Www.internationalregulomeconsortium.ca. Management Structure

www.internationalregulomeconsortium.ca

Management Management StructureStructure

Phase 1 - Genesis - Jul 03-Jan 04Phase 1 - Genesis - Jul 03-Jan 04

• Objective Objective – Consult with scientific, management and political Consult with scientific, management and political

resources in order to ascertain viability of large-scale resources in order to ascertain viability of large-scale international regulome projectinternational regulome project

• Funding Funding – Nil; travel and workshop expenses (<$10K) absorbed by Nil; travel and workshop expenses (<$10K) absorbed by

several existing Rudnicki grantsseveral existing Rudnicki grants

• Accomplishments Accomplishments – Obtained support of Ontario Ministry of Economic Obtained support of Ontario Ministry of Economic

Development and Trade (MEDT) and France's Research Development and Trade (MEDT) and France's Research DirectorateDirectorate

– Established international steering committee (initially, Established international steering committee (initially, Canada and France)Canada and France)

Phase 2 - Initiation (Feb-Jul 04)Phase 2 - Initiation (Feb-Jul 04)

• Objectives Objectives – Register project with Genome Canada as an International Register project with Genome Canada as an International

Consortium Initiative, submission of LOIConsortium Initiative, submission of LOI– Establish International Regulome ConsortiumEstablish International Regulome Consortium– Extend International Steering Committee membershipExtend International Steering Committee membership– Publish project whitepaperPublish project whitepaper– Generate interest in the scientific community and general Generate interest in the scientific community and general

publicpublic

• Funding Funding – Total of $149,600 in support of 1st International Workshop Total of $149,600 in support of 1st International Workshop

and follow-up travel and project management, sourced from and follow-up travel and project management, sourced from Genome Canada, Ontario Genomics Institute, CIHR, MEDT, Genome Canada, Ontario Genomics Institute, CIHR, MEDT, Ottawa Life Sciences Council, Embassies (UK, France, Italy) Ottawa Life Sciences Council, Embassies (UK, France, Italy) and private industry (Sun, Invitrogen, Affymetrix)and private industry (Sun, Invitrogen, Affymetrix)

Phase 2 - Initiation (Feb-Jul 04)Phase 2 - Initiation (Feb-Jul 04)

• Accomplishments Accomplishments – Letter of Intent submitted 18 Feb 04Letter of Intent submitted 18 Feb 04– Held 1st International Workshop, attended by 75 world-Held 1st International Workshop, attended by 75 world-

class scientists from six nations, as well as class scientists from six nations, as well as representatives of the private sector, granting agencies, representatives of the private sector, granting agencies, and embassies and officially struck the consortium on 5 and embassies and officially struck the consortium on 5 May 04May 04

– Expanded Steering Committee membership to include the Expanded Steering Committee membership to include the UK, Netherlands, Singapore and Australia. Other nations UK, Netherlands, Singapore and Australia. Other nations expressing interest in scientific participation include the expressing interest in scientific participation include the US, Italy, and GermanyUS, Italy, and Germany

– Published Ottawa White Paper 5 Jul 04Published Ottawa White Paper 5 Jul 04– Obtained media exposure (12 articles/interviews) for the Obtained media exposure (12 articles/interviews) for the

Consortium and established a public website Consortium and established a public website (http://www.internationalregulomeconsortium.ca)(http://www.internationalregulomeconsortium.ca)

Phase 3 - Ramp-up (Aug 04-Mar 05)Phase 3 - Ramp-up (Aug 04-Mar 05)

• Objectives Objectives – Apply for seed funding to support project management, Apply for seed funding to support project management,

Consortium establishment, proof-of-concept experimentsConsortium establishment, proof-of-concept experiments

– Draft IRC incorporation documentsDraft IRC incorporation documents

– Seek industrial partnerSeek industrial partner

– Apply for major funding through Genome CanadaApply for major funding through Genome Canada

– Apply to Stem Cell Network for hESC research componentApply to Stem Cell Network for hESC research component

– Hold second international workshopHold second international workshop

• Funding Funding – $400K from CIHR and Government of Ontario$400K from CIHR and Government of Ontario

Phase 3 - Ramp-up (Aug 04-Mar 05)Phase 3 - Ramp-up (Aug 04-Mar 05)• Accomplishments Accomplishments

– Application to CIHR "International Opportunity Program – One time Collaborative Research Application to CIHR "International Opportunity Program – One time Collaborative Research Project Grant" completed 3 Mar 05; $200K awarded 18 Mar 05, with the first of 24 monthly Project Grant" completed 3 Mar 05; $200K awarded 18 Mar 05, with the first of 24 monthly installments of $8.3K posted 31 Mar 05 installments of $8.3K posted 31 Mar 05

– $200K in funds committed by Minister Cordiano (Minister, Economic Development and Trade) 27 $200K in funds committed by Minister Cordiano (Minister, Economic Development and Trade) 27 Oct 04 (joint Canada/France IRC funding announcement in Marsailles); application to Oct 04 (joint Canada/France IRC funding announcement in Marsailles); application to Government of Ontario to flow funds submitted 23 Mar 05.Government of Ontario to flow funds submitted 23 Mar 05.

