HIV1 Tat complexes reveal subunit composition of active
HIV-1 Tat complexes reveal subunit composition of active P-TEFb and stable association with 7 SKsn. RNP Bijan Sobhian and Monsef Benkirane (Institute of human genetics, Montpellier, France)
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 (NFk. B, STAT 5, 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 CTD PS-5 P-TEFb CDK 9 PS-2 CTD PS-5 Cyc. T 1 Pol-II NTEFs Latent provirus = stalled LTR Transactivator Pol-II NTEFs Activated provirus
Tat mediated HIV-1 transcriptional activation Tar-RNA PS-5 Pol-II P-TEFb CTD Abortive transcription NTEFs Latency CDK 9 Cyc. T 1 Tat PS-2 CTD PS-5 Pol-II Processive elongation NTEFs Virus production
P-TEFb: Current view CDK 9 HEXIM 1 Cyc. T 1 HEXIM 1 MEPCE 7 SK-RNA CDK 9 Cyc. T 1 LARP 7 CDK 9 Brd 4 Cyc. T 1 Active P-TEFb Inactive P-TEFb complex Transcription elongation (Bensaude O, Zhou Q, Kiss T, Price DH, Coulombe B, Fischer U)
Regulation of P-TEFb by Tat: Current view HEXIM 1 Tat CDK 9 Cyc. T 1 MEPCE 7 SK-RNA CDK 9 Cyc. T 1 CDK 9 Tat Cyc. T 1 LARP 7 Inactive P-TEFb complex Active P-TEFb Transcription elongation (Barboric et al 2007, Sedore et al 2007)
P-TEFb: Current view - BRD 4 complex purification: BRD 4/P-TEFb/Mediator (Ozato K. ) -Ch. IP: BRD 4 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: BRD 4 associates with PIC and dissociates upon elongation (Brady JN) T 29 CDK 9 Brd 4 Cyc. T 1 Active P-TEFb ? ? (Meisheng et al 2009) • BRD 4 recruits P-TEFb to promoters • BRD 4 associated P-TEFb is not the elongating P-TEFb complex
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 Strategy: “Immunoaffinity purification of mammalian protein complexes” (Ogryzko V. and Nakatani Y. , Methods in Enzymology 2003) Tandem affinity chromatography from He. La S 3 cells stably expressing FLAG and HA tagged TAT-101 (e. TAT) or mock cells 1) Growing 4 L of suspension culture 2) Preparing Dignam nuclear extract 3) FLAG-IP followed by HA-IP 4) Visualization of e. TAT and associated proteins by silver staining 5) Mass spectrometry ? ? ? CDK 9 e. Tat Cyc. T 1 FLAG HA Purification of active P-TEFb ?
Tat forms stoichiometric complexe(s) with PTEFb Ta t S 3 MW S 3 FLAG/HA-IP 200 116 97 Cyc. T 1 66 55 CDK 9 37 31 21 14 6 e. TAT
Stoichiometric interactions with different classes of elongation factors (PTEFb, ELLs, PAF 1) and common MLL fusion proteins (AFF 1, ENL, AF 9, AFF 4) involved in Leukemia 200 S 3 MW Ta t FLAG/HA-IP AFF 1 AFF 4 116 97 66 55 Cyc. T 1, ELL 2, MEPCE ENL, AF 9 LARP 7, ELL, PAF 1 CDC 73 CDK 9, EAF 1 37 31 21 14 6 e. TAT
Stoichiometric interactions with the 7 SKsn. RNP 200 S 3 MW Ta t FLAG/HA-IP AFF 1 AFF 4 116 97 66 55 Cyc. T 1, ELL 2, MEPCE ENL, AF 9 LARP 7, ELL, PAF 1 CDC 73 % Protein coverage RNAse protection assay with full length 7 SK Ta S 3 37 S 3 CDK 9, EAF 1 t FLAG/HA-IP 31 7 SK 21 14 6 e. TAT
