Doctoral thesis presentation, Ecole Normale Supérieure, Paris, 2012.

1. PhD defense presentation of
Nina VERSTRAETE
Regulation of the positive
transcription elongation
factor P-TEFb (Cdk9/CycT1)
by HEXIM1 protein
under the supervision of In front of the jury composed of
Olivier Bensaude Michelle Debatisse
Functional Genomics Dpt. Matthias Geyer
Cell biology of Transcription Patricia Uguen
1
Claude Gaillardin
September 28, 2012
Regulation of P-TEFb by HEXIM1 protein

2. Contents
 Introduction
• P-TEFb and the control of transcription elongation
• Hijacking by HIV-1 TAR RNA and Tat protein
• Regulation of P-TEFb (CDK9/CycT1)
o P-TEFb co-factors and gene-specific recruitment
o Inhibition by 7SK ncRNA and HEXIM1 protein
 Problematic
 Methods
 Results
 Discussion
 Conclusions & perspectives
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins

3. Transcription regulates the information flow
from genes to proteins
Proteins and non-coding RNAs are chief actors within the cell,
carrying out the duties specified by the information encoded in genes
Regulation of P-TEFb by HEXIM1 protein

4. Transcription and RNA Polymerase II
• Eukaryotes : RNAPII > protein-coding genes and ncRNA
• Phosphorylation of the CTD of RNAPII Plays Central Roles
in the Integrated Events of Eucaryotic Gene Expression
RNAPII
– Transcriptconserved ( fungi, plants, animals)
Evolutionary RNA
– 52 repeats in mammals (Y1S2P3T4S5P6S7)
– Scaffold for the interaction of nuclear factors
• Transcription, RNA processing DNA
• chromatin structure modification
• DNA damage/repair
• protein degradation
• snRNA modification and snoRNP biogenesis
• Subject to hyperphosphorylation : phosphorylation state
changes as Pol II progresses in the transcription cycle
April 2003 Molecule of the Month by David Goodsell
RCSB Protein Data Bank
Regulation of P-TEFb by HEXIM1 protein

5. RNAPII promoter-proximal pausing
AAAAAAAA
Poly (A)
Regulation of P-TEFb by HEXIM1 protein

6. RNAPII promoter-proximal pausing
Pol II density AAAAAAAA
Poly (A)
Regulation of P-TEFb by HEXIM1 protein

7. RNAPII promoter-proximal pausing
Pol II density AAAAAAAA
Poly (A)
Promoter-proximal
PAUSING
Regulation of P-TEFb by HEXIM1 protein

8. RNAPII promoter-proximal pausing
Pol II density AAAAAAAA
Poly (A)
Regulation of P-TEFb by HEXIM1 protein

9. Functional relevance of RNAPII pausing
1 Establishing permissive chromatin 2 Rapid or synchronous activation
P-TEFb
P NELF
P-TEFb
P P P NELF
DSIF P DSIF
NELF P
P DSIF
Pol II Pol II
GTFs TF2 GTFs
TF1 TF1 Pol II
TF2 GTFs
TF1
3 Checkpoint in early elongation
P-TEFb
P-TEFb P P
RPF
TF2
P P P
DSIF P DSIF P DSIF
NELF
Pol II Pol II Pol II
GTFs
TF1 CEC RPF
Nature Reviews | Genetics
Regulation of P-TEFb by HEXIM1 protein

10. Functional relevance of RNAPII pausing
1 Establishing permissive chromatin 2 Rapid or synchronous activation
P-TEFb
P NELF
P-TEFb
P P P NELF
DSIF P DSIF
NELF P
P DSIF
Pol II Pol II
GTFs TF2 GTFs
TF1 TF1 Pol II
TF2 GTFs
TF1
3 Checkpoint in early elongation
P-TEFb
P-TEFb P P
RPF
TF2
P P P
DSIF P DSIF P DSIF
NELF
Pol II Pol II Pol II
GTFs
TF1 CEC RPF
Nature Reviews | Genetics
Regulation of P-TEFb by HEXIM1 protein

11. Functional relevance of RNAPII pausing
1 Establishing permissive chromatin 2 Rapid or synchronous activation
P-TEFb
P NELF
P-TEFb
P P P NELF
DSIF P DSIF
NELF P
P DSIF
Pol II Pol II
GTFs TF2 GTFs
TF1 TF1 Pol II
TF2 GTFs
TF1
3 Checkpoint in early elongation
P-TEFb
P-TEFb P P
RPF
TF2
P P P
DSIF P DSIF P DSIF
NELF
Pol II Pol II Pol II
GTFs
TF1 CEC RPF
Nature Reviews | Genetics
Regulation of P-TEFb by HEXIM1 protein

