Rac1 signalling modulates transcriptional regulation by BCL-6 and STAT5

Rac1 signalling modulates

transcriptional regulation by BCL-6

and STAT5

Departamento de Genética Humana,

Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal

[email protected] Peter Jordan

IBMC symposium

Rho GTPases and cell signalling June 29, 2012

ECM Plasmamembrane nucleus Focal adhesion actin lamellipodia actin G1 S

Rac1 signaling

-adhesion

-motility

-gene expression/

proliferation

Rac1

Rac1 signalling regulates gene transcription

ROS CycD plasma membrane nuclear membrane IkBαααα P BCL-6 P P P _STAT5 ST AT 5 P MgcRac GAP Rac1 GDP

NFKB1 _transcript



endo Rac1 Myc -Rac 1-L6 1 Myc -vec tor NSC 2376 6 Rac1 α α α α-GFP GFP -BC L-6 GFP -vec tor



Rac1-L61 p50 NFKB1_{/p50 protein} NFkB1/p50 expression is repressed by BCL-6 NFkB1 Pol II RT-PCR Myc -Rac 1-L6 1 Myc -vec tor NSC 2376 6 GFP -BC L-6 GFP -vec tor

F o ld in cr ea se i n lu ci fe ra se a ct iv it y 0,0 0,5 1,0 1,5 2,0 2,5 3,0 F o ld in cr ea se i n lu ci fe ra se a ct iv it y pGL3-5xBCL-6 − −− − −−−− −−−− −−−− −−−− NSC23766 ++++ −−−− −−−− − − − − − − − − − −− − BCL-6 −−−− −−−− −−−− − − − − + + + + − −− − − − − − − −− − −−−− Rac1b-L61 −−−− −−−− − − − − − −− − − − − − − −− − −−−− Rac1-L61 −−−− − − − − − − − − − − − − − − − − siBCL-6 −−−− −−−− −−−− ++++ 0,0 0,5 1,0 1,5 2,0 2,5 3,0 pGL3-vector − −− − −−−− −−−− −−−− −−−− NSC23766 ++++ −−−− −−−− − − − − − − − − − − − − BCL-6 −−−− −−−− −−−− − − − − + + + + − − − − − − − − − −− − −−−− Rac1b-L61 −−−− −−−− − − − − − − − − − − − − − −− − −−−− Rac1-L61 −−−− − −− − − − − − − − − − − −− − siBCL-6 −−−− −−−− −−−− ++++ luc+ SV40 promoter 5x TCGAATTCCTCGAAAG 360 1 BCL-6 reporter

0,0 0,5 1,0 1,5 2,0 pGL3-5xBCL-6 F o ld in cr ea se In lu ci fe ra se a ct iv it y P<0.002 P_<0.001 + - - - - - - - - -+ - -+ + - + + -- - -- - + -- + -+ -- - -+ -- + - - - - - - -P_<0.002 P_<0.003 α-GFP Tubulin -GFP-RhoA-V14 GFP-Rac1b-L61 GFP-Rac1-L61 GFP-PAK-KD GFP-Cdc42-V12 GFP-empty _{+ - - - - - - - -} -+ 25 µµµµM DPI GFP-PAK-CA GFP-JNK-CA The mechanism downstream of Rac1

0,0 0,5 1,0 1,5 2,0 2,5 -+ - + - - + -Ctrl + - - - + - - -siPAK1 -- + - - + - -siPAK2 -IPA-3 10µM - - - - + + -- - + + + - + Rac1-L61 pGL3-5xBCL-6 F o ld in cr ea se in lu ci fe ra se a ct iv it y PAK1  PAK2  anti-PAK1/2/3 Tubulin siC trl siPA K1 siPA K2

B

A

The mechanism downstream of Rac1 relies on PAK1

DsR-vector DsR-Rac1-L61 GFP-BCL-6 GFP-BCL-6 Merge + DAPI Merge + DAPI

DsR-Rac1b-L61 GFP-BCL-6 Merge + DAPI

SW 480 HT 29 BCL-6 200 bp 200 Pol II DL D-1 HE L 697 BCL-6 in colorectal cells BCL= B-Cell lymphoma

