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1 KAROLINSKA INSTITUTET Department of Cell and Molecular Biology Medical Nobel Institute Actin at the crosstalk between gene Actin at the crosstalk between gene transcription and chromatin structure transcription and chromatin structure Piergiorgio Percipalle, PhD Discovery of actomyosin Discovery of actin Actin forms a nuclear gel Injections of anti actin antibodies inhibit RNA synthesis Discovery of actin in chromatin remodelling complexes Discovery of nuclear myosin 1 Actin in pre-mRNP particles Actin functions in gene transcription bound to RNA polymerases I, II and III Discovery of the ATPase activity of myosin 1860s 1939-42 1950s 1970s 1979-81 1984 1998 2000 2001-02 2004-05 More than 100 years of actin More than 100 years of actin The dipteran The dipteran Chironomus tentans Chironomus tentans Nuclear actin in transcriptional puffs Nuclear actin in transcriptional puffs on polytene chromosomes on polytene chromosomes Percipalle et al., 2001 +RNase Chromosome IV Chromosome I -RNase 12 175 83 62 47.5 32.5 25 16.5 BR1 BR2 Nu Nu Cyt Cyt Actin accompanies the mRNA transcript from Actin accompanies the mRNA transcript from gene to polysomes gene to polysomes Elongation of nascent mRNA transcripts In In C. tentans C. tentans hrp65 is present in nuclear hrp65 is present in nuclear actin actin- hnRNP complexes hnRNP complexes DNase I affinity chromatography Percipalle et al., 2003 Hrp65-1 Hrp65-2 Hrp65-3 N,G,Q RRM1 RRM2 +/- N N,G,Q RRM1 RRM2 +/- N N,G,Q RRM1 RRM2 +/- N 499 1 535 aa 517 aa 512 aa hrp 65 is located in the nascent BR RNP

More than 100 years of actin Actin at the crosstalk ... · Actin at the crosstalk between gene transcription and chromatin structure ... on polytene chromosomes Percipalle et al.,

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KAROLINSKA INSTITUTETDepartment of Cell and Molecular Biology

Medical Nobel Institute

Actin at the crosstalk between geneActin at the crosstalk between genetranscription and chromatin structuretranscription and chromatin structure

Piergiorgio Percipalle, PhD

Discovery ofactomyosin

Discoveryof actin

Actin formsa nuclear gel

Injections ofanti actin antibodiesinhibit RNA synthesis

Discovery of actinin chromatin remodellingcomplexes

Discoveryof nuclearmyosin 1

Actin inpre-mRNPparticles

Actin functions ingene transcriptionbound to RNApolymerases I, IIand III

Discovery ofthe ATPase activityof myosin

1860s 1939-42 1950s 1970s 1979-81 1984 1998 2000 2001-02 2004-05

More than 100 years of actinMore than 100 years of actin

The dipteran The dipteran Chironomus tentansChironomus tentans Nuclear actin in transcriptional puffsNuclear actin in transcriptional puffson polytene chromosomeson polytene chromosomes

Percipalle et al., 2001

+RNase

Chromosome IV Chromosome I

-RNase

1 2

1758362

47.5

32.5

25

16.5 BR1 BR2

NuNu

CytCyt

Actin accompanies the mRNA transcript fromActin accompanies the mRNA transcript fromgene to polysomesgene to polysomes

Elongation of nascentmRNA transcripts

Elongation of nascentmRNA transcripts

In In C. tentansC. tentans hrp65 is present in nuclearhrp65 is present in nuclearactinactin--hnRNP complexeshnRNP complexes