12. Contents
 Introduction
• P-TEFb and the control of transcription elongation
• Hijacking by HIV-1 TAR RNA and Tat protein
• Regulation of P-TEFb (CDK9/CycT1)
o P-TEFb co-factors and gene-specific recruitment
o Inhibition by 7SK ncRNA and HEXIM1 protein
 Problematic
 Methods
 Results
 Discussion
 Conclusions & perspectives
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins

13. Usurpation of the transcription elongation machinery
by HIV-1 TAR RNA and Tat protein
Regulation of P-TEFb by HEXIM1 protein

14. Usurpation of the transcription elongation machinery
by HIV-1 TAR RNA and Tat protein
Regulation of P-TEFb by HEXIM1 protein

15. Usurpation of the transcription elongation machinery
by HIV-1 TAR RNA and Tat protein
P-TEFb
Regulation of P-TEFb by HEXIM1 protein

16. Contents
 Introduction
• P-TEFb and the control of transcription elongation
• Hijacking by HIV-1 TAR RNA and Tat protein
• Regulation of P-TEFb (CDK9/CycT1)
o P-TEFb co-factors and gene-specific recruitment
o Inhibition by 7SK ncRNA and HEXIM1 protein
 Problematic
 Methods
 Results
 Discussion
 Conclusions perspectives
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins

17. Positive Transcription Elongation Factor b
• P-TEFb is composed of CDK9 (40 kDa) and Cyclin T (81kDa)
Regulation of P-TEFb by HEXIM1 protein

18. Positive Transcription Elongation Factor b
• P-TEFb is composed of CDK9 (40 kDa) and Cyclin T (81kDa)
• Belongs to the CDK/Cyclin family CDK9
Cyclin T1
• Cyclin-Dependent Kinase :
CDK9 requires the binding of the
Cyclin T partner to become active
ATP
PDB 3BLH
Regulation of P-TEFb by HEXIM1 protein

19. Positive Transcription Elongation Factor b
• P-TEFb is composed of CDK9 (40 kDa) and Cyclin T (81kDa)
• Belongs to the CDK/Cyclin family CDK9
Cyclin T1
• Cyclin-Dependent Kinase :
CDK9 requires the binding of the
Cyclin T partner to become active
• Interacts with several other
transcription factors and co-
activators (C/EBPβ, CIITA, NF-κB, c-
Myc, MyoD, HMGA1, androgen and aryl
hydrocarbon receptors, HIC, B-Myb, GRIP1, ATP
STAT3, AFF4, AF9, ENL, ELL2)
• Can be recruited to chromatin
through BRD4 (Bromodomain-
containing protein 4)
PDB 3BLH
Regulation of P-TEFb by HEXIM1 protein

20. Contents
 Introduction
• P-TEFb and the control of transcription elongation
• Hijacking by HIV-1 TAR RNA and Tat protein
• Regulation of P-TEFb (CDK9/CycT1)
o P-TEFb co-factors and gene-specific recruitment
o Inhibition by 7SK ncRNA and HEXIM1 protein
 Problematic
 Methods
 Results
 Discussion
 Conclusions perspectives
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins

21. Inhibition of P-TEFb
by 7SK RNA and HEXIM1 protein
 7SK non-coding RNA
o 331 nucleotides
o RNAPIII transcript
o Abundant (~2.105 copies per cell)
o Riboregulator of P-TEFb (Nguyen et al. 2001, Yang et al. 2001)
Regulation of P-TEFb by HEXIM1 protein

22. Inhibition of P-TEFb
by 7SK RNA and HEXIM1 protein
 7SK non-coding RNA
o 331 nucleotides
o RNAPIII transcript
o Abundant (~2.105 copies per cell)
o Riboregulator of P-TEFb (Nguyen et al. 2001, Yang et al. 2001)
MePCE
LARP7
o LARP7 binds 7SK 3’ end and protects it from nuclease activity
o MePCE generates a 5’ cap on 7SK that protects it from degradation
Regulation of P-TEFb by HEXIM1 protein

23. Inhibition of P-TEFb
by 7SK RNA and HEXIM1 protein
 HEXIM1 protein
o 359 aa, 41 kDa
o Dimer
o Binds 7SK RNA and Cyclin T1
Hex1 Hex1 o Intrinsically disordered regions
o 3D structure partially defined (Dames et al. 2007, Bigalke et al. 2011)
A B
MePCE
LARP7
A 7SK RNA binding Cyclin T1 binding
Regulatory region Coiled-coil
PYNT
HEXIM1 ARM / NLS
Acidic region
(self-inhibitory) Basic region dimerization domain
1 150 177 211 249 279 352 359
B + - -+
+ - -+
+ - -+
N N
C C
Regulation of P-TEFb by HEXIM1 protein

