Bijan Sobhian and Monsef Benkirane (Institute of human genetics, Montpellier, France)

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. Transactivator CDK9 CycT1 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,… P-TEFb PS-2 CTD Tar-RNA CTD PS-5 PS-5 Pol-II Pol-II NTEFs NTEFs Latent provirus = stalled LTR Activated provirus

3. Tat mediated HIV-1 transcriptional activation Tar-RNA CTD PS-5 Abortive transcription Latency Pol-II NTEFs P-TEFb CDK9 PS-2 CycT1 CTD PS-5 Tat Processive elongation Virus production Pol-II NTEFs

4. MEPCE LARP7 P-TEFb: Current view CDK9 CDK9 CDK9 Brd4 HEXIM1 HEXIM1 CycT1 CycT1 CycT1 7SK-RNA Active P-TEFb Inactive P-TEFb complex Transcription elongation (Bensaude O, Zhou Q, Kiss T, Price DH, Coulombe B, Fischer U)

5. MEPCE LARP7 Regulation of P-TEFb by Tat: Current view HEXIM1 Tat CDK9 CDK9 CDK9 CycT1 Tat CycT1 CycT1 7SK-RNA Active P-TEFb Inactive P-TEFb complex Transcription elongation (Barboric et al 2007, Sedore et al 2007)

6. T29 CDK9 Brd4 CycT1 Active P-TEFb ?? (Meisheng et al 2009) 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 (ELL) associate throughout the coding region(Byun et al) -In vitro transcription: BRD4 associates with PIC and dissociates upon elongation (Brady JN) • 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 ? ? ? CDK9 ? eTat ? CycT1 FLAG HA Purification of active P-TEFb ?

9. Tat forms stoichiometric complexe(s) with PTEFb FLAG/HA-IP S3 S3Tat MW 200 116 97 CycT1 66 55 CDK9 37 31 21 eTAT 14 6

10. Stoichiometric interactions with different classes of elongation factors (PTEFb, ELLs, PAF1) and common MLL fusion proteins (AFF1, ENL, AF9, AFF4) involved in Leukemia FLAG/HA-IP S3 S3Tat MW AFF1 200 AFF4 116 97 CycT1,ELL2,MEPCE ENL,AF9 66 LARP7,ELL,PAF1 CDC73 55 CDK9, EAF1 37 31 21 eTAT 14 6

11. Stoichiometric interactions with the 7SKsnRNP FLAG/HA-IP S3 S3Tat MW AFF1 200 AFF4 116 97 % Protein coverage CycT1,ELL2,MEPCE ENL,AF9 66 LARP7,ELL,PAF1 RNAse protection assay with full length 7SK CDC73 55 FLAG/HA-IP S3 S3Tat CDK9, EAF1 37 31 7SK 21 eTAT 14 6

12. TAT associated complexes S3Tat nuclear extract FLAG-IP Glycerol gradient sedimentation IP against: AF9, ENL, ELL and LARP7

13. CDK9 CDK9 CycT1 CycT1 Tat forms two biochemically and functionally distinct complexes 10% 40% Fraction 7 PAF1 S3Tat nuclear extract 7 11 (Fr) 5 9 13 15 CDC73 AFF1 FLAG-IP AFF4 Glycerol gradient sedimentation ENL Glycerol gradient: Immunoblot PAF1 Tat Tat Tat AF9 CDK9 CycT1 LARP7 CDK9 Fraction 11 HA(eTat) LARP7 MEPCE 7SK-RT-QPCR Fold increase AFF1 Tatcom1 Tatcom2 AFF4 EAF1 MEPCE 7 11 (Fr) 5 13 ELL ENL AF9 CTD-Kinase assay 32P-GST-CTD 7SK

14. Tat associated complexes form in a Jurkat T-cell line FLAG-IP Jurkat Jurkat_Tat AFF1 AFF4 AF9 LARP7 ELL CDK9 HA(eTat)

15. Expression levels and interactions in PBMC GST pull down on activated PBMC Cell extract CycT1-IP IgG-IP + + + + + PHA/IL2 GST-Tat + + CD3/CD28 GST Time (hrs) 0 20 72 72 0 20 72 72 72 AFF4 AFF1 CycT1 AFF4 HEXIM1 55 MEPCE CDK9 42 55 ELL CDK9 42 ERK(1/2) 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 CDK9 siRNA, Flavopiridol and CDK9-DN (He et al 2010) dissociate Tatcom1 S3Tat: CDK9 siRNA FLAG-IP CE S3 S3Tat S3 S3Tat FLAG-IP SCR CDK9 SCR CDK9 siRNA: CycT1 Immunoblot/CTD-kinase CDK9 HA(eTat) Tubulin 32P-[CTD]4 CDK9 is the major Tat associated CTD kinase

