regulation of the positive transcription elongation factor p-tefb (cdk9/cyct1) by hexim1 protein
Post on 13-Jul-2015
309 Views
Preview:
TRANSCRIPT
Regulation of the positive transcription elongation factor P-TEFb (Cdk9/CycT1) by HEXIM1 protein
under the supervision of
Olivier Bensaude Functional Genomics Dpt. Cell biology of Transcription
In front of the jury composed of
Michelle Debatisse Matthias Geyer Patricia Uguen
Claude Gaillardin
PhD defense presentation of
Nina VERSTRAETE
September 28, 2012 1 Regulation of P-TEFb by HEXIM1 protein
Contents
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins
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
Transcription regulates the information flow from genes to proteins
Regulation of P-TEFb by HEXIM1 protein
carrying out the duties specified by the information encoded in genes
Proteins and non-coding RNAs are chief actors within the cell,
Transcription and RNA Polymerase II
Regulation of P-TEFb by HEXIM1 protein
• Eukaryotes : RNAPII > protein-coding genes and ncRNA
• Phosphorylation of the CTD of RNAPII Plays Central Roles in the Integrated Events of Eucaryotic Gene Expression
– Evolutionary conserved ( fungi, plants, animals) – 52 repeats in mammals (Y1S2P3T4S5P6S7)
– Scaffold for the interaction of nuclear factors • Transcription, RNA processing
• 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
RNAPII Transcript RNA
DNA
April 2003 Molecule of the Month by David Goodsell RCSB Protein Data Bank
RNAPII promoter-proximal pausing
Regulation of P-TEFb by HEXIM1 protein
AAAAAAAAPoly (A)
RNAPII promoter-proximal pausing
Regulation of P-TEFb by HEXIM1 protein
AAAAAAAAPoly (A)
Pol I
I den
sity
AAAAAAAAPoly (A)
Pol I
I den
sity
RNAPII promoter-proximal pausing
Regulation of P-TEFb by HEXIM1 protein
Promoter-proximal PAUSING
RNAPII promoter-proximal pausing
Regulation of P-TEFb by HEXIM1 protein
AAAAAAAAPoly (A)
Pol I
I den
sity
Functional relevance of RNAPII pausing
Regulation of P-TEFb by HEXIM1 protein
DSIF
Pol II
NELF
GTFs TF1
DSIF
Pol II GTFs
NELF
TF2 TF1
P
PP-TEFb
2 Rapid or synchronous activation
Pol II
DSIF
Pol II GTFs
NELF
TF2 TF1
P
PP-TEFb
DSIF
Pol II GTFs
NELF
TF2 TF1
P
PP-TEFb
Nature Reviews | Genetics
1 Establishing permissive chromatin
P
P
P
PDSIF
NELF
GTFs
DSIF
Pol II
P
P
P
P-TEFb
RPF Pol II
DSIF
Pol II
P
P
PRPF P-TEFb
TF2
CEC TF1
3 Checkpoint in early elongation
Functional relevance of RNAPII pausing
Regulation of P-TEFb by HEXIM1 protein
DSIF
Pol II
NELF
GTFs TF1
DSIF
Pol II GTFs
NELF
TF2 TF1
P
PP-TEFb
2 Rapid or synchronous activation
Pol II
DSIF
Pol II GTFs
NELF
TF2 TF1
P
PP-TEFb
DSIF
Pol II GTFs
NELF
TF2 TF1
P
PP-TEFb
Nature Reviews | Genetics
1 Establishing permissive chromatin
P
P
P
PDSIF
NELF
GTFs
DSIF
Pol II
P
P
P
P-TEFb
RPF Pol II
DSIF
Pol II
P
P
PRPF P-TEFb
TF2
CEC TF1
3 Checkpoint in early elongation
Functional relevance of RNAPII pausing
Regulation of P-TEFb by HEXIM1 protein
DSIF
Pol II
NELF
GTFs TF1
DSIF
Pol II GTFs
NELF
TF2 TF1
P
PP-TEFb
2 Rapid or synchronous activation
Pol II
DSIF
Pol II GTFs
NELF
TF2 TF1
P
PP-TEFb
DSIF
Pol II GTFs
NELF
TF2 TF1
P
PP-TEFb
Nature Reviews | Genetics
1 Establishing permissive chromatin
P
P
P
PDSIF
NELF
GTFs
DSIF
Pol II
P
P
P
P-TEFb
RPF Pol II
DSIF
Pol II
P
P
PRPF P-TEFb
TF2
CEC TF1
3 Checkpoint in early elongation
Contents
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins
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
Usurpation of the transcription elongation machinery ���by HIV-1 TAR RNA and Tat protein
Regulation of P-TEFb by HEXIM1 protein
Usurpation of the transcription elongation machinery ���by HIV-1 TAR RNA and Tat protein