24. Inhibition of P-TEFb
by 7SK RNA and HEXIM1 protein
 HEXIM1 protein
o 359 aa, 41 kDa
o Dimer
o Binds 7SK RNA and Cyclin T1
Hex1 Hex1 o Intrinsically disordered regions
o 3D structure partially defined (Dames et al. 2007, Bigalke et al. 2011)
A B
Cyclin T1 Cyclin T1
MePCE CDK CDK
9 9
LARP7
A 7SK RNA binding Cyclin T1 binding
Regulatory region Coiled-coil
PYNT
HEXIM1 ARM / NLS
Acidic region
(self-inhibitory) Basic region dimerization domain
1 150 177 211 249 279 352 359
B + - -+
+ - -+
+ - -+
N N
C C
Regulation of P-TEFb by HEXIM1 protein

25. Inhibition of P-TEFb
by 7SK RNA and HEXIM1 protein
Hex1 Hex1
o 7SK non-coding RNA – 331 nt
o LARP7 – 67 kDa
Cyclin T1 o MePCE – 74 kDa
o HEXIM1 – 2 x 41 kDa
MePCE CDK
9
o CycT1 – (2 x) 81 kDa
o CDK9 – (2 x) 40 kDa
LARP7 --------------------------------------------------
o 350 kDa
7SK
snRNP
P-TEFb
CDK9/CycT1
INACTIVE
Regulation of P-TEFb by HEXIM1 protein

26. Inhibition of P-TEFb
by 7SK RNA and HEXIM1 protein
Hex1 Hex1
hnRNP Q/R/A
MePCE
LARP7
7SK
snRNP
Hex1 Hex1
Cyclin T1 Cyclin T1
MePCE CDK CDK
9 9
LARP7
P-TEFb
P-TEFb CDK9/CycT1
CDK9/CycT1 ACTIVE
INACTIVE
Hex1 Hex1
hnRNP Q/R/A
MePCE
LARP7
Regulation of P-TEFb by HEXIM1 protein

27. Inhibition of P-TEFb
by 7SK RNA and HEXIM1 protein
Hex1 Hex1
hnRNP Q/R/A
MePCE
LARP7
7SK
snRNP
Hex1 Hex1
Cyclin T1 Cyclin T1
Transcription
Inhibition CDK
9
CDK
9
Cyclin T1 (UV
exposure,
DRB,
flavopiridol) Cyclin T1
MePCE CDK
9
or
CDK
9
Cyclin T1
LARP7
Cardiac
Hypertrophy CDK
9
P-TEFb
P-TEFb
CDK9/CycT1 CDK9/CycT
INACTIVE ACTIVE
Hex1 Hex1
hnRNP Q/R/A
MePCE
LARP7
Regulation of P-TEFb by HEXIM1 protein

28. Inhibition of P-TEFb
by 7SK RNA and HEXIM1 protein
• Molecular mechanisms
Hex1 Hex1
of HEXIM1-mediated
Hex1 Hex1
inhibition of P-TEFb
Cyclin T1
Cyclin T1 • Biological impact of
MePCE CDK
9
P-TEFb/HEXIM1 interaction
LARP7
7SK
snRNP CDK
INACTIVE 9
Regulation of P-TEFb by HEXIM1 protein

29. Contents
 Introduction
 Problematic - What are the molecular mechanisms
leading to P-TEFb inhibition by HEXIM1?
- What is the biological impact
of P-TEFb / HEXIM1 interaction?
 Methods - Engineering of mutant P-TEFb unable to be inhibited
by HEXIM1 (edgetic approach)
 Results
 Discussion
 Conclusion perspectives
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins

30. Perturbing edges instead of nodes
Nature Methods, 2009
CyclinT1
• Random mutagenesis on Cyclin T1 by error-prone PCR
• Screen for a loss of interaction with HEXIM by reverse two-
hybrid in yeast
Edgetic Perturbation of P-TEFb

31. Perturbing edges instead of nodes
Nature Methods, 2009
HEXIM1
Cyclin T1
• Random mutagenesis on Cyclin T1 by error-prone PCR
• Screen for a loss of interaction with HEXIM1 by reverse
two-hybrid in yeast
Edgetic Perturbation of P-TEFb

32. Mutant screening by Reverse Two-Hybrid
:
toxic for cells able to synthetize uracile
(PCR
Δ)
Edgetic Perturbation of P-TEFb

33. Mutant screening by Reverse Two-Hybrid
 Induction of a counter-selectable gene (ura3)
 Random mutagenous PCR on Cyclin Boxes
 Detect a loss of interaction between CycT1-HEXIM1
:
toxic for cells able to synthetize uracile
(PCR
Δ)
Edgetic Perturbation of P-TEFb

34. Mutant screening by Reverse Two-Hybrid
 Induction of a counter-selectable gene (ura3)
 Random mutagenous PCR on Cyclin Boxes
 Detect a loss of interaction between CycT1-HEXIM1
:
toxic for cells able to synthetize uracile
(PCR
Δ)
Edgetic Perturbation of P-TEFb