17. Tatcom1 displays stronger CTD kinase activity than core PTEFb (CycT1+CDK9) PAF1 CDC73 Tatcom1 S3Tat nuclear extract Core P-TEFb FLAG-IP 10% 40% Glycerol gradient sedimentation (Fr) 5 7 9 11 13 15 Tat Tat CDK9 CDK9 CycT1 CycT1 AFF1 CTD kinase activity AFF4 (Fr) 5 7 Fractions 5 and 7 normalized for CDK9 levels AF9 ENL CycT1 PAF1 CDK9 AF9 HA(eTat) CDK9 32P-[CTD]4 HA(eTat) AFF1 AFF4 EAF1 ELL ENL AF9 Tatcom1 associated factors are required for optimal CDK9 CTD kinase activity

18. AF9 is required for optimal CDK9 CTD-kinase activity FLAG-IP S3Tat: -/+ AF9siRNA S3 S3Tat SCR AF9 siRNA: FLAG-IP AFF1 AFF4 Immunoblot CTD kinase activity CycT1 AF9 ENL ENL (AF9 reprobed) AF9 ELL CDK9 HA(eTat) 32P-[CTD]4 • AF9 knock down results in: • Reduced CTD-kinase activity • Reduced ELL binding

19. The Tat associated PTEFb elongation complex exists in the absence of Tat = PTEFb + [MLL fusion proteins + PAF1] = The active PTEFb complex PAF1 PAF1 PAF1 PAF1 CDC73 CDC73 CDC73 CDC73 IgG CycT1-IP IgG PAF1-IP + AFF1 AFF1 PAF1 CDK9 CDK9 CDK9 CDK9 CycT1 CycT1 CycT1 CycT1 AFF4 CDK9 ENL HA(eTat) Long exposure Core P-TEFb Active/elongating ENL P-TEFb CDK9 HA(eTat) AFF1 AFF1 AFF1 AFF1 AFF4 AFF4 AFF4 AFF4 EAF1 EAF1 EAF1 EAF1 ELL ELL ENL ENL ELL ELL ENL ENL AF9 AF9 AF9 AF9

20. Tat induces formation of: PTEFb + [MLL fusion proteins + PAF1] PAF1 PAF1 PAF1 PAF1 CDC73 CDC73 CDC73 CDC73 IgG CycT1-IP IgG PAF1-IP S3TatK50Q S3Tat S3 S3Tat S3Tat S3 S3Tat + AFF1 Tat Tat AFF1 PAF1 CDK9 CDK9 CDK9 CDK9 CycT1 CycT1 CycT1 CycT1 AFF4 CDK9 HA(eTat) ENL Long exposure Core P-TEFb Active/elongating ENL P-TEFb CDK9 HA(eTat) AFF1 AFF1 AFF1 AFF1 AFF4 AFF4 AFF4 AFF4 EAF1 EAF1 EAF1 EAF1 ELL ENL ELL ENL ELL ELL ENL ENL AF9 AF9 AF9 AF9

21. 31 70000 60000 50000 Arbitrary unit 40000 19 6 30000 12 18 12 20000 4 10000 siRNA: SCR AF9 PAF1 ELL ELL2 EAF1 CDK9 Tatcom1 is required for Tat transactivation Mock eTat siRNA of Tatcom1 subunits reduces Tat mediated transactivation of an integrated LTR-Luciferase reporter

22. 31 70000 60000 50000 Arbitrary unit 40000 19 6 30000 12 18 12 20000 4 10000 siRNA: SCR AF9 PAF1 ELL ELL2 EAF1 CDK9 Tatcom1 is required for Tat transactivation Mock eTat Proximal transcripts Distal transcripts SCR siRNA AF9 siRNA Fold increase siRNA of Tatcom1 subunits reduces Tat mediated transactivation of an integrated LTR-Luciferase reporter AF9 siRNA affects Tat induced elongation This is consistent with AF9 requirement for optimal CDK9 CTD kinase activity

23. Tat assembles and recruits a multifunctional transcriptional elongation complex to the HIV1 promoter RNAPIIPS2 Flag (eTat) % Input eTat CDK9 AF9 ELL Mock LTR Luciferase % Input 1 2 3 4 HP1γ PAF1 IgG % Input Tatcom1 participates in transcription elongation per se 1 1 1 2 2 2 3 3 3 4 4 4 GAPDH GAPDH GAPDH

24. Tat assembles and recruits a multifunctional transcriptional elongation complex to stimulate transcription elongation from the HIV1 promoter PAF1 CDC73 Tatcom1 PTEFb Tat + Tat CDK9 CycT1 Transcription elongation • Conclusions: • Tat forms at least two biochemically and functionally distinct complexes • Tat induces the formation of a complex composed of PTEFb + Leukemia module + PAF1 = Tatcom1 • Tatcom1 is involved in transcription elongation from the HIV1 promoter • AF9 is required for optimal CDK9 CTD-kinase activity and ELL recruitment to Tatcom1 AFF1 AFF4 EAF1 ELL ENL AF9

25. Conclusion and future directions PAF1 CDC73 Activating signals Processive elongation Virus production Latency CDK9 CDK9 CycT1 CycT1 Core P-TEFb Active/elongating P-TEFb • 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 AFF1 AFF4 EAF1 Structure of the Tat associated PTEFb complex may provide opportunities to design inhibitory peptides. ELL ENL AF9

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)