DNase I affinity chromatography

Percipalle et al., 2003

Hrp65-1Hrp65-2

Hrp65-3

N,G,Q RRM1 RRM2 +/- NN,G,Q RRM1 RRM2 +/- N

N,G,Q RRM1 RRM2 +/- N

4991

535 aa517 aa

512 aa

hrp 65 is located in the nascent BR RNP

2

N, G, Q RRM1 RRM2 +/- N

The actinThe actin--binding consensus on hrp65binding consensus on hrp65--22

YRP YVNQRPQKAR YRNGWt-actin-binding 65-2 peptide

517513503

Amino acids required for actin binding

Mutated 65-2 peptide R K

YRP YVNQAPQAAR YRNG

500 517

Percipalle et al., 2003

Peptides nuclear injectionsRun on assay by BrUTP incorporation

In vivo disruption of the actin/hrp65In vivo disruption of the actin/hrp65--2 2 interaction inhibits BrUTP incorporationinteraction inhibits BrUTP incorporation

InjectionsControl +ActinomycinD 65-2CTD 65-2CTD mut-1

Percipalle et al, 2003

Percipalle et al., 2003; Sjölinder et al., 2005

A model for coA model for co--transcriptional recruitment to the transcriptional recruitment to the growing pregrowing pre--mRNPmRNP through through actinactin and hrp65and hrp65--22

p2D10

Histone acetylation

Yestranscription-hnRNP U

Yestranscriptionlonger formCBF-A

??novelhnRNP A3*

??novelhnRNP A3*

??novelhnRNP A3*

??novelhnRNP A2*

ActinActin--bindingbindingFunctionFunctionIsoformIsoformProteinProtein

Mammalian actinMammalian actin--associated hnRNP proteinsassociated hnRNP proteinsidentified by Mass Spectrometryidentified by Mass Spectrometry

Percipalle et al., 2002; Kukalev et al., 2005

hnRNP U

Gene transcription Chromatin modification

Nuclear location

The multifunctional hnRNP U/SAFThe multifunctional hnRNP U/SAF--AA

hnRNP U-N

hnRNP U-M

hnRNP U-C

T71 250

T7250 550

T7551 823

hnRNP U FL

DNA binding(1-161)

RNA pol II binding(269-536)

RNA-binding(683-806)

T71 200 400 600 800

Actin binds a conserved motif in the hnRNP UActin binds a conserved motif in the hnRNP UCC--terminal domainterminal domain

YRP YVNQRPQK A RYRN

KDE DYKQRTQKKA EVEGQR QK A

H-hnRNP U

Ct-hrp65-2504 513

620610

Consensus

Kukalev et al., 2005

3

In vivoIn vivo disruption of the actin/hnRNP U disruption of the actin/hnRNP U interaction inhibits BrUTP incorporationinteraction inhibits BrUTP incorporation

Peptide nuclear microinjection

BrUTP incorporation

BrUTP detection

Confocal microscopy

Kukalev et al., 2005

The actin/hnRNP U complex is required for the synthesis of a run off transcript in vitro

SummarySummary

Actin and hnRNP U are simultaneously associated with the Pol II phopshoCTD in living cells

The actin/hnRNP U complex is required for efficient Pol II transcription in vivo

Actin binds hnRNP U through a conserved amino acid sequence motif

Kukalev et al., 2005

A model for actin/hnRNPA model for actin/hnRNP--mediated recruitmentmediated recruitmentof transcription coof transcription co--activatorsactivators

Percipalle and Visa, 2006

The histone acetyl transferase PCAF The histone acetyl transferase PCAF associates with actin and hnRNP Uassociates with actin and hnRNP U

Immunoprecipitations withanti-PCAF Ab

DNase I AffinityChromatography

Obrdlik et al., 2007a

The histone acetyl transferase PCAF The histone acetyl transferase PCAF associates with actin and hnRNP Uassociates with actin and hnRNP U

1) Conjugate hnRNP U subdomains to beads2) Incubate beads with nuclear extracts 3) Resolve, analyze bound proteins

PCAF facilitates long distance transcriptional enhancement by direct association with enhancer sequences

PCAF requirement as HAT and coactivator has been shown in myogenesis, nuclear receptor mediated and growth factor signaled activation