– Incorporation documents finalized and forwarded to Steering Committee for signature 24 May Incorporation documents finalized and forwarded to Steering Committee for signature 24 May 05.05.

– Initiated negotiations with Invitrogen to participate as a research partner (in addition to Initiated negotiations with Invitrogen to participate as a research partner (in addition to significant in-kind benefits to Consortium). Most recent meeting in San Diego, 25 May 05.significant in-kind benefits to Consortium). Most recent meeting in San Diego, 25 May 05.

– "Study of Transcriptional Regulation in hESC" submitted as 2005 SCN Core proposal 29 May 05"Study of Transcriptional Regulation in hESC" submitted as 2005 SCN Core proposal 29 May 05

– Second IRC Workshop hosted by French Steering Committee representative Dr. Irwin Davidson Second IRC Workshop hosted by French Steering Committee representative Dr. Irwin Davidson 30-31 May 0530-31 May 05

– Participated in French IRC application, review is pendingParticipated in French IRC application, review is pending

– Submit Notification of Intent letter, CFI International Joint Ventures Fund 2005Submit Notification of Intent letter, CFI International Joint Ventures Fund 2005

– Opened discussions with Sun Microsystems regarding partnership with IRCagOpened discussions with Sun Microsystems regarding partnership with IRCag

– Greenblatt and Rossant labs optimizes tag for tandem affinity purificationGreenblatt and Rossant labs optimizes tag for tandem affinity purification

Phase 3 - Ramp-up (Aug 04-Mar 05)Phase 3 - Ramp-up (Aug 04-Mar 05)• Accomplishments (continued)Accomplishments (continued)

– International IRC working groups use multiple tools to identify approximately 2,500 International IRC working groups use multiple tools to identify approximately 2,500 DNA-binding transcription factors from genomic sequence dataDNA-binding transcription factors from genomic sequence data

– Rudnicki lab employs recombineering to engineer tag in multiple gene targeting Rudnicki lab employs recombineering to engineer tag in multiple gene targeting constructs in BACsconstructs in BACs

– Rudnicki lab introduces tag into endogenous Oct4 gene in mESC by homologous Rudnicki lab introduces tag into endogenous Oct4 gene in mESC by homologous recombinationrecombination

– Rudnicki employs tandem affinity purification to identify protein components of a Rudnicki employs tandem affinity purification to identify protein components of a tagged TFtagged TF

– Singapore group uses di-tag library approach to sequence binding sites for Oct4, Singapore group uses di-tag library approach to sequence binding sites for Oct4, Sox2 and Nanog.Sox2 and Nanog.

– First Board of Directors Meeting to occur November, 2005First Board of Directors Meeting to occur November, 2005

Phase 4 - Establishment (April 05-Mar 06)Phase 4 - Establishment (April 05-Mar 06)• Funding Funding

– $500K requested from Genome Canada to support this phase$500K requested from Genome Canada to support this phase

• Objectives Objectives – Participate in EU IRC applicationsParticipate in EU IRC applications– Retool/regroup Ontario Genomics Innovation Centre to support IRC activitiesRetool/regroup Ontario Genomics Innovation Centre to support IRC activities– Undertake proof-of-concept experiments (scalability of high-throughput Undertake proof-of-concept experiments (scalability of high-throughput

techniques)techniques)– Apply for Genome Canada International Consortium Initiative fundingApply for Genome Canada International Consortium Initiative funding– Complete IRC incorporation and hold first (interim) Board meetingComplete IRC incorporation and hold first (interim) Board meeting– Independent scientific review, jointly sponsored by Genome Canada and Independent scientific review, jointly sponsored by Genome Canada and

other partnersother partners– Submit application, Ontario Research Fund Research Excellence competitionSubmit application, Ontario Research Fund Research Excellence competition– Submit project outline, CFI International Joint Ventures Fund 2005Submit project outline, CFI International Joint Ventures Fund 2005– Complete development agreement with InvitrogenComplete development agreement with Invitrogen– Complete development agreement with Sun MicrosystemsComplete development agreement with Sun Microsystems

Phase 5 – Implementation (April 06 – Dec 06)Phase 5 – Implementation (April 06 – Dec 06)

• FundingFunding– Up to $1.5M from Genome Canada to implement the Up to $1.5M from Genome Canada to implement the

project.project.

