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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