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

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


•  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


AAAAAAAAPoly (A)



AAAAAAAAPoly (A)

Pol I

I den

sity

AAAAAAAAPoly (A)

Pol I

I den

sity



Promoter-proximal PAUSING



AAAAAAAAPoly (A)

Pol I

I den

sity

Functional relevance of RNAPII pausing


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



DSIF

Pol II

NELF

GTFs TF1

DSIF

Pol II GTFs

NELF

TF2 TF1

P

PP-TEFb


Pol II

DSIF

Pol II GTFs

NELF

TF2 TF1

P

PP-TEFb

DSIF

Pol II GTFs

NELF

TF2 TF1

P

PP-TEFb



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




DSIF

Pol II

NELF

GTFs TF1

DSIF

Pol II GTFs

NELF

TF2 TF1

P

PP-TEFb


Pol II

DSIF

Pol II GTFs

NELF

TF2 TF1

P

PP-TEFb

DSIF

Pol II GTFs

NELF

TF2 TF1

P

PP-TEFb



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


Contents




  Problematic

  Methods

  Results

  Discussion


Usurpation of the transcription elongation machinery ��by HIV-1 TAR RNA and Tat protein






P-TEFb

Contents




  Problematic

  Methods

  Results

  Discussion


Positive Transcription Elongation Factor b


•  P-TEFb is composed of CDK9 (40 kDa) and Cyclin T (81kDa)



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




CDK9 Cyclin T1

ATP

PDB 3BLH


•  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




  Problematic

  Methods

  Results

  Discussion


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)



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)



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



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)



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



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/CycT1

ACTIVE

7SK snRNP



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


7SK snRNP



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


  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


Cyclin T1

Nature Methods, 2009

HEXIM1

Mutant screening by Reverse Two-Hybrid


: toxic for cells able to synthetize uracile

(PCR Δ)



  Induction of a counter-selectable gene (ura3)   Random mutagenous PCR on Cyclin Boxes   Detect a loss of interaction between CycT1-HEXIM1


(PCR Δ)





(PCR Δ)





(PCR Δ)

Mutant Library��Random Mutagenesis by error-prone PCR


Reverse two-hybrid in Yeast


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


  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



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,





-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,





-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


Random mutations

CycT1

HEXIM1

Gal4BD

Gal4AD

GAL4 PROMOTER REPORTER GENE (ura3)

Interaction between mutant CycT1 and HEXIM1 / CDK9

GROWTH ON MEDIA LACKING URACIL

?

1.



+++ + -

STRONGinteraction

weakinteraction

NOinteraction

Yeast growth on media lacking uracil

multiple interaction phenotypes 69 CycT1 point mutations

Random mutations

CycT1

HEXIM1

Gal4BD

Gal4AD




?

1.



HEXIM1

+++ (11)

-

+

+++

-

+

+++ + -

STRONGinteraction

weakinteraction

NOinteraction



Random mutations

CycT1

HEXIM1

Gal4BD

Gal4AD




?

1.



HEXIM1

+++ (11)

-

+

+++

-

+

+++ + -

STRONGinteraction

weakinteraction

NOinteraction



Random mutations

CycT1

HEXIM1

CDK9

Gal4BD

Gal4AD

Gal4AD




?

1. 2.



HEXIM1 CDK9

- ; - (7)

+++ (11)

- ; + (8)

+ ; + (3)

- ; +++ (11)

+ ; +++ (13)

+ ; - (0)

+++

-

-

-

+

+ +

-

-

+

+++

+++

+

+++ + -

STRONGinteraction

weakinteraction

NOinteraction



Random mutations

CycT1

HEXIM1

CDK9

Gal4BD

Gal4AD

Gal4AD




?

1. 2.



HEXIM1 CDK9

- ; - (7)

+++ (11)

- ; + (8)

+ ; + (3)

- ; +++ (11)

+ ; +++ (13)

+ ; - (0)

+++

-

-

-

+

+ +

-

-

+

+++

+++

+

+++ + -

STRONGinteraction

weakinteraction

NOinteraction



Random mutations

CycT1

HEXIM1

CDK9

Gal4BD

Gal4AD

Gal4AD




?

1. 2.



HEXIM1 CDK9

- ; - (7)

+++ (11)

- ; + (8)

+ ; + (3)

- ; +++ (11)

+ ; +++ (13)

+ ; - (0)

+++

-

-

-

+

+ +

-

-

+

+++

+++

+

Random mutations

CycT1

HEXIM1

CDK9

Gal4BD

Gal4AD

Gal4AD




?