Regulation of P-TEFb by HEXIM1 protein
Usurpation of the transcription elongation machinery ���by HIV-1 TAR RNA and Tat protein
Regulation of P-TEFb by HEXIM1 protein
P-TEFb
Contents
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins
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
Positive Transcription Elongation Factor b
Regulation of P-TEFb by HEXIM1 protein
• P-TEFb is composed of CDK9 (40 kDa) and Cyclin T (81kDa)
Positive Transcription Elongation Factor b
Regulation of P-TEFb by HEXIM1 protein
CDK9 Cyclin T1
ATP
• Belongs to the CDK/Cyclin family
• Cyclin-Dependent Kinase : CDK9 requires the binding of the Cyclin T partner to become active
PDB 3BLH
• P-TEFb is composed of CDK9 (40 kDa) and Cyclin T (81kDa)
Positive Transcription Elongation Factor b
Regulation of P-TEFb by HEXIM1 protein
CDK9 Cyclin T1
ATP
PDB 3BLH
• P-TEFb is composed of CDK9 (40 kDa) and Cyclin T (81kDa)
• Belongs to the CDK/Cyclin family
• 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, STAT3, AFF4, AF9, ENL, ELL2)
• Can be recruited to chromatin through BRD4 (Bromodomain-containing protein 4)
Contents
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins
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
Inhibition of P-TEFb ���by 7SK RNA and HEXIM1 protein
Regulation of P-TEFb by 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)
Inhibition of P-TEFb ���by 7SK RNA and HEXIM1 protein
Regulation of P-TEFb by HEXIM1 protein
LARP7
MePCE
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
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)
Inhibition of P-TEFb ���by 7SK RNA and HEXIM1 protein
Regulation of P-TEFb by HEXIM1 protein
LARP7
MePCE
Hex1 Hex1
HEXIM1 protein o 359 aa, 41 kDa o Dimer o Binds 7SK RNA and Cyclin T1 o Intrinsically disordered regions o 3D structure partially defined (Dames et al. 2007, Bigalke et al. 2011)
A B C D
Coiled-coil dimerization domain
Cyclin T1 binding7SK RNA binding
HEXIM1 Acidic region
PYN
T1 359150 177 211 249 352279
Regulatory region (self-inhibitory)
ARM / NLSBasic region
N
C
+++
---
N
C
+++---
A
B
Inhibition of P-TEFb ���by 7SK RNA and HEXIM1 protein
Regulation of P-TEFb by HEXIM1 protein
LARP7
MePCE
Hex1 Hex1
Cyclin T1
CDK9
Cyclin T1
CDK9
A B C D
Coiled-coil dimerization domain
Cyclin T1 binding7SK RNA binding
HEXIM1 Acidic region
PYN
T1 359150 177 211 249 352279
Regulatory region (self-inhibitory)
ARM / NLSBasic region
N
C
+++
---
N
C
+++---
A
B
HEXIM1 protein o 359 aa, 41 kDa o Dimer o Binds 7SK RNA and Cyclin T1 o Intrinsically disordered regions o 3D structure partially defined (Dames et al. 2007, Bigalke et al. 2011)
Inhibition of P-TEFb ���by 7SK RNA and HEXIM1 protein
Regulation of P-TEFb by HEXIM1 protein
LARP7
MePCE
Hex1 Hex1
Cyclin T1
CDK9
7SK snRNP
P-TEFbCDK9/CycT1INACTIVE
o 7SK non-coding RNA – 331 nt o LARP7 – 67 kDa o MePCE – 74 kDa o HEXIM1 – 2 x 41 kDa o CycT1 – (2 x) 81 kDa o CDK9 – (2 x) 40 kDa
-------------------------------------------------- o 350 kDa
Inhibition of P-TEFb ���by 7SK RNA and HEXIM1 protein
Regulation of P-TEFb by HEXIM1 protein
LARP7
MePCE
Hex1 Hex1
Cyclin T1
CDK9
Cyclin T1
CDK9
LARP7
MePCE
hnRNP Q/R/A
LARP7
MePCE
hnRNP Q/R/A
Hex1 Hex1
Hex1 Hex1
P-TEFbCDK9/CycT1INACTIVE
P-TEFbCDK9/CycT1
ACTIVE
7SK snRNP
Inhibition of P-TEFb ���by 7SK RNA and HEXIM1 protein
Regulation of P-TEFb by HEXIM1 protein
LARP7
MePCE
Hex1 Hex1
Cyclin T1
CDK9
Cyclin T1
CDK9
Cyclin T1
CDK9
Cyclin T1
CDK9
Cyclin T1
CDK9
LARP7
MePCE
hnRNP Q/R/A
LARP7
MePCE
hnRNP Q/R/A
Hex1 Hex1
Hex1 Hex1
Transcription Inhibition(UV exposure, DRB, flavopiridol)
or
Cardiac Hypertrophy
P-TEFb
CDK9/CycT
ACTIVE
P-TEFbCDK9/CycT1INACTIVE