35. Mutant screening by Reverse Two-Hybrid
 Induction of a counter-selectable gene (ura3)
 Random mutagenous PCR on Cyclin Boxes
 Detect a loss of interaction between CycT1-HEXIM1
:
toxic for cells able to synthetize uracile
(PCR
Δ)
Edgetic Perturbation of P-TEFb

36. Mutant Library
Random Mutagenesis by error-prone PCR
Edgetic Perturbation of P-TEFb

37. Reverse two-hybrid in Yeast
Transformation in Yeast
Mav103 : + HEXIM1-AD (-Leu)
MATa SPAL10::URA3 leu2-3,
112 trp1-901 his3200 ade2-101
gal4 gal80 can1r cyh2r + PCR Δ
GAL1::HIS3@LYS2 + CycT-BD // (-Trp)
GAL1::lacZ@URA3
-L -T -L -T -U -L -T +5FOA
PCR on colony
?
sequencing
HEXIM-AD interaction no interaction
CycT-BD HEXIM-AD x CycT-BD FOA resistance
homologous ura3 ↑
recombination
Edgetic Perturbation of P-TEFb

38. Contents
 Introduction
 Problematic
 Methods
 Results
• Identification of Cyclin T1 residues involved in HEXIM1 binding
• Visualization of HEXIM1 putative binding surface on Cyclin T1
• Functional impact on P-TEFb activity
 Discussion
 Conclusion perspectives
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins

39. Cyclin T1 mutant library analysis
• Identification of mutations by -L -T +5FOA
200 sequences
analyzed PCR on colonies FOAr and
sequencing
%)#
!#
')#
.32819#6#6;;391.;1,
!)#
+)#
(%#
*)#
$%#
()#
'# %#
$)#
#
)#
)#
,-./01# 763801# 49-:01# :91254391# 9521,=-4# .6#
23454-6.# 23454-6.,# 23454-6.,# ,46:#;676.# 23454-6.#
Regulation of P-TEFb by HEXIM1 protein

40. Cyclin T1 mutant library analysis
• Identification of mutations by -L -T +5FOA
200 sequences
analyzed PCR on colonies FOAr and
sequencing
%)#
!#
')#
.32819#6#6;;391.;1,
!)#
+)#
(%#
*)#
$%#
()#
'# %#
$)#
#
)#
)#
,-./01# 763801# 49-:01# :91254391# 9521,=-4# .6#
23454-6.# 23454-6.,# 23454-6.,# ,46:#;676.# 23454-6.#
Regulation of P-TEFb by HEXIM1 protein

41. Cyclin T1 mutant library analysis
• Identification of mutations by -L -T +5FOA
200 sequences
analyzed PCR on colonies FOAr and
sequencing
%)#
!#
')#
.32819#6#6;;391.;1,
!)#
+)#
(%#
*)#
$%#
()#
'# %#
$)#
#
)#
)#
!
,-./01# 763801# 49-:01# :91254391# 9521,=-4# .6#
23454-6.# 23454-6.,# 23454-6.,# ,46:#;676.# 23454-6.#
Regulation of P-TEFb by HEXIM1 protein

42. Validation of 69 Cyclin T1 point mutations
in forward two-hybrid for interaction with HEXIM1 and CDK9
AD
l4
1.
a G
1
IM
H EX
Random
? Interaction between
mutations mutant CycT1 and HEXIM1 / CDK9
CycT1 GROWTH ON MEDIA LACKING URACIL
Gal4BD
GAL4 PROMOTER REPORTER GENE (ura3)
Regulation of P-TEFb by HEXIM1 protein

43. Validation of 69 Cyclin T1 point mutations
in forward two-hybrid for interaction with HEXIM1 and CDK9
AD
l4
1.
a G
1
IM
H EX
Random
? Interaction between
mutations mutant CycT1 and HEXIM1 / CDK9
CycT1 GROWTH ON MEDIA LACKING URACIL
Gal4BD
GAL4 PROMOTER REPORTER GENE (ura3)
Yeast growth on media lacking uracil
+++ + -
STRONG weak NO
interaction interaction interaction
69 CycT1 point mutations
 multiple interaction phenotypes
Regulation of P-TEFb by HEXIM1 protein

44. Validation of 69 Cyclin T1 point mutations
in forward two-hybrid for interaction with HEXIM1 and CDK9
HEXIM1
AD
l4
1.
a G
1
IM
H EX +++
(11)
Random
? Interaction between
+++
mutations mutant CycT1 and HEXIM1 / CDK9 -
CycT1 GROWTH ON MEDIA LACKING URACIL -
Gal4BD
GAL4 PROMOTER REPORTER GENE (ura3)
Yeast growth on media lacking uracil
+++ + -
+
+
STRONG weak NO
interaction interaction interaction
69 CycT1 point mutations
 multiple interaction phenotypes
Regulation of P-TEFb by HEXIM1 protein