• ObjectivesObjectives– Appoint IRC Board of DirectorsAppoint IRC Board of Directors– Staff and equip the teamStaff and equip the team– Implement high throughput approachesImplement high throughput approaches– Pursue additional partnersPursue additional partners– Apply for CFI International Joint Ventures FundApply for CFI International Joint Ventures Fund– Apply for Ontario Research Fund and other provinial Apply for Ontario Research Fund and other provinial

agenciesagencies– Implement high-throughput methodology.Implement high-throughput methodology.

Phase 6-10- Execution Y1-Y4 (Jan 07 – Dec 10)Phase 6-10- Execution Y1-Y4 (Jan 07 – Dec 10)

• FundingFunding– Up to $5 provided in annual disbursements Up to $5 provided in annual disbursements

– (up to 25% of international contributions)(up to 25% of international contributions)

• ObjectivesObjectives– Conduct experimental/analytical portion of IRC. Conduct experimental/analytical portion of IRC.

– Milestones and deliverables to be negotiated in previous Milestones and deliverables to be negotiated in previous cycle.cycle.

– Progress will be reviewed annually, six months out of Progress will be reviewed annually, six months out of phase with the funding cycle.phase with the funding cycle.

High Throughput Gene TargetingHigh Throughput Gene Targeting

• Recombinational vector generation in BACsRecombinational vector generation in BACs

• TAP-tag and flox up to 2000 genes in miceTAP-tag and flox up to 2000 genes in mice

• ES/embryo aggregationES/embryo aggregation

NeoNeo6His-TEV-3FLAG6His-TEV-3FLAG

FRT SiteFRT Site

Wild-type Oct4 AlleleWild-type Oct4 Allele5.16 kb5.16 kb

Targeted Oct4 AlleleTargeted Oct4 Allele

Targeted Oct4 Allele (after FLP Recombinase)Targeted Oct4 Allele (after FLP Recombinase)

6His-TEV-3FLAG6His-TEV-3FLAG

http://www.ensembl.org/Mus_musculus/transview?transcript=ENSMUST00000025271&db=core

-Flag-Flag

64-64-

49-49-

-Tubulin-Tubulin

C2C1

C2C1

22JJ

11Oct

4CHF#

Oct4C

HF#

1164-64-

49-49--Oct4-Oct4

-Tubulin-Tubulin

C2C1

C2C1

22JJ

11Oct

4CHF#

Oct4C

HF#

11-Oct4-cTAG-Oct4-cTAG

C-TAP Tagging of Oct4C-TAP Tagging of Oct4

Mass SpectroscopyMass Spectroscopy

• Tandem affinity purification of complexesTandem affinity purification of complexes

• LC MS MSLC MS MS

TranscriptionTranscriptionComplexComplex

TAP Tag MethodologyTAP Tag MethodologyTAP Tag MethodologyTAP Tag Methodology

6xHis6xHis6xHis6xHis TEVTEVTEVTEV 3xFLAG3xFLAG3xFLAG3xFLAGN-N-N-N- -C-C-C-C

C-terminal TAP tagC-terminal TAP tagC-terminal TAP tagC-terminal TAP tag

Pax7Pax7Pax7Pax7

Associated proteinsAssociated proteinsAssociated proteinsAssociated proteins

A A – Build construct– Build constructA A – Build construct– Build construct

B B – Express construct in cells– Express construct in cellsB B – Express construct in cells– Express construct in cells

