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

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. Many ways to activate but a common requirement: P-TEFb Pol-II Tar-RNACTD PS-5 NTEFs Pol-II CTD PS-5 Transactivator CDK9 CycT1 PS-2 P-TEFb NTEFs Drugs:TSA, Prostratin,… Latent provirus = stalled LTRActivated provirus HMBA activates the LTR by increasing P-TEFb activity (Contreras et al 2007)

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

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

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

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 Active P-TEFb ?? CDK9 CycT1 T29 (Meisheng et al 2009) BRD4 associated P-TEFb is not the elongating P-TEFb complex BRD4 recruits P-TEFb to promoters

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

? ? ? Purification of Tat associated proteins 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 “Immunoaffinity purification of mammalian protein complexes” (Ogryzko V. and Nakatani Y., Methods in Enzymology 2003) eTat Purification of active P-TEFb ? CDK9 CycT1 FLAGHA ? ? Strategy:

Tat forms stoichiometric complexe(s) with PTEFb MW CycT1 CDK9 eTAT S3TatS3 FLAG/HA-IP

Stoichiometric interactions with different classes of elongation factors (PTEFb, ELLs, PAF1) and common MLL fusion proteins (AFF1, ENL, AF9, AFF4) involved in Leukemia LARP7,ELL,PAF1 MW AFF1 AFF4 CycT1,ELL2,MEPCE ENL,AF9 CDK9, EAF1 eTAT S3TatS3 FLAG/HA-IP CDC73

Stoichiometric interactions with the 7SKsnRNP LARP7,ELL,PAF1 MW AFF1 AFF4 CycT1,ELL2,MEPCE ENL,AF9 CDK9, EAF1 eTAT S3TatS3 FLAG/HA-IP CDC73 S3 S3Tat 7SK FLAG/HA-IP RNAse protection assay with full length 7SK % Protein coverage

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

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

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

ELL Expression levels and interactions in PBMC Time (hrs) PHA/IL2 CD3/CD Cell extractCycT1-IPIgG-IP AFF4 CycT1 HEXIM1 CDK 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 GST-Tat GST AFF1 AFF4 MEPCE CDK GST pull down on activated PBMC

Tatcom1 assembly is PTEFb dependent FLAG-IP S3Tat: CDK9 siRNA Immunoblot/CTD-kinase 32 P-[CTD] 4 CycT1 HA(eTat) CDK9 siRNA: SCR FLAG-IP S3S3Tat CDK9SCR CE S3S3Tat CDK9 Tubulin CDK9 is the major Tat associated CTD kinase Tatcom1 formation is abolished in Cyclin T1 depleted extracts CDK9 siRNA, Flavopiridol and CDK9-DN (He et al 2010) dissociate Tatcom1 A Cyclin T1 binding-defective Tat (C22G) can’t form Tatcom1

Tatcom1 displays stronger CTD kinase activity than core PTEFb (CycT1+CDK9) AF9 CycT1 CDK9 HA(eTat) 32 P-[CTD] 4 HA(eTat) PAF1 ENL AFF1 AF9 AFF4 CDK (Fr) 10%40% S3Tat nuclear extract FLAG-IP Glycerol gradient sedimentation 57(Fr) Fractions 5 and 7 normalized for CDK9 levels CTD kinase activity Tatcom1 ELL AF9 CDC73 PAF1 CycT1 Tat CDK9 ENL AFF4 AFF1 EAF1 CycT1TatCDK9 Tatcom1 associated factors are required for optimal CDK9 CTD kinase activity Core P-TEFb

FLAG-IP S3Tat: -/+ AF9siRNA Immunoblot CTD kinase activity AFF1 HA(eTat) siRNA: SCR FLAG-IP S3S3Tat AF9 AFF4 CycT1 ELL CDK9 AF9 ENL AF9 (AF9 reprobed) 32 P-[CTD] 4 AF9 is required for optimal CDK9 CTD-kinase activity AF9 knock down results in: Reduced CTD-kinase activity Reduced ELL binding

CycT1-IP IgG HA(eTat) ENL CDK9 ENL AFF1 AFF4 Long exposure ELL AF9 CDC73 PAF1 ENL AFF4 AFF1 EAF1 The Tat associated PTEFb elongation complex exists in the absence of Tat = PTEFb + [MLL fusion proteins + PAF1] = The active PTEFb complex PAF1-IPIgG CDK9 HA(eTat) AFF1 PAF1 ELL AF9 ENL AFF4 AFF1 EAF1 CDC73 PAF1 CycT1 CDK9 CycT1 CDK9 CycT1CDK9 ELL AF9 CDC73 PAF1 ENL AFF4 AFF1 EAF1 CycT1CDK9 Core P-TEFbActive/elongating P-TEFb ELL AF9 CDC73 PAF1 ENL AFF4 AFF1 EAF1 +

S3Tat CycT1-IP IgG HA(eTat) ENL CDK9 ENL AFF1 S3TatS3TatK50QS3 AFF4 Long exposure Tat induces formation of: PTEFb + [MLL fusion proteins + PAF1] S3Tat S3 PAF1-IPIgG CDK9 HA(eTat) AFF1 PAF1 ELL AF9 CDC73 PAF1 ENL AFF4 AFF1 EAF1 CycT1 CDK9 ELL AF9 ENL AFF4 AFF1 EAF1 CDC73 PAF1 CycT1 CDK9 ELL AF9 CDC73 PAF1 CycT1 Tat CDK9 ENL AFF4 AFF1 EAF1 Tat CycT1CDK9 Core P-TEFbActive/elongating P-TEFb ELL AF9 CDC73 PAF1 ENL AFF4 AFF1 EAF1 +

siRNA: SCRAF9PAF1ELLELL2CDK9EAF Arbitrary unit Mock eTat Tatcom1 is required for Tat transactivation siRNA of Tatcom1 subunits reduces Tat mediated transactivation of an integrated LTR-Luciferase reporter

SCR siRNA AF9 siRNA Fold increase Proximal transcripts Distal transcripts AF9 siRNA affects Tat induced elongation This is consistent with AF9 requirement for optimal CDK9 CTD kinase activity siRNA: SCRAF9PAF1ELLELL2CDK9EAF Arbitrary unit Mock eTat siRNA of Tatcom1 subunits reduces Tat mediated transactivation of an integrated LTR-Luciferase reporter Tatcom1 is required for Tat transactivation

Mock eTat Luciferase 1234 LTR % Input 1234 GAPDH RNAPIIPS2 Flag (eTat) CDK9ELLAF9 HP1γ IgG 1234 GAPDH 1234 PAF1 % Input Tat assembles and recruits a multifunctional transcriptional elongation complex to the HIV1 promoter Tatcom1 participates in transcription elongation per se

Tatcom1 Transcription elongation Tat PTEFb + ELL AF9 CDC73 PAF1 CycT1 Tat CDK9 ENL AFF4 AFF1 EAF1 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 Tat assembles and recruits a multifunctional transcriptional elongation complex to stimulate transcription elongation from the HIV1 promoter

Conclusion and future directions ELL AF9 CDC73 PAF1 CycT1CDK9 ENL AFF4 AFF1 EAF1 CycT1CDK9 Core P-TEFbActive/elongating P-TEFb Activating Latency Processive elongation Virus production signals 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.

Acknowledgments Qiang Zhou (UC Berkeley) 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 FRM, ERC, ANRS, SIDACTION, ANR (funding)