45. Validation of 69 Cyclin T1 point mutations
in forward two-hybrid for interaction with HEXIM1 and CDK9
HEXIM1
AD
AD
l4
1. 2.
a
l4
G
1
a
IM
G
HE
X
CD
K9 +++
(11)
Random
? Interaction between
+++
mutations mutant CycT1 and HEXIM1 / CDK9 -
CycT1 GROWTH ON MEDIA LACKING URACIL -
Gal4BD
GAL4 PROMOTER REPORTER GENE (ura3)
Yeast growth on media lacking uracil
+++ + -
+
+
STRONG weak NO
interaction interaction interaction
69 CycT1 point mutations
 multiple interaction phenotypes
Regulation of P-TEFb by HEXIM1 protein

46. Validation of 69 Cyclin T1 point mutations
in forward two-hybrid for interaction with HEXIM1 and CDK9
HEXIM1 CDK9
AD
AD
l4
1. 2.
a
l4
G
1
a
IM
G
HE
X
CD
K9 +++
(11)
Random
? Interaction between
+++
mutations mutant CycT1 and HEXIM1 / CDK9 -
;
+++
(11)
CycT1 GROWTH ON MEDIA LACKING URACIL - +++
Gal4BD
-
;
+
(8)
GAL4 PROMOTER REPORTER GENE (ura3)
- +
Yeast growth on media lacking uracil
-
;
-
(7)
+++ + -
- -
+
;
+++
(13)
+ +++
STRONG weak NO
interaction interaction interaction
+
;
+
(3)
+ +
69 CycT1 point mutations
+
;
-
(0)
 multiple interaction phenotypes + -
Regulation of P-TEFb by HEXIM1 protein

47. Validation of 69 Cyclin T1 point mutations
in forward two-hybrid for interaction with HEXIM1 and CDK9
HEXIM1 CDK9
AD
AD
l4
1. 2.
a
l4
G
1
a
IM
G
HE
X
CD
K9 +++
(11)
Random
? Interaction between
+++
mutations mutant CycT1 and HEXIM1 / CDK9 -
;
+++
(11)
CycT1 GROWTH ON MEDIA LACKING URACIL - +++
Gal4BD
-
;
+
(8)
GAL4 PROMOTER REPORTER GENE (ura3)
- +
Yeast growth on media lacking uracil
-
;
-
(7)
+++ + -
- -
+
;
+++
(13)
+ +++
STRONG weak NO
interaction interaction interaction
+
;
+
(3)
+ +
69 CycT1 point mutations
+
;
-
(0)
 multiple interaction phenotypes + -
Regulation of P-TEFb by HEXIM1 protein

48. Validation of 69 Cyclin T1 point mutations
in forward two-hybrid for interaction with HEXIM1 and CDK9
HEXIM1 CDK9
X AD
AD
l4
1. 2.
a
l4
G
1
a
IM
G
HEX
CD
K9 +++
(11)
Random
? Interaction between
+++
mutations mutant CycT1 and HEXIM1 / CDK9 -
;
+++
(11)
CycT1 GROWTH ON MEDIA LACKING URACIL - +++
Gal4BD
-
;
+
(8)
GAL4 PROMOTER REPORTER GENE (ura3)
- +
Yeast growth on media lacking uracil
-
;
-
(7)
+++ + -
- -
+
;
+++
(13)
+ +++
STRONG weak NO
interaction interaction interaction
+
;
+
(3)
+ +
69 CycT1 point mutations
+
;
-
(0)
 multiple interaction phenotypes + -
Regulation of P-TEFb by HEXIM1 protein

49. Validation of 69 Cyclin T1 point mutations
in forward two-hybrid for interaction with HEXIM1 and CDK9
HEXIM1 CDK9
X AD
AD
l4
1. 2.
a
l4
G
1
a
IM
G
HEX
CD
K9 +++
(11)
Random
? Interaction between
+++
mutations mutant CycT1 and HEXIM1 / CDK9 -
;
+++
(11)
CycT1 GROWTH ON MEDIA LACKING URACIL - +++
Gal4BD
-
;
+
(8)
GAL4 PROMOTER REPORTER GENE (ura3)
- +
Yeast growth on media lacking uracil
-
;
-
(7)
+++ + -
- -
+
;
+++
(13)
+ +++
STRONG weak NO
interaction interaction interaction
+
;
+
(3)
+ +
69 CycT1 point mutations
+
;
-
(0)
 multiple interaction phenotypes + -
Regulation of P-TEFb by HEXIM1 protein

