Bijan Sobhian and Monsef Benkirane

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
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)
DrugsTSA, Prostratin,
P-TEFb
PS-2
Tar-RNA
CTD
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
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
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
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
S3Tat
S3
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
S3Tat
S3
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
S3Tat
S3
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
Tat forms two biochemically and functionally
distinct complexes
10
40
Fraction 7
S3Tat nuclear extract
7
11
(Fr)
5
9
13
15
AFF1
FLAG-IP
AFF4
Glycerol gradient sedimentation
ENL
Glycerol gradient Immunoblot
PAF1
AF9
CDK9
Fraction 11
HA(eTat)
LARP7
MEPCE
7SK-RT-QPCR
Fold increase
7
11
(Fr)
5
13
CTD-Kinase assay
32P-GST-CTD
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) cant
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-CTD4
CDK9 is the major Tat associated CTD kinase
17
Tatcom1 displays stronger CTD kinase activity
than core PTEFb (CycT1CDK9)
S3Tat nuclear extract
Tatcom1
Core P-TEFb
FLAG-IP
10
40
Glycerol gradient sedimentation
5
7
9
11
13
15
(Fr)
AFF1
CTD kinase activity
AFF4
5
7
(Fr)
Fractions 5 and 7 normalized for CDK9 levels
AF9
ENL
CycT1
PAF1
CDK9
AF9
HA(eTat)
CDK9
32P-CTD4
HA(eTat)
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-CTD4

• 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
IgG
CycT1-IP
PAF1-IP
IgG

AFF1
AFF1
PAF1
AFF4
CDK9
ENL
HA(eTat)
Long exposure
Core P-TEFb
Active/elongating
ENL
P-TEFb
CDK9
HA(eTat)
20
Tat induces formation of PTEFb MLL fusion
proteins PAF1
IgG
CycT1-IP
PAF1-IP
IgG
S3TatK50Q
S3Tat
S3
S3Tat
S3Tat
S3
S3Tat

AFF1
AFF1
PAF1
AFF4
CDK9
HA(eTat)
ENL
Long exposure
Core P-TEFb
Active/elongating
ENL
P-TEFb
CDK9
HA(eTat)
21
Tatcom1 is required for Tat transactivation
siRNA of Tatcom1 subunits reduces Tat mediated
transactivation of an integrated LTR-Luciferase
reporter
22
Tatcom1 is required for Tat transactivation
Proximal transcripts
Distal transcripts
SCR siRNA
AF9 siRNA
Fold increase
AF9 siRNA affects Tat induced elongation This is
consistent with AF9 requirement for optimal CDK9
CTD kinase activity
siRNA of Tatcom1 subunits reduces Tat mediated
transactivation of an integrated LTR-Luciferase
reporter
23
Tat assembles and recruits a multifunctional
transcriptional elongation complex to the HIV1
promoter
RNAPIIPS2
Flag (eTat)
Input
CDK9
ELL
AF9
Input
HP1?
IgG
PAF1
Input
Tatcom1 participates in transcription elongation
per se
24
Tat assembles and recruits a multifunctional
transcriptional elongation complex to stimulate
transcription elongation from the HIV1 promoter
Tatcom1
PTEFb
Tat

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

25
Conclusion and future directions
Activating
signals
Processive elongation Virus production
Latency
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

Structure of the Tat associated PTEFb complex may
provide opportunities to design inhibitory
peptides.
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)