7SK snRNP
Inhibition of P-TEFb ���by 7SK RNA and HEXIM1 protein
Regulation of P-TEFb by HEXIM1 protein
LARP7
MePCE
Hex1 Hex1
Cyclin T1
CDK9
Hex1 Hex1
Cyclin T1
CDK97SK snRNPINACTIVE
• Molecular mechanisms of HEXIM1-mediated inhibition of P-TEFb
• Biological impact of P-TEFb/HEXIM1 interaction
Contents
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins
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
Perturbing edges instead of nodes
• 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
CyclinT1
Nature Methods, 2009
Perturbing edges instead of nodes
• 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
Cyclin T1
Nature Methods, 2009
HEXIM1
Mutant screening by Reverse Two-Hybrid
Edgetic Perturbation of P-TEFb
: toxic for cells able to synthetize uracile
(PCR Δ)
Mutant screening by Reverse Two-Hybrid
Edgetic Perturbation of P-TEFb
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)
Mutant screening by Reverse Two-Hybrid
Edgetic Perturbation of P-TEFb
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)
Mutant screening by Reverse Two-Hybrid
Edgetic Perturbation of P-TEFb
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)
Mutant Library���Random Mutagenesis by error-prone PCR
Edgetic Perturbation of P-TEFb
Reverse two-hybrid in Yeast
Edgetic Perturbation of P-TEFb
Transformation in Yeast Mav103 : MATa SPAL10::URA3 leu2-3, 112 trp1-901 his3200 ade2-101 gal4 gal80 can1r cyh2r GAL1::HIS3@LYS2 GAL1::lacZ@URA3
+ HEXIM1-AD
+ PCR Δ + CycT-BD // (-Trp)
(-Leu)
-L -T -L -T -U -L -T +5FOA
HEXIM-AD CycT-BD
interaction HEXIM-AD x CycT-BD
ura3 ↑
no interaction FOA resistance
homologous recombination
PCR on colony
sequencing
?
Contents
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins
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 P-TEFb by HEXIM1 protein
Cyclin T1 mutant library analysis
• Identification of mutations by PCR on colonies FOAr and sequencing
>200 sequences analyzed
-L -T +5FOA
!"#
$%#
&&#&'# &%#
(%#
)#
&)#
$)#
()#
*)#
+)#
!)#
')#
%)#
,-./01#23454-6.#
763801#23454-6.,#
49-:01#23454-6.,#
:91254391#,46:#;676.#
<9521,=-<4# .6#23454-6.#
.3281
9#6<#6;;
391.
;1,
Regulation of P-TEFb by HEXIM1 protein
Cyclin T1 mutant library analysis
• Identification of mutations by PCR on colonies FOAr and sequencing
>200 sequences analyzed
-L -T +5FOA
!"#
$%#
&&#&'# &%#
(%#
)#
&)#
$)#
()#
*)#
+)#
!)#
')#
%)#
,-./01#23454-6.#
763801#23454-6.,#
49-:01#23454-6.,#
:91254391#,46:#;676.#
<9521,=-<4# .6#23454-6.#
.3281
9#6<#6;;
391.
;1,
Regulation of P-TEFb by HEXIM1 protein
Cyclin T1 mutant library analysis
• Identification of mutations by PCR on colonies FOAr and sequencing
>200 sequences analyzed
-L -T +5FOA
!"#
$%#
&&#&'# &%#
(%#
)#
&)#
$)#
()#
*)#
+)#
!)#
')#
%)#
,-./01#23454-6.#
763801#23454-6.,#
49-:01#23454-6.,#
:91254391#,46:#;676.#
<9521,=-<4# .6#23454-6.#
.3281
9#6<#6;;
391.
;1,
!
Validation of 69 Cyclin T1 point mutations ���in forward two-hybrid for interaction with HEXIM1 and CDK9
Regulation of P-TEFb by HEXIM1 protein
Random mutations
CycT1
HEXIM1
Gal4BD
Gal4AD
GAL4 PROMOTER REPORTER GENE (ura3)
Interaction between mutant CycT1 and HEXIM1 / CDK9
GROWTH ON MEDIA LACKING URACIL
?
1.
Validation of 69 Cyclin T1 point mutations ���in forward two-hybrid for interaction with HEXIM1 and CDK9
Regulation of P-TEFb by HEXIM1 protein
+++ + -
STRONGinteraction
weakinteraction
NOinteraction
Yeast growth on media lacking uracil
multiple interaction phenotypes 69 CycT1 point mutations
Random mutations
CycT1
HEXIM1
Gal4BD
Gal4AD
GAL4 PROMOTER REPORTER GENE (ura3)
Interaction between mutant CycT1 and HEXIM1 / CDK9
GROWTH ON MEDIA LACKING URACIL
?
1.