50. Random mutagenesis combined with reverse two-hybrid
Mutation
L19P
HEXIM1 CDK9
- -
Mutation
L44M
HEXIM1 CDK9
+ +++
Mutation
R38S
HEXIM1 CDK9
- +++
 Cyclin T1 residues critical for
V104E - - Q50L + +++ Q56R
(2) - +++ HEXIM1 interaction (2-hybrid)
V157E - - Y70S + +++ I59F
(2) - +++
L182R - - V104M + +++ P85L - +++
C200R
L203P
-
-
-
-
S123C
V130A
+
+
+++
+++
I105F
(2)
V107E
-
-
+++
+++
 Mutations concerning the
F241S
(2) - - L144R + +++ C111R - +++ same residues  our screen
N60K
Q97K
-
-
+
+
C160R
K168R
+
+
+++
+++
L133R
H154R
-
-
+++
+++
reached good coverage
V104G - + S181G + +++ Y175H
(2) - +++
L170W - + S188G + +++ T179A - +++
M177K - + A197T + +++
V196E - + C198G
(2) + +++
W221R - +
P249L - +
W12R
(2) + +
F75S + +
F146L + +
Yeast growth on media lacking uracil
+++ + -
STRONG weak NO
interaction interaction interaction
Regulation of P-TEFb by HEXIM1 protein

51. Random mutagenesis combined with reverse two-hybrid
Mutation
L19P
HEXIM1 CDK9
- -
Mutation
L44M
HEXIM1 CDK9
+ +++
Mutation
R38S
HEXIM1 CDK9
- +++
 Cyclin T1 residues critical for
V104E - - Q50L + +++ Q56R
(2) - +++ HEXIM1 interaction (2-hybrid)
V157E - - Y70S + +++ I59F
(2) - +++
L182R - - V104M + +++ P85L - +++
C200R
L203P
-
-
-
-
S123C
V130A
+
+
+++
+++
I105F
(2)
V107E
-
-
+++
+++
 Mutations concerning the
F241S
(2) - - L144R + +++ C111R - +++ same residues  our screen
N60K
Q97K
-
-
+
+
C160R
K168R
+
+
+++
+++
L133R
H154R
-
-
+++
+++
reached good coverage
V104G - + S181G + +++ Y175H
(2) - +++
L170W - + S188G + +++ T179A - +++
M177K - + A197T + +++  40% of the mutations
V196E
W221R
-
-
+
+
C198G
(2) + +++
altering HEXIM1 binding were
P249L - + also deficient for CDK9
W12R
(2) + +
F75S + +
interaction
F146L + +
Yeast growth on media lacking uracil  11 Cyclin T1 point mutants
+++ + -
unable to bind HEXIM1 while
conserving CDK9 (2-hybrid)
STRONG weak NO
interaction interaction interaction
Regulation of P-TEFb by HEXIM1 protein

52. Contents
 Introduction
 Problematic
 Methods
 Results
• Identification of Cyclin T1 residues involved in HEXIM1 binding
• Visualization of HEXIM1 putative binding surface on Cyclin T1
• Functional impact on P-TEFb activity
 Discussion
 Conclusion perspectives
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins

53. Visualization of HEXIM1 putative binding surface on Cyclin T1
Cyclin T1 residues
important for HEXIM1
binding are mainly
located:
 on one side
of the Cyclin
PDB 3BLH
 in the
groove
between the !#$% '()*
Cyclin folds + +
+ ,
, ,
, ,,,
+ ,,,
Regulation of P-TEFb by HEXIM1 protein

54. Visualization of HEXIM1 putative binding surface on Cyclin T1
PDB 3BLH
CycT1 Y175
!#$% '()*
+ +
+ ,
, ,
, ,,,
+ ,,,
Regulation of P-TEFb by HEXIM1 protein

55. Cyclin T1 Tyrosine 175
- Structural role -
 Tyrosine = aromatic residue
 Surface
 Direct interaction with HEXIM1
Regulation of P-TEFb by HEXIM1 protein

56. Cyclin T1 Tyrosine 175
- Structural role -
 Tyrosine = aromatic residue
H154
 Surface Y175
T179
 Direct interaction with HEXIM1
N60
Y175 H183
 Hydrogen bond network Q56
 Orientation of Y175 on surface Q46
 Residues identified in our screen
Q50
Regulation of P-TEFb by HEXIM1 protein

57. Cyclin T1 Tyrosine 175
- Structural role -
 Tyrosine = aromatic residue
H154
 Surface Y175
T179
 Direct interaction with HEXIM1
N60
Y175 H183
 Hydrogen bond network Q56
 Orientation of Y175 on surface Q46
 Residues identified in our screen
Q50
 Mutations of CycT1 Y175 impair
HEXIM1 binding to P-TEFb in human cells
WT
WT Y175H Y175E Y175L Y175R Y175S
Regulation of P-TEFb by HEXIM1 protein