C–C– Purify complex Purify complexC–C– Purify complex Purify complex

Pax7Pax7Pax7Pax7 -FLAG-FLAGM2M2

-FLAG-FLAGM2M2

TEV ProteaseTEV Protease3xFLAG Peptide3xFLAG Peptide

TEV ProteaseTEV Protease3xFLAG Peptide3xFLAG Peptide

EDTA EDTA EDTA EDTA

Pax7Pax7Pax7Pax7

Pax7Pax7Pax7Pax7 NiNi++

resinresinNiNi++

resinresin

Mass Spec of Pax7-cTAP ComplexMass Spec of Pax7-cTAP Complexm

Pax7

d-C

TAP-

lysa

te

mPa

x7d-

CTA

P-ly

sate

mPa

x7d-

CTA

P-IP

mPa

x7d-

CTA

P-IP

FLA

GFL

AG

mPa

x7d-

CTA

P-TE

V-IP

Ni+

resi

n

mPa

x7d-

CTA

P-TE

V-IP

Ni+

resi

n

mPa

x7d-

CTA

P-TE

V

mPa

x7d-

CTA

P-TE

V

Western blotWestern blot-Pax7-Pax7

mPa

x7d-

CTA

P- fi

nal e

luat

e

mPa

x7d-

CTA

P- fi

nal e

luat

e

His

-FLA

G ta

g –

final

elu

ate

His

-FLA

G ta

g –

final

elu

ate

64kD-64kD-82kD-82kD-

115kD-115kD-181kD-181kD-

48kD-48kD-

35kD-35kD-

10kD-10kD-

19kD-19kD-

Octamer binding proteinOctamer binding protein

Wdr5 proteinWdr5 protein

RNA splicing factorRNA splicing factor

Putative CDKPutative CDK

PTB-assoc. splicing factorPTB-assoc. splicing factor

Putative KH-domain Putative KH-domain containing proteincontaining protein

Silver stainSilver stain

BBAA

Pax7Pax7 Lix1Lix1 Rnf30Rnf30 Rik221Rik221

HisFLAG tagHisFLAG tag

mPax7d-CTAPmPax7d-CTAP

NTAP-mPax7dNTAP-mPax7d

Fo

ld A

ctiv

atio

nF

old

Act

ivat

ion

Real Time PCRReal Time PCRReal Time PCRReal Time PCR

Normal Activity of Pax7-CTAPNormal Activity of Pax7-CTAPNormal Activity of Pax7-CTAPNormal Activity of Pax7-CTAP

1.1. LysateLysate2.2. Lysate IP Lysate IP -FLAG -FLAG 3.3. Eluate from Eluate from -FLAG beads -FLAG beads

(3xFLAG peptide+ TEV)(3xFLAG peptide+ TEV)4.4. Eluate from Ni+ resinEluate from Ni+ resin

1.1. LysateLysate2.2. Lysate IP Lysate IP -FLAG -FLAG 3.3. Eluate from Eluate from -FLAG beads -FLAG beads

(3xFLAG peptide+ TEV)(3xFLAG peptide+ TEV)4.4. Eluate from Ni+ resinEluate from Ni+ resin

Purification of mPax7 CTAPPurification of mPax7 CTAPPurification of mPax7 CTAPPurification of mPax7 CTAP

Pax7 Western BlotPax7 Western BlotPax7 Western BlotPax7 Western Blot

1111 2222 3333 4444

mPa

x7d-

CTA

P

mPa

x7d-

CTA

P

mPa

x7d-

CTA

P

mPa

x7d-

CTA

P

His

-FLA

G ta

g

His

-FLA

G ta

g

His

-FLA

G ta

g

His

-FLA

G ta

g

64kD-64kD-64kD-64kD-82kD-82kD-82kD-82kD-

115kD-115kD-115kD-115kD-

181kD-181kD-181kD-181kD-





Mass Spec of Pax7-cTAP ComplexMass Spec of Pax7-cTAP ComplexMass Spec of Pax7-cTAP ComplexMass Spec of Pax7-cTAP Complex

Silver StainSilver StainSilver StainSilver Stain

Pax7 and NonO/p54Pax7 and NonO/p54nrb nrb Pax7 and NonO/p54Pax7 and NonO/p54nrb nrb

Wdr5Wdr5Wdr5Wdr5

PTB-assoc. splicing factor (PSF)PTB-assoc. splicing factor (PSF)PTB-assoc. splicing factor (PSF)PTB-assoc. splicing factor (PSF)

Putative CDK domain proteinPutative CDK domain proteinPutative CDK domain proteinPutative CDK domain protein

Putative KH-domain containing proteinPutative KH-domain containing proteinPutative KH-domain containing proteinPutative KH-domain containing protein

HSP 70HSP 70HSP 70HSP 70

Pax7 Interacting ProteinsPax7 Interacting Proteins

• Pax7-CTAP Pax7-CTAP – Protein phosphatase 1cgamma Protein phosphatase 1cgamma – Gelsolin Gelsolin – Tropomodulin Tropomodulin – Gamma-actinGamma-actin– GAPDHGAPDH– gi|51829802gi|51829802

– PSFPSF– Molecular chaperone HSP70.2/HSP70 Molecular chaperone HSP70.2/HSP70

– NonO/P54nrbNonO/P54nrb – Unnamed Putative CDK domain proteinUnnamed Putative CDK domain protein– Hypothetical protein MGC34648 Hypothetical protein MGC34648

– Wdr5 proteinWdr5 protein – Solute carrier family 25, member 5 Solute carrier family 25, member 5 – Thioredoxin domain containing 4 Thioredoxin domain containing 4

– Pax7Pax7 – gi|12847921 unnamed protein product gi|12847921 unnamed protein product

– Molecular chaperone grp78 precursorMolecular chaperone grp78 precursor

• His-FLAG tagHis-FLAG tag– Protein phosphatase 1cgamma Protein phosphatase 1cgamma

– GelsolinGelsolin

– TropomodulinTropomodulin

– Gamma-actinGamma-actin

– GAPDH GAPDH

– gi|51829802gi|51829802

– Ribosomal protein L27a Ribosomal protein L27a

– Nucleoside-diphosphate kinaseNucleoside-diphosphate kinase

– ADP-ribosylation factor ADP-ribosylation factor

– Fscn1 protein Fscn1 protein

– ActinActin

– Coronin-3 Coronin-3

– Hnrp-L Hnrp-L

– Ckap4 protein Ckap4 protein

Validation of Pax7 InteractionsValidation of Pax7 Interactions

-Pax7-Pax7-Pax7-Pax7 -Wdr5-Wdr5-Wdr5-Wdr5 -NonO-NonO-NonO-NonO -Ash2L-Ash2L-Ash2L-Ash2L

