SKsnRNP bound P-TEFb complexes increase. Tatcom1 and Tatcom2 form in activated T-cells

16 Tatcom1 assembly is PTEFb dependent Tatcom1 formation is abolished in Cyclin T1 depleted extracts A Cyclin T1 binding-defective Tat (C22G) can t form Tatcom1 sirna, Flavopiridol and -DN (He et al 2010) dissociate Tatcom1 S3Tat: sirna FLAG-IP sirna: FLAG-IP S3 S3Tat SCR CE S3 S3Tat SCR Immunoblot/CTD-kinase HA(eTat) Tubulin 32 P-[CTD] 4 is the major Tat associated CTD kinase

17 Tatcom1 displays stronger CTD kinase activity than core PTEFb (+) S3Tat nuclear extract FLAG-IP Glycerol gradient sedimentation (Fr) AFF1 10% 40% Core P-TEFb Tat Tatcom1 Tat EAF1 AFF1 AFF4 CTD kinase activity AFF4 (Fr) HA(eTat) 32 P-[CTD] Fractions 5 and 7 normalized for levels HA(eTat) Tatcom1 associated factors are required for optimal CTD kinase activity

18 is required for optimal CTD-kinase activity S3Tat: -/+ sirna FLAG-IP Immunoblot CTD kinase activity sirna: AFF1 AFF4 ( reprobed) FLAG-IP S3 S3Tat SCR HA(eTat) 32 P-[CTD] 4 knock down results in: Reduced CTD-kinase activity Reduced binding

19 The Tat associated PTEFb elongation complex exists in the absence of Tat = PTEFb + [MLL fusion proteins + ] = The active PTEFb complex EAF1 AFF1 AFF4 -IP IgG AFF1 AFF4 Long exposure IgG -IP AFF1 HA(eTat) + Core P-TEFb EAF1 AFF1 AFF4 Active/elongating P-TEFb HA(eTat) EAF1 AFF1 AFF4 EAF1 AFF1 AFF4

20 Tat induces formation of: PTEFb + [MLL fusion proteins + ] -IP IgG S3 S3Tat S3TatK50Q S3Tat AFF1 AFF4 Long exposure IgG -IP S3Tat S3 S3Tat AFF1 HA(eTat) EAF1 AFF1 AFF4 EAF1 + AFF1 AFF4 Core P-TEFb Tat EAF1 Tat AFF1 AFF4 Active/elongating P-TEFb HA(eTat) EAF1 AFF1 AFF4

21 Tatcom1 is required for Tat transactivation Arbitrary unit Mock etat sirna: SCR 2 EAF1 sirna of Tatcom1 subunits reduces Tat mediated transactivation of an integrated LTR-Luciferase reporter

22 Tatcom1 is required for Tat transactivation Arbitrary unit Mock etat Fold increase Proximal transcripts Distal transcripts SCR sirna sirna sirna: SCR 2 EAF1 sirna of Tatcom1 subunits reduces Tat mediated transactivation of an integrated LTR-Luciferase reporter sirna affects Tat induced elongation This is consistent with requirement for optimal CTD kinase activity

23 Tat assembles and recruits a multifunctional transcriptional elongation complex to the HIV1 promoter etat Mock Flag (etat) RNAPIIPS2 LTR Luciferase % Input % Input % Input HP1γ IgG GAPDH GAPDH GAPDH Tatcom1 participates in transcription elongation per se

24 Tat assembles and recruits a multifunctional transcriptional elongation complex to stimulate transcription elongation from the HIV1 promoter PTEFb Tat Transcription elongation Tat Tatcom1 EAF1 AFF1 AFF4 + Conclusions: Tat forms at least two biochemically and functionally distinct complexes Tat induces the formation of a complex composed of PTEFb + Leukemia module + = Tatcom1 Tatcom1 is involved in transcription elongation from the HIV1 promoter is required for optimal CTD-kinase activity and recruitment to Tatcom1

25 Conclusion and future directions Core P-TEFb Activating signals Latency EAF1 AFF1 AFF4 Active/elongating P-TEFb Processive elongation Virus production Which signals/pathways are required to form active PTEFb? Expression/Stability of the identified cofactors Association of the activating module Exploring the mechanism by which Tat induces this complex Structure of the Tat associated PTEFb complex may provide opportunities to design inhibitory peptides.

26 Acknowledgments Monsef Benkirane and Rosemary Kiernan for amazing tutorship Nadine Laguette, Ahmad Yatim, Mirai Nakamura, Daniel Latreille, Yamina Bennasser, Oussama Meziane, Christine Chable-Bessia, Alexandre Wagschal, Ke Zhang Qiang Zhou (UC Berkeley) FRM, ERC, ANRS, SIDACTION, ANR (funding)