58. Cyclin T1 Tyrosine 175
- Structural role -
 Tyrosine = aromatic residue
H154
 Surface Y175
T179
 Direct interaction with HEXIM1
N60
Y175 H183
 Hydrogen bond network Q56
 Orientation of Y175 on surface Q46
 Residues identified in our screen
Q50
 Mutations of CycT1 Y175 impair
HEXIM1 binding to P-TEFb in human cells
WT
WT Y175H Y175E Y175L Y175R Y175S
 Cyclin T1 Y175 might
be an important interfacial
residue directly involved
in HEXIM1 binding
Regulation of P-TEFb by HEXIM1 protein

59. Cyclin T1 Tyrosine 175
- Functional role -
endogenous
CycT1 CDK9
Ga
l4
Gal4 sites
G5-38-HIV TATA Luc
-38 +84
Regulation of P-TEFb by HEXIM1 protein

60. Cyclin T1 Tyrosine 175
- Functional role -
endogenous
CycT1 CDK9
Ga
l4
Gal4 sites
G5-38-HIV TATA Luc
-38 +84
16
14
12
fold activation
10
8
6
4
2
0
1 2 3 4 5
CycT1 WT - - - - +
Regulation of P-TEFb by HEXIM1 protein

61. Cyclin T1 Tyrosine 175
- Functional role -
endogenous
CycT1 CDK9
Ga
l4
Gal4 sites
G5-38-HIV TATA Luc
-38 +84
16
14
12
fold activation
10
8
6
4
2
0
1 2 3 4 5 6 7 8
CycT1 WT - - - - + + + +
Flag-HEXIM - -
Regulation of P-TEFb by HEXIM1 protein

62. Cyclin T1 Tyrosine 175
- Functional role -
endogenous
CycT1 CDK9
Ga
l4
Gal4 sites
G5-38-HIV TATA Luc
-38 +84
16
14
12
fold activation
10
8
6
4
2
0
1 2 3 4 5 6 7 8 9
CycT1 WT - - - - + + + + -
CycT1 Y175E - - - - - - - - +
Flag-HEXIM - - -
Regulation of P-TEFb by HEXIM1 protein

63. Cyclin T1 Tyrosine 175
- Functional role -
endogenous
CycT1 CDK9
Ga
l4
Gal4 sites
G5-38-HIV TATA Luc
-38 +84
16
14
12
fold activation
10
8
6
4
2
0
1 2 3 4 5 6 7 8 9 10 11 12
CycT1 WT - - - - + + + + - - - -
CycT1 Y175E - - - - - - - - + + + +
Flag-HEXIM - - -
Regulation of P-TEFb by HEXIM1 protein

64. Cyclin T1 Tyrosine 175
- Functional role -
endogenous
CycT1 CDK9
Ga
l4
Gal4 sites
G5-38-HIV TATA Luc
-38 +84
16
14 100
12
80
fold activation
10
60
8
6 40
4
20
2
0 0
1 2 3 4 5 6 7 8 9 10 11 12
CycT1 WT - - - - + + + + - - - -
CycT1 Y175E - - - - - - - - + + + +
Flag-HEXIM - - -
Regulation of P-TEFb by HEXIM1 protein

65. 11 Cyclin T1 point mutations
- Functional role -
Mutation HEXIM1 CDK9
R38S - +++
Q56R
(2) - +++
I59F
(2) - +++
P85L - +++
I105F
(2) - +++
V107E - +++
C111R - +++
L133R - +++
H154R - +++
Y175H
(2) - +++
T179A - +++
11 Cyclin T1 point
mutations abolishing
HEXIM1 interaction and
conserving CDK9
interaction (2-hybrid)
Regulation of P-TEFb by HEXIM1 protein

66. 11 Cyclin T1 point mutations
- Functional role -
Mutation HEXIM1 CDK9
16 R38S - +++
14 * Q56R
(2) - +++
12 I59F
(2) - +++
fold activation
10 P85L - +++
* I105F
(2) - +++
8
V107E - +++
6
C111R - +++
4
L133R - +++
**
2 **
** ** ** H154R - +++
0 Y175H
(2) - +++
1 2 3 4 5 6 7 8 9 10 11 12 13
T179A - +++
G5-38-HIV + + + + + + + + + + + + +
H
E
R
R
A
R
5F
6R
Gal4-CycTm
8S
5L
07
75
F
54
11
33
T
79
I59
W
-
I10
Q5
P8
R3
V1
H1
Y1
C1
L1
T1
Regulation of P-TEFb by HEXIM1 protein

67. 11 Cyclin T1 point mutations
- Functional role -
Mutation HEXIM1 CDK9
16 R38S - +++
14 * Q56R
(2) - +++
12 I59F
(2) - +++
fold activation
10 P85L - +++
* I105F
(2) - +++
8
V107E - +++
6
C111R - +++
4
L133R - +++
**
2 **
** ** ** H154R - +++
0 Y175H
(2) - +++
1 2 3 4 5 6 7 8 9 10 11 12 13
T179A - +++
G5-38-HIV + + + + + + + + + + + + +
R
R
A
H
E
R
8S
5F
6R
Gal4-CycTm
F
5L
54
11
79
07
33
75
T
I59
W
-
I10
R3
Q5
P8
H1
C1
V1
T1
L1
Y1
Regulation of P-TEFb by HEXIM1 protein