Validation of 69 Cyclin T1 point mutations ���in forward two-hybrid for interaction with HEXIM1 and CDK9
Regulation of P-TEFb by HEXIM1 protein
HEXIM1
+++ (11)
-
+
+++
-
+
+++ + -
STRONGinteraction
weakinteraction
NOinteraction
Yeast growth on media lacking uracil
multiple interaction phenotypes 69 CycT1 point mutations
Random mutations
CycT1
HEXIM1
Gal4BD
Gal4AD
GAL4 PROMOTER REPORTER GENE (ura3)
Interaction between mutant CycT1 and HEXIM1 / CDK9
GROWTH ON MEDIA LACKING URACIL
?
1.
Validation of 69 Cyclin T1 point mutations ���in forward two-hybrid for interaction with HEXIM1 and CDK9
Regulation of P-TEFb by HEXIM1 protein
HEXIM1
+++ (11)
-
+
+++
-
+
+++ + -
STRONGinteraction
weakinteraction
NOinteraction
Yeast growth on media lacking uracil
multiple interaction phenotypes 69 CycT1 point mutations
Random mutations
CycT1
HEXIM1
CDK9
Gal4BD
Gal4AD
Gal4AD
GAL4 PROMOTER REPORTER GENE (ura3)
Interaction between mutant CycT1 and HEXIM1 / CDK9
GROWTH ON MEDIA LACKING URACIL
?
1. 2.
Validation of 69 Cyclin T1 point mutations ���in forward two-hybrid for interaction with HEXIM1 and CDK9
Regulation of P-TEFb by HEXIM1 protein
HEXIM1 CDK9
- ; - (7)
+++ (11)
- ; + (8)
+ ; + (3)
- ; +++ (11)
+ ; +++ (13)
+ ; - (0)
+++
-
-
-
+
+ +
-
-
+
+++
+++
+
+++ + -
STRONGinteraction
weakinteraction
NOinteraction
Yeast growth on media lacking uracil
multiple interaction phenotypes 69 CycT1 point mutations
Random mutations
CycT1
HEXIM1
CDK9
Gal4BD
Gal4AD
Gal4AD
GAL4 PROMOTER REPORTER GENE (ura3)
Interaction between mutant CycT1 and HEXIM1 / CDK9
GROWTH ON MEDIA LACKING URACIL
?
1. 2.
Validation of 69 Cyclin T1 point mutations ���in forward two-hybrid for interaction with HEXIM1 and CDK9
Regulation of P-TEFb by HEXIM1 protein
HEXIM1 CDK9
- ; - (7)
+++ (11)
- ; + (8)
+ ; + (3)
- ; +++ (11)
+ ; +++ (13)
+ ; - (0)
+++
-
-
-
+
+ +
-
-
+
+++
+++
+
+++ + -
STRONGinteraction
weakinteraction
NOinteraction
Yeast growth on media lacking uracil
multiple interaction phenotypes 69 CycT1 point mutations
Random mutations
CycT1
HEXIM1
CDK9
Gal4BD
Gal4AD
Gal4AD
GAL4 PROMOTER REPORTER GENE (ura3)
Interaction between mutant CycT1 and HEXIM1 / CDK9
GROWTH ON MEDIA LACKING URACIL
?
1. 2.
Validation of 69 Cyclin T1 point mutations ���in forward two-hybrid for interaction with HEXIM1 and CDK9
Regulation of P-TEFb by HEXIM1 protein
HEXIM1 CDK9
- ; - (7)
+++ (11)
- ; + (8)
+ ; + (3)
- ; +++ (11)
+ ; +++ (13)
+ ; - (0)
+++
-
-
-
+
+ +
-
-
+
+++
+++
+
Random mutations
CycT1
HEXIM1
CDK9
Gal4BD
Gal4AD
Gal4AD
GAL4 PROMOTER REPORTER GENE (ura3)
Interaction between mutant CycT1 and HEXIM1 / CDK9
GROWTH ON MEDIA LACKING URACIL
?
1. 2.X
+++ + -
STRONGinteraction
weakinteraction
NOinteraction
Yeast growth on media lacking uracil
multiple interaction phenotypes 69 CycT1 point mutations
Validation of 69 Cyclin T1 point mutations ���in forward two-hybrid for interaction with HEXIM1 and CDK9
Regulation of P-TEFb by HEXIM1 protein
- ; - (7)
+++ (11)
- ; + (8)
+ ; + (3)
- ; +++ (11)
+ ; +++ (13)
+ ; - (0)
HEXIM1 CDK9
+++
-
-
-
+
+ +
-
-
+
+++
+++
+
Random mutations
CycT1
HEXIM1
CDK9
Gal4BD
Gal4AD
Gal4AD
GAL4 PROMOTER REPORTER GENE (ura3)
Interaction between mutant CycT1 and HEXIM1 / CDK9
GROWTH ON MEDIA LACKING URACIL
?