H2B Myc -Rac 1-L6 1 S NS S NS Myc -PAK 1 S NS S NS Myc -vec tor S NS S NS S NS S NS α-GFP α-Myc Tubulin Myc -PAK -KD Myc- PAK-KD GFP-BCL-6 Myc- PAK1 GFP-BCL-6

Effect of PAK1 on BCL-6 in colorectal cells

subcellular fractionation

S= soluble

+ -+ -+ GFP-BCL-6 GFP-vector rPAK1 -+ + rPAK1 α-GFP

Kinase assay p-BCL-6_p-PAK1

PAK1 phosphorylates BCL-6 in vitro

Rac1/PAK1 signalling promotes the release of repressor BCL-6 from chromatin.

M o ck R a c1 -L 6 1 P A K 1 -C A α_-GFP α_-tubulin GFP-PAK1 GFP-Rac1-L61 pGL3-5xBCL-6 F o ld i n cr e a se i n L u ci fe ra se a ct iv it y

BCL-6 and STAT5 recognise a similar core binding motif

TTCNNNGAA

Genes reported to be regulated by both factors: cyclin D2, blimp-1, BCL-XL, Socs2, prolactin

BCL-6 S T A T 5 P S T A T 5 P

pGL3-5xBCL-6 M o ck R a c1 -L 6 1 P A K 1 -C A S T A T 5 A P A K 1 -C A + S T A T 5 A α_-GFP α_-tubulin GFP-STAT5A GFP-PAK1 GFP-Rac1-L61 F o ld i n cr e a se i n L u ci fe ra se a ct iv it y Rac1 = PAK1+STAT5

Active Rac1 promotes a switch from BCL-6 to STAT5 at the promoter. In p u t N S I g G αααα -S T A T 5 αααα -B C L-6 Rac1-L61 − −− − 253 bp − −− − 253 bp O D (a b st ra ct u n it s) N S I g G αααα -S T A T 5 αααα -B C L-6 Input Mock BCL-6 repeats luc+ promoter 5x TCGAATTCCTCGAAAG 360 1 BCL-6 reporter ChIP

Does BCL-6/STAT5 transcription switching

happen in cells?

D LD -1 S W 4 8 0 H T 2 9 α α α α-BCL-6 α α α α-phospho-STAT5 α α α α-STAT5 α α α α-phospho-PAK1/2 α α α α-PAK1/2 α α α α-Rac1 BCL-6 phospho-BCL-6 phospho-PAK2 phospho-PAK1 PAK2 PAK1 Total Rac1 (active) GTP-Rac1

Which endogenous genes might be targets of

the transcription factor switch?

+1 -2500 -2474 TGGGTTTCAAGGA CDKN2B +1 -2500 -896 TCCTAGAAAGCTG CCND2 +1 -2500 -146 TCCTTGAAGGAGC SUMO1 si B C L-6 si C tr l H T 2 9 D LD -1 BCL-6 si S T A T 5 A si C tr l STAT5A si S T A T 5 A si B C L-6 MW POL II si C tr l Up Down

Cell cycle PCR array (84 genes)

O D (a b st ra ct u n it s) SUMO1 promoter −−−− 327 bp O D (a b st ra ct u n it s) CDKN2B promoter −−−− 340 bp O D (a b st ra ct u n it s) CCND2 promoter −−−− 344 bp SW480 +++ Active Rac1 0 Active PAK1 + Active STAT5 + Active BCL-6 HT29 ++ Active Rac1 ++ Active PAK1 ++ Active STAT5 ++ Active BCL-6 DLD-1 + Active Rac1 + Active PAK1 + Active STAT5 + Active BCL-6 N S I g G αααα -S T A T 5 αααα -B C L-6 In p u t N S I g G αααα -S T A T 5 αααα -B C L-6 In p u t N S I g G αααα -S T A T 5 αααα -B C L-6 Input DLD-1 SW480 HT29 N S I g G αααα -S T A T 5 αααα -B C L-6 In p u t N S I g G αααα -S T A T 5 αααα -B C L-6 In p u t N S I g G αααα -S T A T 5 αααα -B C L-6 Input DLD-1 SW480 HT29

‘Steady state’ promoter occupancies in colorectal cell lines

Do the observed promoter occupancy rates

reflect changes in gene expression?