68. 11 Cyclin T1 point mutations
- Functional role -
Mutation HEXIM1 CDK9
16 R38S - +++
14 * Q56R
(2) - +++
12 I59F
(2) - +++
fold activation
10 P85L - +++
* I105F
(2) - +++
8
V107E - +++
6
C111R - +++
4
L133R - +++
**
2 **
** ** ** H154R - +++
0 Y175H
(2) - +++
1 2 3 4 5 6 7 8 9 10 11 12 13
T179A - +++
G5-38-HIV + + + + + + + + + + + +
R
9A
Gal4-CycTm
F
54
I59
7
T
T
H1
T1
W
W
GFP-CycT1 Possible impacts on:
co-immunoprecipitation
in human cells
HEXIM1  CDK9 activation
CDK9
 Substrate recognition
+ + +
Regulation of P-TEFb by HEXIM1 protein

69. 11 Cyclin T1 point mutations
- Functional role -
Mutation HEXIM1 CDK9 Activity
16 R38S - +++ -
14 * Q56R
(2) - +++ +
12 I59F
(2) - +++ -
fold activation
10 P85L - +++ +
* I105F
(2) - +++ +
8
V107E - +++ +++
6
C111R - +++ -
4
L133R - +++ +
**
2 **
** ** ** H154R - +++ -
0 Y175H
(2) - +++ +++
1 2 3 4 5 6 7 8 9 10 11 12 13
T179A - +++ -
G5-38-HIV + + + + + + + + + + + + +
H
R
E
R
A
R
8S
5F
6R
Gal4-CycTm
F
5L
07
54
11
75
79
33
T
I59
W
-
I10
R3
Q5
P8
V1
H1
C1
Y1
T1
L1
Possible impacts on:
Same interaction phenotype  CDK9 activation
but different functional effects on P-TEFb activity  Substrate recognition
Regulation of P-TEFb by HEXIM1 protein

70. 11 Cyclin T1 point mutations
- Structure / Function relationships -
H154 Y175
Mutation HEXIM1 CDK9 Activity
T179
N60
R38S - +++ -
H183
Q56 Q56R
(2) - +++ +
Q46 I59F
(2) - +++ -
Q50 P85L - +++ +
I105F
(2) - +++ +
V107E - +++ +++
C111R - +++ -
L133R - +++ +
H154R - +++ -
Y175H
(2) - +++ +++
T179A - +++ -
 Mutations ⇗ activity: surface
 Mutations ⇘ activity: internal
and in the vicinity
 Signal transduction pathway?
Regulation of P-TEFb by HEXIM1 protein

71. Regulation of P-TEFb by HEXIM1 protein
Hex1 Hex1
Cyclin T1
Molecular mechanisms
of HEXIM1-mediated
CDK inhibition of P-TEFb
9
 Signal transduction pathway?
Regulation of P-TEFb by HEXIM1 protein

72. Regulation of P-TEFb by HEXIM1 protein
Hex1 Hex1
Cyclin T1
Molecular mechanisms
of HEXIM1-mediated
CDK inhibition of P-TEFb
9
 Signal transduction pathway?
Regulation of P-TEFb by HEXIM1 protein

73. Regulation of P-TEFb by HEXIM1 protein
Hex1 Hex1
Cyclin T1
Molecular mechanisms
of HEXIM1-mediated
CDK inhibition of P-TEFb
9
 Signal transduction pathway?
Regulation of P-TEFb by HEXIM1 protein

74. Contents
 Introduction
 Problematic
 Methods
 Results
 Discussion
 Conclusion perspectives
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins

75. HEXIM1 might bind Cyclin T1
in a rigid groove between the cyclin folds
Residues involved in HEXIM1
flexible binding are:
1. mainly located in the groove
between the Cyclin folds.
2. Enriched in rigid regions
3. Overlap with predicted ligand
binding regions
4. Overlap with HIV-1 Tat
binding surface
rigid
Crystallographic temperature factors
(B-factors)
Regulation of P-TEFb by HEXIM1 protein

76. HEXIM1 might bind Cyclin T1
in a rigid groove between the cyclin folds
Residues involved in HEXIM1
flexible binding are:
1. mainly located in the groove
between the Cyclin folds.
2. Enriched in rigid regions
Conserved rigid surface residues
 role in stabilizing the structure
of Cyclin T1 and in the core
interface CycT1/HEXIM1
(Lakshmipuram
et
al.
2012)
rigid
Crystallographic temperature factors
(B-factors)
Regulation of P-TEFb by HEXIM1 protein