1. 2.X
+++ + -
STRONGinteraction
weakinteraction
NOinteraction
Yeast growth on media lacking uracil
multiple interaction phenotypes 69 CycT1 point mutations
+++ + -
STRONGinteraction
weakinteraction
NOinteraction
Yeast growth on media lacking uracil
Random mutagenesis combined with reverse two-hybrid
Regulation of P-TEFb by HEXIM1 protein
Mutation HEXIM1 CDK9 Mutation HEXIM1 CDK9 Mutation HEXIM1 CDK9
L19P - - L44M + +++ R38S - +++
V104E - - Q50L + +++ Q56R (2) - +++
V157E - - Y70S + +++ I59F (2) - +++
L182R - - V104M + +++ P85L - +++
C200R - - S123C + +++ I105F (2) - +++
L203P - - V130A + +++ V107E - +++
F241S (2) - - L144R + +++ C111R - +++
N60K - + C160R + +++ L133R - +++
Q97K - + K168R + +++ H154R - +++
V104G - + S181G + +++ Y175H (2) - +++
L170W - + S188G + +++ T179A - +++
M177K - + A197T + +++
V196E - + C198G (2) + +++
W221R - +
P249L - +
W12R (2) + +
F75S + +
F146L + +
Cyclin T1 residues critical for HEXIM1 interaction (2-hybrid)
Mutations concerning the same residues our screen reached good coverage
+++ + -
STRONGinteraction
weakinteraction
NOinteraction
Yeast growth on media lacking uracil
Random mutagenesis combined with reverse two-hybrid
Regulation of P-TEFb by HEXIM1 protein
Mutation HEXIM1 CDK9 Mutation HEXIM1 CDK9 Mutation HEXIM1 CDK9
L19P - - L44M + +++ R38S - +++
V104E - - Q50L + +++ Q56R (2) - +++
V157E - - Y70S + +++ I59F (2) - +++
L182R - - V104M + +++ P85L - +++
C200R - - S123C + +++ I105F (2) - +++
L203P - - V130A + +++ V107E - +++
F241S (2) - - L144R + +++ C111R - +++
N60K - + C160R + +++ L133R - +++
Q97K - + K168R + +++ H154R - +++
V104G - + S181G + +++ Y175H (2) - +++
L170W - + S188G + +++ T179A - +++
M177K - + A197T + +++
V196E - + C198G (2) + +++
W221R - +
P249L - +
W12R (2) + +
F75S + +
F146L + +
Cyclin T1 residues critical for HEXIM1 interaction (2-hybrid)
Mutations concerning the same residues our screen reached good coverage
40% of the mutations altering HEXIM1 binding were also deficient for CDK9 interaction
11 Cyclin T1 point mutants unable to bind HEXIM1 while conserving CDK9 (2-hybrid)
Contents
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins
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
Visualization of HEXIM1 putative binding surface on Cyclin T1
Regulation of P-TEFb by HEXIM1 protein
!"#$%& '()*+ ++ ,, ,, ,,,+ ,,,
PDB 3BLH
Cyclin T1 residues important for HEXIM1 binding are mainly located:
on one side of the Cyclin
in the groove between the Cyclin folds
Visualization of HEXIM1 putative binding surface on Cyclin T1
Regulation of P-TEFb by HEXIM1 protein
!"#$%& '()*+ ++ ,, ,, ,,,+ ,,,
CycT1 Y175 PDB 3BLH
Cyclin T1 Tyrosine 175���- Structural role -
Regulation of P-TEFb by HEXIM1 protein
Tyrosine = aromatic residue Surface Direct interaction with HEXIM1
Cyclin T1 Tyrosine 175���- Structural role -
Regulation of P-TEFb by HEXIM1 protein
Y175
Tyrosine = aromatic residue Surface Direct interaction with HEXIM1
N60
Q56
Q50
Q46
T179
H154
H183
Y175
Hydrogen bond network Orientation of Y175 on surface Residues identified in our screen
WTWT Y175H Y175E Y175L Y175R Y175S
Cyclin T1 Tyrosine 175���- Structural role -
Regulation of P-TEFb by HEXIM1 protein
Y175
Tyrosine = aromatic residue Surface Direct interaction with HEXIM1
N60
Q56
Q50
Q46
T179
H154
H183
Y175
Hydrogen bond network Orientation of Y175 on surface Residues identified in our screen
Mutations of CycT1 Y175 impair HEXIM1 binding to P-TEFb in human cells
Cyclin T1 Tyrosine 175���- Structural role -
Regulation of P-TEFb by HEXIM1 protein
Y175
Tyrosine = aromatic residue Surface Direct interaction with HEXIM1
N60
Q56
Q50
Q46
T179
H154
H183
Y175
Hydrogen bond network Orientation of Y175 on surface Residues identified in our screen
Mutations of CycT1 Y175 impair HEXIM1 binding to P-TEFb in human cells
WTWT Y175H Y175E Y175L Y175R Y175S
Cyclin T1 Y175 might be an important interfacial residue directly involved in HEXIM1 binding
Cyclin T1 Tyrosine 175���- Functional role -
Regulation of P-TEFb by HEXIM1 protein
LucGal4 sites
G5-38-HIV TATA
-38 +84
CycT1endogenous
CDK9Gal4
Cyclin T1 Tyrosine 175���- Functional role -
Regulation of P-TEFb by HEXIM1 protein