TAT associated complexes S 3 Tat nuclear extract FLAG-IP Glycerol gradient sedimentation IP against: AF 9, ENL, ELL and LARP 7
Tat forms two biochemically and functionally distinct complexes 10% S 3 Tat nuclear extract (Fr) 40% 5 7 9 11 13 15 Fraction 7 Tatcom 1 AFF 1 FLAG-IP AFF 4 CDC 73 ENL Glycerol gradient: Immunoblot ELL ENL AF 9 Cyc. T 1 PAF 1 Tat CDK 9 AF 9 CDK 9 Fraction 11 HA(e. Tat) Tatcom 2 LARP 7 MEPCE Cyc. T 1 MEPCE Tat CDK 9 7 SK-RT-QPCR Cyc. T 1 Fold increase Glycerol gradient sedimentation EAF 1 AFF 4 PAF 1 Tat LARP 7 (Fr) CTD-Kinase assay 5 32 P-GST-CTD 7 11 13 CDK 9
Tat associated complexes form in a Jurkat T-cell line _T at rk Ju Ju rk at at FLAG-IP AFF 1 AFF 4 AF 9 LARP 7 ELL CDK 9 HA(e. Tat)
Expression levels and interactions in PBMC ++ + 0 20 72 72 ++ + GST pull down on activated PBMC + T GS -Ta T t PHA/IL 2 CD 3/CD 28 Time (hrs) Cyc. T 1 -IP Ig. G-IP GS Cell extract 0 20 72 72 72 AFF 4 Cyc. T 1 AFF 4 HEXIM 1 55 CDK 9 42 ELL MEPCE 55 CDK 9 ERK(1/2) 42 Active P-TEFb and 7 SKsn. RNP subunits are induced upon T cell activation. Both, active and 7 SKsn. RNP bound P-TEFb complexes increase. Tatcom 1 and Tatcom 2 form in activated T-cells
Tatcom 1 assembly is PTEFb dependent Tatcom 1 formation is abolished in Cyclin T 1 depleted extracts A Cyclin T 1 binding-defective Tat (C 22 G) can’t form Tatcom 1 CDK 9 si. RNA, Flavopiridol and CDK 9 -DN (He et al 2010) dissociate Tatcom 1 Immunoblot/CTD-kinase Cyc. T 1 32 P-[CTD] SCR si. RNA: CE S 3 Tat CDK 9 FLAG-IP SCR FLAG-IP S 3 Tat CDK 9 S 3 Tat: CDK 9 si. RNA 4 CDK 9 HA(e. Tat) Tubulin CDK 9 is the major Tat associated CTD kinase
Tatcom 1 displays stronger CTD kinase activity than core PTEFb (Cyc. T 1+CDK 9) AF 9 S 3 Tat nuclear extract Tatcom 1 PAF 1 Core P-TEFb FLAG-IP 10% Glycerol gradient sedimentation AFF 1 CTD kinase activity AFF 4 (Fr) 5 CDC 73 ELL AF 9 40% 7 Cyc. T 1 EAF 1 AFF 4 ENL CDK 9 HA(e. Tat) Cyc. T 1 9 11 13 15 Tat Cyc. T 1 CDK 9 (Fr) 5 Tat CDK 9 7 Fractions 5 and 7 normalized for CDK 9 levels ENL PAF 1 AF 9 CDK 9 HA(e. Tat) 32 P-[CTD] 4 Tatcom 1 associated factors are required for optimal CDK 9 CTD kinase activity
AF 9 is required for optimal CDK 9 CTD-kinase activity S 3 Tat: -/+ AF 9 si. RNA FLAG-IP Immunoblot CTD kinase activity si. RNA: AFF 1 AFF 4 Cyc. T 1 AF 9 ENL (AF 9 reprobed) AF 9 knock down results in: • Reduced CTD-kinase activity • Reduced ELL binding ENL AF 9 ELL CDK 9 HA(e. Tat) 32 P-[CTD] 4 SC R AF 9 FLAG-IP S 3 Tat
The Tat associated PTEFb elongation complex exists in the absence of Tat = PTEFb + [MLL fusion proteins + PAF 1] = The active PTEFb complex PAF 1 Cyc. T 1 -IP Ig. G PAF 1 -IP AFF 1 PAF 1 AFF 4 CDK 9 ENL CDK 9 HA(e. Tat) CDC 73 ELL AF 9 ENL Cyc. T 1 CDK 9 AF 9 ENL + Cyc. T 1 CDK 9 Core P-TEFb ENL EAF 1 AFF 4 ELL PAF 1 CDC 73 EAF 1 AFF 4 ELL AF 9 ENL Cyc. T 1 CDK 9 HA(e. Tat) Long exposure PAF 1 CDC 73 EAF 1 AFF 4 PAF 1 EAF 1 AFF 4 CDC 73 ELL AF 9 ENL Cyc. T 1 CDK 9 Active/elongating P-TEFb