-Erk1/2-Erk1/2-Erk1/2-Erk1/2

-Pax7-Pax7-Pax7-Pax7

-Wdr5-Wdr5-Wdr5-Wdr5

-NonO-NonO-NonO-NonO

-Ash2L-Ash2L-Ash2L-Ash2L

IPIPIPIP

WesternWesternWesternWestern

1111 2222 3333 1111 2222 3333 1111 2222 3333 1111 2222 3333

1111 2222 3333 1111 2222 3333 1111 2222 3333

1111 2222 3333 1111 2222 3333 1111 2222 3333

1111 2222 3333 1111 2222 3333 1111 2222 3333

1111 2222 3333

1.1. LysateLysate2.2. IP Protein GIP Protein G3.3. IP AntibodyIP Antibody

1.1. LysateLysate2.2. IP Protein GIP Protein G3.3. IP AntibodyIP Antibody

The Pax7 Transcriptional ComplexThe Pax7 Transcriptional Complex

• PSF + p54nrb/NonO + HSP 70PSF + p54nrb/NonO + HSP 70 – Multi-functional interacting nuclear proteinsMulti-functional interacting nuclear proteins– Involved in DNA binding and transcription as heterodimerInvolved in DNA binding and transcription as heterodimer– NonO enhances association of DBD to cognate sitesNonO enhances association of DBD to cognate sites

• Ishitani et al., (2003)Ishitani et al., (2003)• Emili et al., (2002)Emili et al., (2002)• Otto et al., (2001)Otto et al., (2001)

• Wdr5 & Ash2LWdr5 & Ash2L

– Components of histone methyltransferase complex with Menin, Components of histone methyltransferase complex with Menin, MLL, RbBP5, HCF1/2MLL, RbBP5, HCF1/2

– Directs H3K4 di- and tri-methylationDirects H3K4 di- and tri-methylation– Marks sites of gene activation (Hox in DM ) Marks sites of gene activation (Hox in DM )

• Yokoyama et al., (2004)Yokoyama et al., (2004)• Milne et al., (2005)Milne et al., (2005)

Identification of Target GenesIdentification of Target Genes

• Tandem Affinity Purification of TF-cTAP bound chromatinTandem Affinity Purification of TF-cTAP bound chromatin

• Di-Tag DNA sequencing of purified TF-binding sitesDi-Tag DNA sequencing of purified TF-binding sites

• Tandem Affinity Purification of TF-cTAP bound chromatinTandem Affinity Purification of TF-cTAP bound chromatin

• Di-Tag DNA sequencing of purified TF-binding sitesDi-Tag DNA sequencing of purified TF-binding sites

Pax7Pax7Pax7Pax7Associated Associated proteinsproteins

Associated Associated proteinsproteins

A A – Express construct in cells– Express construct in cellsA A – Express construct in cells– Express construct in cells

Pax7Pax7Pax7Pax7

Bound genomic DNABound genomic DNABound genomic DNABound genomic DNA

Pax7Pax7Pax7Pax7

B B – Crosslink proteins to DNA – Crosslink proteins to DNA (1% formaldehyde)(1% formaldehyde)B B – Crosslink proteins to DNA – Crosslink proteins to DNA (1% formaldehyde)(1% formaldehyde)

C C – Purify complex – Purify complex C C – Purify complex – Purify complex

-FLAG-FLAG-FLAG-FLAG

TEV ProteaseTEV Protease3xFLAG peptide3xFLAG peptide

TEV ProteaseTEV Protease3xFLAG peptide3xFLAG peptide

Pax7Pax7Pax7Pax7NiNi++

resinresinNiNi++

resinresin

DNA fragmented by sonicationDNA fragmented by sonicationDNA fragmented by sonicationDNA fragmented by sonication

EDTAEDTAEDTAEDTA

DNA PurificationDNA PurificationDNA PurificationDNA Purification

TF Binding sitesTF Binding sitesTF Binding sitesTF Binding sites

Chromatin Tandem Affinity PurificationChromatin Tandem Affinity PurificationChromatin Tandem Affinity PurificationChromatin Tandem Affinity Purification

• Cells fixed with 1% Cells fixed with 1% formaldehyde for 10’ @ r.t.formaldehyde for 10’ @ r.t.

• Lysis in SDS bufferLysis in SDS buffer

• Sonication time: 10sSonication time: 10s

• First IP: First IP: -FLAG (poly)-FLAG (poly)

• Cells fixed with 1% Cells fixed with 1% formaldehyde for 10’ @ r.t.formaldehyde for 10’ @ r.t.