LucGal4 sites
G5-38-HIV TATA
-38 +84
CycT1endogenous
CDK9Gal4
0
2
4
6
8
10
12
14
16
1 2 3 4 5 CycT1 WT - - - - +
fold
act
ivat
ion
Cyclin T1 Tyrosine 175���- Functional role -
Regulation of P-TEFb by HEXIM1 protein
LucGal4 sites
G5-38-HIV TATA
-38 +84
CycT1endogenous
CDK9Gal4
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8 CycT1 WT - - - - + + + +
Flag-HEXIM - -
fold
act
ivat
ion
Cyclin T1 Tyrosine 175���- Functional role -
Regulation of P-TEFb by HEXIM1 protein
LucGal4 sites
G5-38-HIV TATA
-38 +84
CycT1endogenous
CDK9Gal4
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8 9
CycT1 Y175E - - - - - - - - + CycT1 WT - - - - + + + + -
Flag-HEXIM - - -
fold
act
ivat
ion
Cyclin T1 Tyrosine 175���- Functional role -
Regulation of P-TEFb by HEXIM1 protein
LucGal4 sites
G5-38-HIV TATA
-38 +84
CycT1endogenous
CDK9Gal4
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8 9 10 11 12
CycT1 Y175E - - - - - - - - + + + + CycT1 WT - - - - + + + + - - - -
Flag-HEXIM - - -
fold
act
ivat
ion
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8 9 10 11 12
CycT1 Y175E - - - - - - - - + + + + CycT1 WT - - - - + + + + - - - -
Flag-HEXIM - - -
fold
act
ivat
ion
Cyclin T1 Tyrosine 175���- Functional role -
Regulation of P-TEFb by HEXIM1 protein
LucGal4 sites
G5-38-HIV TATA
-38 +84
CycT1endogenous
CDK9Gal4
0
20
40
60
80
100
11 Cyclin T1 point mutations���- Functional role -
Regulation of P-TEFb by HEXIM1 protein
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)
11 Cyclin T1 point mutations���- Functional role -
Regulation of P-TEFb by HEXIM1 protein
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8 9 10 11 12 13
Gal4-CycTm - WT
R38S
Q56
R I59
F P8
5L
I105F
V107E C11
1R
L133
R H15
4R
Y175H
G5-38-HIV + + + + + + + + + + + +
T179
A
+
fold
act
ivat
ion
* * * ** *
* ** *
*
*
Mutation HEXIM1 CDK9
R38S - +++
Q56R (2) - +++
I59F (2) - +++
P85L - +++
I105F (2) - +++
V107E - +++
C111R - +++
L133R - +++
H154R - +++
Y175H (2) - +++
T179A - +++
11 Cyclin T1 point mutations���- Functional role -
Regulation of P-TEFb by HEXIM1 protein
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8 9 10 11 12 13
Gal4-CycTm - WT
R38S Q56
R I5
9F P8
5L
I105F
V10
7E
C111R L1
33R
H154R Y1
75H
G5-38-HIV + + + + + + + + + + + +
T179A
+
fold
act
ivat
ion
* * * ** *
* ** *
*
*
Mutation HEXIM1 CDK9
R38S - +++
Q56R (2) - +++
I59F (2) - +++
P85L - +++
I105F (2) - +++
V107E - +++
C111R - +++
L133R - +++
H154R - +++
Y175H (2) - +++
T179A - +++
11 Cyclin T1 point mutations���- Functional role -
Regulation of P-TEFb by HEXIM1 protein
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8 9 10 11 12 13
Gal4-CycTm
I59F
H154R
WT
WT
G5-38-HIV + + + + + + + + + + +
T179A
+
fold
act
ivat
ion
* * * ** *
* ** *
*
*
co-immunoprecipitationin human cells
GFP-CycT1
HEXIM1
CDK9
+ + +
Mutation HEXIM1 CDK9
R38S - +++
Q56R (2) - +++
I59F (2) - +++
P85L - +++
I105F (2) - +++
V107E - +++
C111R - +++
L133R - +++
H154R - +++
Y175H (2) - +++
T179A - +++
Possible impacts on: CDK9 activation
Substrate recognition
11 Cyclin T1 point mutations���- Functional role -
Regulation of P-TEFb by HEXIM1 protein
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8 9 10 11 12 13
Gal4-CycTm - WT
R38S Q56
R I5
9F P8
5L
I105F
V107E C111R L1
33R
H154R Y175H
G5-38-HIV + + + + + + + + + + + +
T179A
+
fold
act
ivat
ion
* * * ** *
* ** *
*
*
Mutation HEXIM1 CDK9
R38S - +++
Q56R (2) - +++
I59F (2) - +++
P85L - +++
I105F (2) - +++
V107E - +++
C111R - +++
L133R - +++
H154R - +++
Y175H (2) - +++
T179A - +++
Activity
-
+
-
+
+
+++
-
+
-
+++
+++
-
-
-
-
-
Same interaction phenotype but different functional effects on P-TEFb activity
Possible impacts on: CDK9 activation
Substrate recognition
11 Cyclin T1 point mutations���- Structure / Function relationships -
Regulation of P-TEFb by HEXIM1 protein
Mutation HEXIM1 CDK9
R38S - +++
Q56R (2) - +++
I59F (2) - +++
P85L - +++
I105F (2) - +++
V107E - +++
C111R - +++
L133R - +++
H154R - +++
Y175H (2) - +++
T179A - +++
Activity
-
+
-
+
+
+++
-
+
-
+++
+++
-
-
-
-
-
Mutations ⇗ activity: surface
Mutations ⇘ activity: internal and in the vicinity
Signal transduction pathway?