Tat induces formation of: PTEFb + [MLL fusion proteins + PAF 1] PAF 1 S 3 Tat Ig. G PAF 1 -IP S 3 Tat. K 50 Q S 3 Cyc. T 1 -IP Ig. G CDC 73 AFF 1 PAF 1 AFF 4 CDK 9 ENL HA(e. Tat) Long exposure ENL CDK 9 PAF 1 CDC 73 ELL AF 9 ENL Cyc. T 1 EAF 1 AFF 4 CDC 73 ELL AF 9 ENL Cyc. T 1 CDK 9 ELL AF 9 ENL + Cyc. T 1 CDK 9 Core P-TEFb EAF 1 AFF 4 HA(e. Tat) PAF 1 EAF 1 AFF 4 CDK 9 Tat EAF 1 AFF 4 PAF 1 CDC 73 ELL AF 9 ENL Cyc. T 1 Tat CDK 9 Active/elongating P-TEFb
Tatcom 1 is required for Tat transactivation Arbitrary unit 70000 31 Mock 60000 e. Tat 50000 40000 30000 12 19 6 4 10000 si. RNA: SCR 18 12 20000 AF 9 PAF 1 ELL 2 EAF 1 CDK 9 si. RNA of Tatcom 1 subunits reduces Tat mediated transactivation of an integrated LTR-Luciferase reporter
Tatcom 1 is required for Tat transactivation 35 35 30 30 25 25 20 31 60000 50000 40000 30000 12 e. Tat 5 5 0 -5 Distal transcripts SCR si. RNA AF 9 si. RNA 0 Mock e. Tat 4 10000 si. RNA: SCR 15 10 18 12 20000 20 10 19 6 Proximal transcripts Mock Fold increase Arbitrary unit 70000 15 AF 9 PAF 1 ELL 2 EAF 1 CDK 9 si. RNA of Tatcom 1 subunits reduces Tat mediated transactivation of an integrated LTR-Luciferase reporter AF 9 si. RNA affects Tat induced elongation This is consistent with AF 9 requirement for optimal CDK 9 CTD kinase activity
Tat assembles and recruits a multifunctional transcriptional elongation complex to the HIV 1 promoter Mock LTR Luciferase 1 2 3 RNAPIIPS 2 Flag (e. Tat) % Input e. Tat 4 6 CDK 9 4 % Input 5 2 PAF 1 0 2 10 HP 1γ Ig. G 5 1 0 2 H PD 4 GA 3 2 H 4 PD GA 3 1 4 1 0 -1 0 2 % Input 0 0 -5 1 4 2 ELL 10 2 1 6 15 AF 9 1 Tatcom 1 participates in transcription elongation per se 0 0
Tat assembles and recruits a multifunctional transcriptional elongation complex to stimulate transcription elongation from the HIV 1 promoter Tatcom 1 PTEFb EAF 1 AFF 4 PAF 1 CDC 73 Tat ELL AF 9 Cyc. T 1 Tat ENL + CDK 9 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 + PAF 1 = Tatcom 1 • Tatcom 1 is involved in transcription elongation from the HIV 1 promoter • AF 9 is required for optimal CDK 9 CTD-kinase activity and ELL recruitment to Tatcom 1
Conclusion and future directions Cyc. T 1 Activating signals PAF 1 CDC 73 EAF 1 AFF 4 ELL AF 9 ENL CDK 9 Latency Core P-TEFb Processive elongation Cyc. T 1 CDK 9 Virus production 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 Structure of the Tat associated PTEFb complex may provide opportunities to design inhibitory peptides.
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)
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