• Lysis in SDS bufferLysis in SDS buffer

• Sonication time: 10sSonication time: 10s

• First IP: First IP: -FLAG (poly)-FLAG (poly)

• Pax7-TAP not recovered following binding to Pax7-TAP not recovered following binding to -FLAG beads!-FLAG beads!

• Residual detection of complex on protein-A beads (not shown)Residual detection of complex on protein-A beads (not shown)

• Formaldehyde interactions with His-tag interfering with antibody recognition of Formaldehyde interactions with His-tag interfering with antibody recognition of FLAG epitope?FLAG epitope?

• Metz et al, 2004 - Metz et al, 2004 - Identification of formaldehyde-induced modifications in Identification of formaldehyde-induced modifications in proteins: reactions with model peptidesproteins: reactions with model peptides

• Pax7-TAP not recovered following binding to Pax7-TAP not recovered following binding to -FLAG beads!-FLAG beads!

• Residual detection of complex on protein-A beads (not shown)Residual detection of complex on protein-A beads (not shown)

• Formaldehyde interactions with His-tag interfering with antibody recognition of Formaldehyde interactions with His-tag interfering with antibody recognition of FLAG epitope?FLAG epitope?

• Metz et al, 2004 - Metz et al, 2004 - Identification of formaldehyde-induced modifications in Identification of formaldehyde-induced modifications in proteins: reactions with model peptidesproteins: reactions with model peptides

Ch-TAP: Standard ChIP ConditionsCh-TAP: Standard ChIP ConditionsCh-TAP: Standard ChIP ConditionsCh-TAP: Standard ChIP Conditions

Standard ChIP: Standard ChIP: -MyoD Antibody-MyoD Antibody

LaneLane FormaldehydeFormaldehyde ChIPChIP

1.1. 1%1% 2.2. 0.1% 0.1% 3.3. 0.01% 0.01%

4.4. 0.001%0.001%

5.5. 0.0001%0.0001%

1111 2222 3333 4444 5555

Myogenin Promoter (150bp PCR product)Myogenin Promoter (150bp PCR product)Myogenin Promoter (150bp PCR product)Myogenin Promoter (150bp PCR product)

ChIP of MyoD-cTAP with ChIP of MyoD-cTAP with -FLAG-FLAG

Myogenin Promoter (150bp PCR product)Myogenin Promoter (150bp PCR product)Myogenin Promoter (150bp PCR product)Myogenin Promoter (150bp PCR product)

•MyoD-6His-TEV-3FLAGMyoD-6His-TEV-3FLAG

•Crosslink 0.1% formaldehydeCrosslink 0.1% formaldehyde

•ChIP with ChIP with -FLAG -FLAG

•Detect E-box in myogenin promoterDetect E-box in myogenin promoter

Ch-TAP: Formaldehyde TitrationCh-TAP: Formaldehyde TitrationCh-TAP: Formaldehyde TitrationCh-TAP: Formaldehyde Titration

• Titration of formaldehyde Titration of formaldehyde concentration reveals ability to IP concentration reveals ability to IP Pax7-cTAPPax7-cTAP

• Pax7 complex is recoverable Pax7 complex is recoverable through TEV cleavage and second through TEV cleavage and second affinity purificationaffinity purification

Ch-TAP: DNA Recovery and AnalysisCh-TAP: DNA Recovery and AnalysisCh-TAP: DNA Recovery and AnalysisCh-TAP: DNA Recovery and Analysis

Input

Input

Input

Input

Input

Input

Ch-TAPCh-TAP •0.1% formaldehyde0.1% formaldehyde•PCR detection of binding sitePCR detection of binding site

Ch-TAP/W

GA

Ch-TAP/W

GA

Ch-TAP/W

GA

Ch-TAP/W

GA

Ch-TAP/W

GA

Ch-TAP/W

GA

WGA WGA •whole genome amplificationwhole genome amplification•PCR libraryPCR library

Transcriptional Regulation in mESCsTranscriptional Regulation in mESCs

Assumptions:

ES EB

selfself--renewalrenewal

totipotencytotipotency

lineage lineage

commitmentcommitment

Hypothesis:

Comparative analysis of ESComparative analysis of ES--EB differentiation timeEB differentiation time--course course data will allow the identification of genes that are data will allow the identification of genes that are important for stem cell identity and lineage commitment.important for stem cell identity and lineage commitment.

Oct4

Assumptions:

ES EB

selfself--renewalrenewal

totipotencytotipotency

lineage lineage

commitmentcommitment

Hypothesis:

Comparative analysis of ESComparative analysis of ES--EB differentiation timeEB differentiation time--course course data will allow the identification of genes that are data will allow the identification of genes that are important for stem cell identity and lineage commitment.important for stem cell identity and lineage commitment.