N60
Q56
Q50
Q46
T179
H154
H183
Y175
Regulation of P-TEFb by HEXIM1 protein
Regulation of P-TEFb by HEXIM1 protein
Hex1 Hex1
Cyclin T1
CDK9
Molecular mechanisms of HEXIM1-mediated inhibition of P-TEFb
Signal transduction pathway?
Regulation of P-TEFb by HEXIM1 protein
Regulation of P-TEFb by HEXIM1 protein
Hex1 Hex1
Cyclin T1
CDK9
Molecular mechanisms of HEXIM1-mediated inhibition of P-TEFb
Signal transduction pathway?
Regulation of P-TEFb by HEXIM1 protein
Regulation of P-TEFb by HEXIM1 protein
Hex1 Hex1
Cyclin T1
CDK9
Molecular mechanisms of HEXIM1-mediated inhibition of P-TEFb
Signal transduction pathway?
Contents
Regulation of the positive transcription elongation factor P-TEFb by HEXIM1 and HIV-1 Tat proteins
Introduction
Problematic
Methods
Results
Discussion
Conclusion & perspectives
HEXIM1 might bind Cyclin T1 ���in a rigid groove between the cyclin folds
Regulation of P-TEFb by HEXIM1 protein
Residues involved in HEXIM1 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
!exible
rigid
Crystallographic temperature factors (B-factors)
HEXIM1 might bind Cyclin T1 ���in a rigid groove between the cyclin folds
Regulation of P-TEFb by HEXIM1 protein
Residues involved in HEXIM1 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)
!exible
rigid
Crystallographic temperature factors (B-factors)
Regulation of P-TEFb by HEXIM1 protein
Residues involved in HEXIM1 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
!exible
rigid
Crystallographic temperature factors (B-factors)
HEXIM1 might bind Cyclin T1 ���in a rigid groove between the cyclin folds
Regulation of P-TEFb by HEXIM1 protein
Residues involved in HEXIM1 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
!exible
rigid
Crystallographic temperature factors (B-factors)
HEXIM1 might bind Cyclin T1 ���in a rigid groove between the cyclin folds
HEXIM1 might bind Cyclin T1 ���in predicted ligand binding regions
Regulation of P-TEFb by HEXIM1 protein
Residues involved in HEXIM1 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
Laurie et al. Bioinformatics (2005)
The Q-SiteFinder algorithm
• The protein surface is coated with a layer of methyl (-CH3) probes to calculate van der Waals interaction energies between the protein and probes.
• Clusters with most favorable interaction energies are retained as potential ligand-binding sites.
HEXIM1 might bind Cyclin T1 ���in predicted ligand binding regions
Regulation of P-TEFb by HEXIM1 protein
Residues involved in HEXIM1 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
Positioning Cyclin T1 residues important for HEXIM1 binding on P-TEFb available 3D structures
Regulation of P-TEFb by HEXIM1 protein
Residues involved in HEXIM1 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
PDB 3MI9
Conservation of P-TEFb ���and its regulation by HEXIM1 and 7SK ncRNA
Regulation of P-TEFb by HEXIM1 protein
P-TEFb HEXIM1 7SK
H. sapiens ! ! !D. rerio ! ! !D. melanogaster ! ! !C. elegans ! ? ?S. cerevisiae !S. pombe !
Animal innovation?
Conservation of P-TEFb ���and its regulation by HEXIM1 and 7SK ncRNA
Regulation of P-TEFb by HEXIM1 protein
P-TEFb HEXIM1 7SK
H. sapiens ! ! !D. rerio ! ! !D. melanogaster ! ! !C. elegans ! ? ?S. cerevisiae !S. pombe !
Animal innovation?
