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
MEPCELARP7
CycT1
CDK9
CycT1HEXIM1
CDK9
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 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 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
S3
FLAG/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
Fol
d in
crea
se
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
AFF4
CycT1
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-
Tat
GST
AFF1
AFF4
MEPCE
CDK9
55
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
at
S3T
atK
50Q
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 siRNA
AF9 siRNA
Fol
d in
crea
se
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 4G
APD
H
RNAPIIPS2Flag (eTat)
CDK9 ELLAF9
HP1γ IgG
1 2 3 4G
APD
H 1 2 3 4G
APD
H
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)