Oct4Oct4

Pluripotency

What are the transcriptional regulatory networks that What are the transcriptional regulatory networks that guide self-renewal, pluripotency and early lineage guide self-renewal, pluripotency and early lineage

commitment?commitment?

What are the transcriptional regulatory networks that What are the transcriptional regulatory networks that guide self-renewal, pluripotency and early lineage guide self-renewal, pluripotency and early lineage

commitment?commitment?

Differentiation

pluripotencypluripotency

Oct4 Correlation Analysis-RationaleOct4 Correlation Analysis-RationaleOct4 Correlation Analysis-RationaleOct4 Correlation Analysis-Rationale

• Diverse set of data from StemBaseDiverse set of data from StemBase– http://www.scgp.ca:8080/StemBasehttp://www.scgp.ca:8080/StemBase– 45 Stem Cell, Putative Stem Cell, and Differentiated 45 Stem Cell, Putative Stem Cell, and Differentiated

Derivatives were collected in biological triplicate and Derivatives were collected in biological triplicate and analyzed as part of the Stem Cell Genomics Projectanalyzed as part of the Stem Cell Genomics Project

– Affymetrix MOE430 GeneChip SetAffymetrix MOE430 GeneChip Set

• Oct4 used as a ‘marker’ for self-renewal and Oct4 used as a ‘marker’ for self-renewal and pluripotencypluripotency

• Genes highly correlated to Oct4 may lead to the Genes highly correlated to Oct4 may lead to the identification ofidentification of– Genes that play an important role in maintaining ‘ES’Genes that play an important role in maintaining ‘ES’– Oct4 target genesOct4 target genes– Genes that regulate Oct4 expressionGenes that regulate Oct4 expression

• Diverse set of data from StemBaseDiverse set of data from StemBase– http://www.scgp.ca:8080/StemBasehttp://www.scgp.ca:8080/StemBase– 45 Stem Cell, Putative Stem Cell, and Differentiated 45 Stem Cell, Putative Stem Cell, and Differentiated

Derivatives were collected in biological triplicate and Derivatives were collected in biological triplicate and analyzed as part of the Stem Cell Genomics Projectanalyzed as part of the Stem Cell Genomics Project

– Affymetrix MOE430 GeneChip SetAffymetrix MOE430 GeneChip Set

• Oct4 used as a ‘marker’ for self-renewal and Oct4 used as a ‘marker’ for self-renewal and pluripotencypluripotency

• Genes highly correlated to Oct4 may lead to the Genes highly correlated to Oct4 may lead to the identification ofidentification of– Genes that play an important role in maintaining ‘ES’Genes that play an important role in maintaining ‘ES’– Oct4 target genesOct4 target genes– Genes that regulate Oct4 expressionGenes that regulate Oct4 expression

Oct4 Correlation Analysis-MethodOct4 Correlation Analysis-MethodOct4 Correlation Analysis-MethodOct4 Correlation Analysis-Method

• Method to assign statistical significance to genes Method to assign statistical significance to genes that cluster togetherthat cluster together– Pearson Correlation Coefficient (rho) calculated for all Pearson Correlation Coefficient (rho) calculated for all

probesets to Oct4 probeset (|rho| ≥ 0.75 cutoff)probesets to Oct4 probeset (|rho| ≥ 0.75 cutoff)

– 10,000 iterative random resamplings including 65-70% of 10,000 iterative random resamplings including 65-70% of the data were performedthe data were performed

– Probesets included on the list pass the rho cutoff in at Probesets included on the list pass the rho cutoff in at least 40% of the trialsleast 40% of the trials

– Computation time is about 3 days depending on computerComputation time is about 3 days depending on computer

• Method to assign statistical significance to genes Method to assign statistical significance to genes that cluster togetherthat cluster together– Pearson Correlation Coefficient (rho) calculated for all Pearson Correlation Coefficient (rho) calculated for all

probesets to Oct4 probeset (|rho| ≥ 0.75 cutoff)probesets to Oct4 probeset (|rho| ≥ 0.75 cutoff)

– 10,000 iterative random resamplings including 65-70% of 10,000 iterative random resamplings including 65-70% of the data were performedthe data were performed

– Probesets included on the list pass the rho cutoff in at Probesets included on the list pass the rho cutoff in at least 40% of the trialsleast 40% of the trials

– Computation time is about 3 days depending on computerComputation time is about 3 days depending on computer

Correlation Analysis-Results and ValidationCorrelation Analysis-Results and ValidationCorrelation Analysis-Results and ValidationCorrelation Analysis-Results and Validation

• ResultsResults– 74 probesets negatively correlated74 probesets negatively correlated– 1225 probesets positively correlated1225 probesets positively correlated