PsiBlast single unannotated putative C. elegans protein Y39E4B.6 for HEXIM homolog. Marz et al. 2009
Conservation of P-TEFb ���and its regulation by HEXIM1 and 7SK ncRNA
Regulation of P-TEFb by HEXIM1 protein
-L-T
-L-T-U
ceHEXIM
(putative)WT WT WTF156L WTWT
ceCyclin T1 WT Y193S
huCycT1
Y175
huHEXIM1
F267
Y193E WTWT
P-TEFb HEXIM1 7SK
H. sapiens ! ! !D. rerio ! ! !D. melanogaster ! ! !C. elegans ! ? ?S. cerevisiae !S. pombe !
Animal innovation?
PsiBlast single unannotated putative C. elegans protein Y39E4B.6 for HEXIM homolog.
ceCyclin T1 and (putative) ceHEXIM1:
• interact in two-hybrid • show conservation of binding residues
Marz et al. 2009
Conservation of P-TEFb ���and its regulation by HEXIM1 and 7SK ncRNA
Regulation of P-TEFb by HEXIM1 protein
ceCyclin T1 and (putative) ceHEXIM1:
• interact in two-hybrid • show conservation of binding residues
-L-T
-L-T-U
ceHEXIM
(putative)WT WT WTF156L WTWT
ceCyclin T1 WT Y193S
huCycT1
Y175
huHEXIM1
F267
Y193E WTWT
P-TEFb HEXIM1 7SK
H. sapiens ! ! !D. rerio ! ! !D. melanogaster ! ! !C. elegans ! ? ?S. cerevisiae !S. pombe !
Animal innovation?
PsiBlast single unannotated putative C. elegans protein Y39E4B.6 for HEXIM homolog.
Existence of a true HEXIM1 homology and conservation of Hex1/CycT1 binding residues from human to nematodes
Marz et al. 2009
Conclusions & perspectives
Cyclin T1 residues involved in HEXIM1 binding:
Are located on HIV-1 Tat binding surface Tat and HEXIM1 competitive and mutual exclusive binding to Cyclin T1 reported in biochemical studies
Conclusions & perspectives
Cyclin T1 residues involved in HEXIM1 binding:
Are located on HIV-1 Tat binding surface Tat and HEXIM1 competitive and mutual exclusive binding to Cyclin T1 reported in biochemical studies
Have different impact on CDK9 transcriptional activity signal transduction pathway at the molecular level
Hex1 Hex1
Cyclin T1
CDK9
Conclusions & perspectives
Cyclin T1 residues involved in HEXIM1 binding:
Are located on HIV-1 Tat binding surface Tat and HEXIM1 competitive and mutual exclusive binding to Cyclin T1 reported in biochemical studies
Have different impact on CDK9 transcriptional activity signal transduction pathway at the molecular level
40% are also involved in CDK9 binding Cyclin folds stabilization? CycT1/CDK9 contact relevant for HEXIM1 recognition?
50% mutations ⇘ P-TEFb transcriptional activity Role of CDK9/CycT1 active conformational state in recognition and regulation by HEXIM1?
Hex1 Hex1
Cyclin T1
CDK9
Conclusions & perspectives
Edgetic approach…
o Define HEXIM1-binding regions on CycT1
o Build experimental tools to
• map protein-protein interactions
Hex1 Hex1
Cyclin T1
CDK9
Conclusions & perspectives
• map protein-protein interactions
• investigate HEXIM1 role on P-TEFb functions:
P-TEFb recruitment (microscopy, ChIP) Gene-specific activity Mobility (microscopy)
Hex1 Hex1
Cyclin T1
CDK9
gene A gene B
gene C
gene Dgene E
Hex1 Hex1
Cyclin T1
CDK9
Edgetic approach…
o Define HEXIM1-binding regions on CycT1
o Build experimental tools to
Conclusions & perspectives
• map protein-protein interactions
Hex1 Hex1
Cyclin T1
CDK9
HIV-1 LTR / TAR HIV-1 genes
HIV-1 LTR / TAR luciferase gene
+ + +- + +- - +
HIV-LucLucTat proteinTat protein
huCycT1huCycT1
0
3
6
9
12
15
P-TEFbHex1 Hex1 • investigate HEXIM1 role on P-TEFb
functions:
P-TEFb recruitment (microscopy, ChIP) Gene-specific activity Mobility (microscopy)
Tat transactivation of HIV-1 promoter
…
Edgetic approach…
o Define HEXIM1-binding regions on CycT1
o Build experimental tools to
Olivier Bensaude Anne Catherine Dock Bregeon Van Trung Nguyen Gaëlle Diribarne Lydia Kobbi Bo Gu Marc Faltot Solène Ivaha Anthony Drecourt Anne-Laure Lefevre Olivier Liau
The Functional Genomics Dpt.
SPIBens
Acknowledgements
September 28, 2012 92
top related