PCAF acetylates free histones and nucleosome, preferentially on H3K14

4

A novel peptideA novel peptide--specific antibody that specific antibody that disrupts the actindisrupts the actin--hnRNP U interactionhnRNP U interaction

DNase I beads hnRNP U-C beads IP

PCAFPCAF--mediated HAT activity is dependent mediated HAT activity is dependent on the actinon the actin--hnRNP U interactionhnRNP U interaction

Nuclear extract

DNase I affinity chromatography+/- anti-hnRNP U antibody CED17

for competition

+ purified histones

+ 3H-CoA

Liquid scintillation

HAT activity assay

CTD phosphorylation state reflects the CTD phosphorylation state reflects the phase of transcriptionphase of transcription

phosphoSer5 >>>> phopshoSer5 / Ser2 >>>>> phosphoSer2

Robert J. Sims 3rd, Rimma Belotserkovskaya, and Danny ReinbergGenes & Dev., Oct 2004; 18: 2437 - 2468.

Anton Meinhart et al. Genes Dev. 2005; 19: 1401-1415

Heptapetide repeat: Y1S2P3T4S5P6S7

Modifications: Phosphorylation,Isomerisation..Glycosylation

CarboxyterminalCarboxyterminal domain (CTD) domain (CTD) of RNAof RNA--Pol IIPol II

Actin, hnRNP U and PCAF associate with Actin, hnRNP U and PCAF associate with phosphorylated pol II CTD phosphorylated pol II CTD

CTD

CTD phosphorylation(cdk7, cdk9)

Conjugate phosphorylatedand unphosphorylated

CTD to beads

CTD beads incubationwith nuclear extracts

Analysis of CTD-boundproteins on immunoblots

Actin, hnRNP U and PCAF are present on Actin, hnRNP U and PCAF are present on promoter and coding region of pol II genes promoter and coding region of pol II genes

5

Actin, hnRNP U and PCAF are integrated inActin, hnRNP U and PCAF are integrated inribonucleoprotein complexesribonucleoprotein complexes

Actin, hnRNP U and PCAF are associatedActin, hnRNP U and PCAF are associatedwith nascent prewith nascent pre--mRNA transcriptsmRNA transcripts

Disruption of the actinDisruption of the actin--hnRNP U interaction hnRNP U interaction represses pol II transcriptionrepresses pol II transcription

Antibody nuclear microinjection

BrUTP incorporation

BrUTP detection

Confocal microscopy

Statistical quantification

SummarySummary

Disruption of the actin-hnRNP U interaction in living cells represses pol II transcription in a HAT-dependent manner

In vitro disruption of the actin-hnRNP U complex releases PCAF protein and its HAT activity

The HAT PCAF associates with actin and hnRNP U

Obrdlik et al., 2007a

Actin, hnRNP U and PCAF associate with the phopshorylated pol II CTD

ActinActin--hnRNP U mediate recruitment of hnRNP U mediate recruitment of PCAFPCAF

Obrdlik et al., 2007b

Actin and myosin in Actin and myosin in ribosomal gene transcriptionribosomal gene transcription

6

A nuclear form of myosin 1 in A nuclear form of myosin 1 in gene transcriptiongene transcription

Pestic-Dragovich et al., 2000

Nuclear myosin 1 (NM1) is a Nuclear myosin 1 (NM1) is a bona fidebona fidenucleolar proteinnucleolar protein

Fomproix and Percipalle, 2004

+Act D

NM1 is present on actively transcribingNM1 is present on actively transcribingribosomal genesribosomal genes

Fomproix and Percipalle, 2004

Living HeLa cells -incubation with BrUTP

Fixation/Permeabilization

Detection of NM1 andincorporated BrUTP

NM1 is present on actively transcribingNM1 is present on actively transcribingribosomal genesribosomal genes