1. 2.X

+++ + -

STRONGinteraction

weakinteraction

NOinteraction





- ; - (7)

+++ (11)

- ; + (8)

+ ; + (3)

- ; +++ (11)

+ ; +++ (13)

+ ; - (0)

HEXIM1 CDK9

+++

-

-

-

+

+ +

-

-

+

+++

+++

+

Random mutations

CycT1

HEXIM1

CDK9

Gal4BD

Gal4AD

Gal4AD




?

1. 2.X

+++ + -

STRONGinteraction

weakinteraction

NOinteraction



+++ + -

STRONGinteraction

weakinteraction

NOinteraction


Random mutagenesis combined with reverse two-hybrid


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


Random mutagenesis combined with reverse two-hybrid


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


  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


Visualization of HEXIM1 putative binding surface on Cyclin T1


!"#$%& '()*+ ++ ,, ,, ,,,+ ,,,

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


!"#$%& '()*+ ++ ,, ,, ,,,+ ,,,

CycT1 Y175 PDB 3BLH

Cyclin T1 Tyrosine 175��- Structural role -


  Tyrosine = aromatic residue   Surface   Direct interaction with HEXIM1



Y175


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



Y175


N60

Q56

Q50

Q46

T179

H154

H183

Y175


  Mutations of CycT1 Y175 impair HEXIM1 binding to P-TEFb in human cells



Y175


N60

Q56

Q50

Q46

T179

H154

H183

Y175


  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 -


LucGal4 sites

G5-38-HIV TATA

-38 +84

CycT1endogenous

CDK9Gal4



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



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



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



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



LucGal4 sites

G5-38-HIV TATA

-38 +84

CycT1endogenous

CDK9Gal4

0

20

40

60

80

100

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)



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

* * * ** *

* ** *

*

*


R38S - +++

Q56R (2) - +++

I59F (2) - +++

P85L - +++

I105F (2) - +++

V107E - +++

C111R - +++

L133R - +++

H154R - +++

Y175H (2) - +++

T179A - +++



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

* * * ** *

* ** *

*

*


R38S - +++

Q56R (2) - +++

I59F (2) - +++

P85L - +++

I105F (2) - +++

V107E - +++

C111R - +++

L133R - +++

H154R - +++

Y175H (2) - +++

T179A - +++



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

+ + +


R38S - +++

Q56R (2) - +++

I59F (2) - +++

P85L - +++

I105F (2) - +++

V107E - +++

C111R - +++

L133R - +++

H154R - +++

Y175H (2) - +++

T179A - +++

Possible impacts on:   CDK9 activation

  Substrate recognition



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

* * * ** *

* ** *

*

*


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 -



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



Hex1 Hex1

Cyclin T1

CDK9

Molecular mechanisms of HEXIM1-mediated inhibition of P-TEFb




Hex1 Hex1

Cyclin T1

CDK9





Hex1 Hex1

Cyclin T1

CDK9



Contents


  Introduction

  Problematic

  Methods

  Results

  Discussion


HEXIM1 might bind Cyclin T1 ��in a rigid groove between the cyclin folds


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)






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








!exible

rigid









!exible

rigid



HEXIM1 might bind Cyclin T1 ��in predicted ligand binding regions







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







Positioning Cyclin T1 residues important for HEXIM1 binding on P-TEFb available 3D structures







PDB 3MI9

Conservation of P-TEFb ��and its regulation by HEXIM1 and 7SK ncRNA


P-TEFb HEXIM1 7SK

H. sapiens ! ! !D. rerio ! ! !D. melanogaster ! ! !C. elegans ! ? ?S. cerevisiae !S. pombe !

Animal innovation?



P-TEFb HEXIM1 7SK


Animal innovation?

PsiBlast single unannotated putative C. elegans protein Y39E4B.6 for HEXIM homolog. Marz et al. 2009



-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


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



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


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




  Have different impact on CDK9 transcriptional activity signal transduction pathway at the molecular level

Hex1 Hex1

Cyclin T1

CDK9




  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


  Edgetic approach…

o  Define HEXIM1-binding regions on CycT1

o  Build experimental tools to

•  map protein-protein interactions

Hex1 Hex1

Cyclin T1

CDK9



•  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






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

  …




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

Health & Medicine

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