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-RNA CTDPS-5

NTEFs Pol-II

CTDPS-5

Transactivator

CDK9

CycT1

PS-2P-TEFb

NTEFs

Drugs:TSA, Prostratin,…

Latent provirus = stalled LTR Activated provirus

HMBA activates the LTR by increasing P-TEFb activity (Contreras et al 2007)

Pol-II

Tar-RNA CTDPS-5

Abortive transcription

Latency

Pol-II

CTDPS-5

Processive elongation

Virus production

Tat

CDK9

CycT1 PS-2P-TEFb

NTEFs

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

CycT1HEXIM1CDK9

CycT1HEXIM1

(Bensaude O, Zhou Q, Kiss T, Price DH, Coulombe B, Fischer U)

Regulation of P-TEFb by Tat: Current view

Tat

Active P-TEFbInactive P-TEFb complex

7SK-RNA

Transcription

elongation

CDK9

MEPCELARP7

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 culture2) Preparing Dignam nuclear extract3) FLAG-IP followed by HA-IP4) Visualization of eTAT and associated proteins by silver staining5) 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 FLAG HA

??

Strategy:

Tat forms stoichiometric complexe(s) with PTEFb

MW

200

116

97

66

55

37

31

21

14

6

CycT1

CDK9

eTAT

S3Tat

S3FLAG/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

200

116

97

66

55

37

31

21

14

6

AFF1

AFF4

CycT1,ELL2,MEPCEENL,AF9

CDK9, EAF1

eTAT

S3Tat

S3FLAG/HA-IP

CDC73

Stoichiometric interactions with the 7SKsnRNP

LARP7,ELL,PAF1

MW

200

116

97

66

55

37

31

21

14

6

AFF1

AFF4

CycT1,ELL2,MEPCEENL,AF9

CDK9, EAF1

eTAT

S3Tat

S3

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

CDK9

5 7 9 1113 15(Fr)10% 40%

7SK-RT-QPCR

(Fr) 5 7 11 13

32P-GST-CTD

Fold

incr

ease

Tat forms two biochemically and functionally distinct complexes

S3Tat nuclear extract

FLAG-IP

Glycerol gradient

sedimentation Glycerol gradient: Immunoblot

CTD-Kinase assay

Tatcom1

ELL AF9CDC73

PAF1

CycT1

Tat CDK9

ENL

AFF4AFF1EAF1

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

Jurk

atJu

rkat

_Tat

ELL

AFF1

ELL

Expression levels and interactions in PBMC

Time (hrs) 0 20 72 72 0 20 72 72 72

PHA/IL2CD3/CD28 +

+++

++ +Cell extract CycT1-IP IgG-IP

AFF4CycT1

HEXIM1

CDK955

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

GST-Ta

tGST

AFF1

AFF4

MEPCE

CDK955

42

GST pull down on activated PBMC

Tatcom1 assembly is PTEFb dependent

FLAG-IP

S3Tat: CDK9 siRNA

Immunoblot/CTD-kinase

32P-[CTD]4

CycT1

HA(eTat)

CDK9

siRNA: SC

R

FLAG-IPS3 S3Tat

CD

K9

SC

R

CES3 S3Tat

CD

K9

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)

32P-[CTD]4

HA(eTat)

PAF1

ENL

AFF1

AF9

AFF4

CDK9

5 7 9 11 13 15(Fr)

10% 40%

S3Tat nuclear extract

FLAG-IP

Glycerol gradient

sedimentation

5 7(Fr)

Fractions 5 and 7 normalized for CDK9 levels

CTD kinase activity

Tatcom1

ELL AF9CDC73

PAF1

CycT1

Tat CDK9

ENL

AFF4AFF1EAF1

CycT1

Tat CDK9

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-IPS3 S3Tat

AF9

AFF4

CycT1

ELL

CDK9

AF9

ENL ENL

AF9(AF9 reprobed)

32P-[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 AF9CDC73

PAF1

ENL

AFF4AFF1EAF1

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

AFF4AFF1EAF1

CDC73

PAF1

CycT1

CDK9

CycT1

CDK9

CycT1

CDK9

ELL AF9CDC73

PAF1

ENL

AFF4AFF1EAF1

CycT1

CDK9

Core P-TEFb Active/elongatingP-TEFb

ELL AF9CDC73

PAF1

ENL

AFF4AFF1EAF1

+

S3T

atCycT1-IP IgG

HA(eTat)

ENL

CDK9

ENL

AFF1

S3T

atS

3Tat

K50

Q

S3

AFF4

Long exposure

Tat induces formation of: PTEFb + [MLL fusion proteins + PAF1]

S3T

at

S3T

at

S3

PAF1-IPIgG

CDK9

HA(eTat)

AFF1

PAF1

ELL AF9CDC73

PAF1

ENL

AFF4AFF1EAF1

CycT1

CDK9

ELL AF9 ENL

AFF4AFF1EAF1

CDC73

PAF1

CycT1

CDK9

ELL AF9CDC73

PAF1

CycT1

Tat

CDK9

ENL

AFF4AFF1EAF1

TatCycT1

CDK9

Core P-TEFb Active/elongatingP-TEFb

ELL AF9CDC73

PAF1

ENL

AFF4AFF1EAF1

+

siRNA: SCR AF9 PAF1 ELL ELL2 CDK9EAF1

10000

20000

30000

40000

50000

60000

7000031

126

124

19

18Arb

itrar

y un

it

Mock eTat

Tatcom1 is required for Tat transactivation

siRNA of Tatcom1 subunits reduces Tat mediated transactivation of an integrated LTR-Luciferase reporter

SCR siRNAAF9 siRNA

Fold

incr

ease

Proximal transcripts

Distal transcripts

AF9 siRNA affects Tat induced elongation

This is consistent with AF9 requirement for optimal CDK9 CTD kinase activity

siRNA: SCR AF9 PAF1 ELL ELL2 CDK9EAF1

10000

20000

30000

40000

50000

60000

7000031

126

124

19

18Arb

itrar

y un

it

Mock eTat

siRNA of Tatcom1 subunits reduces Tat mediated transactivation of an integrated LTR-Luciferase reporter

Tatcom1 is required for Tat transactivation

MockeTat

Luciferase

1 2 3 4

LTR

% In

put

% In

put

1 2 3 4GAP

DH

RNAPIIPS2Flag (eTat)

CDK9 ELLAF9

HP1γ IgG

1 2 3 4GAP

DH 1 2 3 4GAP

DH

PAF1

% In

put

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 AF9CDC73

PAF1

CycT1

Tat CDK9

ENL

AFF4AFF1EAF1

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 AF9CDC73

PAF1

CycT1

CDK9

ENL

AFF4AFF1EAF1

CycT1

CDK9

Core P-TEFb Active/elongating P-TEFb

Activating

LatencyProcessive 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)