Fomproix and Percipalle, 2004;Philimonenko et al., 2005;Reviewed by Visa, 2005

NM1 is involved in pol I transcriptionNM1 is involved in pol I transcription

• NM1 co-localizes with endogeneous pol I

• NM1 is required for pol I transcription

NM1

Pol 1

NM1 is associated with the chromatin NM1 is associated with the chromatin remodelling complex WSTF/SNF2hremodelling complex WSTF/SNF2h

BB--WICHWICH1) Assembly is transcription and RNase-dependent2) NM1-WSTF-ISWI contains rRNA processing

factors – RNA helicase II/Gua3) NM1-WSTF-ISWI associates with chromatin

7

In living cells, NM1, WSTF and SNF2h are In living cells, NM1, WSTF and SNF2h are associated with pol Iassociated with pol I

Nuclei

Immunoprecipitation with S57299

Detection of associatedproteins

Cross-linker

Adduct

NM1, WSTF, SNF2h are present all NM1, WSTF, SNF2h are present all along the ribosomal genealong the ribosomal gene

rRNArRNA

Pol I

Pol I

Pol I TIF-IAP

5’

ACU

Pol I AssemblyTranscription Initiation

Transcription Elongation

18S

18S 28S

28S

The different phases of RNA polymerase I The different phases of RNA polymerase I mediated transcriptionmediated transcription

Adapted from Visa, 2005

Nuclear extract

+ anti-NM1 antibodyor

+ anti-WSTF antibody

+ ATP/CTP+ rDNA template

+UTP+ UTP+ GTP

ACU trimer(abortiveinitiation)

Run off transcript(transcriptionelongation)

NM1 and WSTF are not required for pol I NM1 and WSTF are not required for pol I transcription initiationtranscription initiation

Abortive initiation assay

WSTF is required for rRNA transcript WSTF is required for rRNA transcript elongation on chromatin templates elongation on chromatin templates

Naked DNA ChromatinTemplate DNA

WSTF gene silencing by siRNA inhibits WSTF gene silencing by siRNA inhibits synthesis of the 45 S presynthesis of the 45 S pre--rRNA rRNA

WSTF siRNA

Total RNApreparation

Densitometricquantification

RT-PCR

8

B-WICH is a novel chromatin remodelling complex thatcontains nuclear myosin 1

B-WICH is required for the activation and maintenanceof productive pol I transcription

SummarySummary

B-WICH activates pol I transcription through a chromatinremodelling-based mechanism

• Pol I assembly on promoter• Transcription initiation

NM1 PTIF-IA Pol I

Actin

rDNA

WSTF SNF2h

SL1/TIF-IB

A model for the function of actin and NM1 in A model for the function of actin and NM1 in rDNA transcriptionrDNA transcription

Percipalle and Östlund Farrants, 2006

• Promoter clearance• Transcription elongation

5’

pre- rRNA

UCAPol I

Actin

NM1 P

Pol IActin

NM1WSTF SNF2h

Chromatin remodellingthrough WICH

Transcription

WSTF SNF2h

TIF-IA

AcknowledgementsAcknowledgementsLab membersLab members

Ales ObrdlikS. Chandrasekhar RajuAlexander KukalevEmilie LouvetYlva Nord

Nathalie Fomproix

CMB, Karolinska Institute

CollaborationsCollaborationsBertil Daneholt

(CMB, Karolinska Institute)

Neus Visa(Stockholm University)

Martti Tammi(National University of Singapore)

Ann-Kristin Östlund FarrantsErica Cavellan

(Stockolm University)

Uli Scheer(University of Wurzburg)

Ingrid GrummtRenate Voit

(University of Heidelberg)

Tomas BergmanCarina Palmberg

(MBB, Karolinska Institute)

Apostolia Guialis(National Hellenic Research Foundation)

Elena KiselevaInstitute of Cytology and Genetics

FundingFundingSwedish Research CouncilKarolinska Institute