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
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Regulation of the Positive Transcription Elongation Factor P-TEFb (CDK9/CycT1) by HEXIM1 protein
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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
– 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
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