Do promoter occupancy and gene expression

respond to Rac1 signalling?

CCND2 P ro m o te r o cc u p an cy (O D i n ab st rac t u n it s) DLD-1 Bound STAT5 Bound BCL-6 mRNA level SW480 Bound STAT5 Bound BCL-6 mRNA level HT29 T ran sc rip t e x p re ss io n (F o ld o v e r m o ck ) Bound STAT5 Bound BCL-6 mRNA level phospho-STAT5 STAT5 GFP-PAK1 GFP-Rac1 M o ck R a c1 -L 6 1 P A K 1 si P A K 1 M o ck R a c1 -L 6 1 P A K 1 si P A K 1 M o ck R a c1 -L 6 1 P A K 1 si P A K 1 -α α α α-GFP

Summary

Novel insights into the modulation of gene transcription by Rac1 signalling.

 Rac1 signalling activates gene transcription by inducing a transcriptional switch from repressor BCL-6 to activator STAT5 at gene promoters.

 Rac1/PAK1 signalling promotes the release of repressor BCL-6 from chromatin.

cyclin D2, CDKN2B and SUMO1 are endogenous genes, which are inversely regulated by BCL-6 and STAT5 in colorectal cells.

Rac1 GDP GEF TK S T A T 5 P S T A T 5 P MgcRac GAP Rac1 GTP Rac1 GTP PAK1 PAK1 plasma membrane nuclear membrane BCL-6 P P STAT5 P ST AT 5 P _MgcRac GAP Rac1 GDP

Proposed mechanism

Acknowledgements

INSA

Patrícia Barros

Paulo Matos

Funding

Eric Lam, Imperial College London

Mock Rac1-L61 PAK1 PAK1-KD p h o sp h o -S T A T 5 S T A T 5 G F P -P A K 1 G F P -R a c1 C D K N 2 B Promoter occupancy

(OD in abstract units)

Tra nsc rip t e xp re ssi on (Fo ld o ve r m ock ) S U M O 1 Tra nsc rip t e xp re ssi on (Fo ld o ve r m ock ) Promoter occupancy

(OD in abstract units)

C C N D 2 D LD -1 S W 4 8 0 H T 2 9 B o u n d S T A T 5 B o u n d B C L-6 m R N A le v e l B o u n d S T A T 5 B o u n d B C L-6 m R N A le v e l B o u n d S T A T 5 B o u n d B C L-6 m R N A le v e l Tra nsc rip t e xp re ssi on (Fo ld o ve r m ock ) Promoter occupancy

(OD in abstract units)

Mock Rac1-L61 PAK1 PAK1-KD Mock Rac1-L61 PAK1 PAK1-KD αααα -G F P

Fig S1 Whole cell lysates STrEP-BCL-6 GFP -STA T5A α α α α-GFP α α α α-Tubulin α α α α-BCL-6 GFP-STAT5 GFP-PAK1 GFP -PA K1 GFP -STA T5A GFP -PA K1 beads STrEP-vector BCL-6

Myc-Rac1-L61 Myc-empty S NS α α α α-GFP α α α α-Tubulin α αα α-Myc S NS GFP-STAT5 Myc-Rac1-L61 Histone H2B

ROI1 ROI2 G ra y v a lu e Distance (pixel) G ra y v a lu e Distance (pixel) Fig 2B

α-GFP α-Myc Myc -Rac 1-L6 1 S NS BCL-6 Myc -Rac 1b-L 61 S NS Myc -vec tor S NS Tubulin Histone H2B



Rac1-L61



Rac1b-L61

α-GFP α-Myc Myc -Rac 1-L6 1 S NS BCL-6 Myc -Rac 1b-L 61 S NS Myc -vec tor S NS Tubulin Histone H2B



Rac1-L61



Rac1b-L61 BCL-6 in colorectal cells subcellular fractionation S= soluble NS= non-soluble, chromatin-bound