• Correlations of known Oct4 target genesCorrelations of known Oct4 target genes– UTF1UTF1 100%100%– FGF4FGF4 99% 99%– NanogNanog 97% 97%– Sox2Sox2 49% 49%

• Sox2 correlation is lower because expression is not Sox2 correlation is lower because expression is not restricted to ESrestricted to ES

• Absence of lineage committed genesAbsence of lineage committed genes• Limitation of MethodLimitation of Method

– Relevant genes that have non-ES restricted expression may Relevant genes that have non-ES restricted expression may be excluded due to stringency of cutoffsbe excluded due to stringency of cutoffs

• ResultsResults– 74 probesets negatively correlated74 probesets negatively correlated– 1225 probesets positively correlated1225 probesets positively correlated

• Correlations of known Oct4 target genesCorrelations of known Oct4 target genes– UTF1UTF1 100%100%– FGF4FGF4 99% 99%– NanogNanog 97% 97%– Sox2Sox2 49% 49%

• Sox2 correlation is lower because expression is not Sox2 correlation is lower because expression is not restricted to ESrestricted to ES

• Absence of lineage committed genesAbsence of lineage committed genes• Limitation of MethodLimitation of Method

– Relevant genes that have non-ES restricted expression may Relevant genes that have non-ES restricted expression may be excluded due to stringency of cutoffsbe excluded due to stringency of cutoffs

GeneGene CorrelationCorrelation

Nanog +99%Nanog +99%

Sox2 +49%Sox2 +49%

Tdrd7 -95%Tdrd7 -95%

Mef2a -49%Mef2a -49%

Myog 0%Myog 0%

GeneGene CorrelationCorrelation

Nanog +99%Nanog +99%

Sox2 +49%Sox2 +49%

Tdrd7 -95%Tdrd7 -95%

Mef2a -49%Mef2a -49%

Myog 0%Myog 0%

11

1010

100100

10001000

1000010000

100000100000

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Sox2Sox2Oct4 (P/A)Oct4 (P/A) MyogMyog Tdrd7Tdrd7NanogNanog Mef2aMef2a

ECECECEC mESCmESCmESCmESC

HematopoieticHematopoieticHematopoieticHematopoieticNeuronalNeuronalNeuronalNeuronal

RetinalRetinalRetinalRetinalMMuscuscleleMMuscusclele

EpithelialEpithelialEpithelialEpithelialAdiposeAdiposeAdiposeAdipose

DifferentiatedDifferentiatedDifferentiatedDifferentiatedmESCmESCmESCmESC

Sig

na

l In

ten

sit

yS

ign

al

Inte

ns

ity

transcriptional regulation

125

intracellular signalling

54protein

modification27

protein biosynthesis

21

apoptosis25

cell cycle39

chromatin35

DNA repair30

unknown304

ubiquitination24

tRNA processing20

transport29

DNA replication22

metabolism35

mitochondria19

mRNA splicing46

nucleic acid binding

31

transcriptional regulation

125


54protein

modification27

protein biosynthesis

21

apoptosis25

cell cycle39

chromatin35

DNA repair30

unknown304

ubiquitination24

tRNA processing20

transport29

DNA replication22

metabolism35

mitochondria19

mRNA splicing46

nucleic acid binding

31

AAAA

protein modification

11


4 transcriptional regulation

9

transport7

unknown16

apoptosis4

chromatin3

protein modification

11


4 transcriptional regulation

9

transport7

unknown16

apoptosis4

chromatin3

BBBB

Positively Correlated GenesPositively Correlated GenesPositively Correlated GenesPositively Correlated Genes Negatively CorrelatedNegatively Correlated GenesGenesNegatively CorrelatedNegatively Correlated GenesGenesUnknownUnknown

Transcriptional regulationTranscriptional regulation

Intracellular signalingIntracellular signaling

mRNA splicingmRNA splicing

Cell cycleCell cycle

ChromatinChromatin

UnknownUnknown



mRNA splicingmRNA splicing

Cell cycleCell cycle

ChromatinChromatin

UnknownUnknown


Protein modificationProtein modification

TransportTransport


ApoptosisApoptosis

UnknownUnknown


Protein modificationProtein modification

TransportTransport


ApoptosisApoptosis

Highly Represented Gene Ontology CategoriesHighly Represented Gene Ontology CategoriesHighly Represented Gene Ontology CategoriesHighly Represented Gene Ontology Categories

Binding Site Analysis of Oct4 Correlated GenesBinding Site Analysis of Oct4 Correlated GenesBinding Site Analysis of Oct4 Correlated GenesBinding Site Analysis of Oct4 Correlated Genes

• Putative binding sites selected by the presence of Putative binding sites selected by the presence of neighboring Oct4 and Sox2 binding sites scanning -neighboring Oct4 and Sox2 binding sites scanning -2kb from the TSS to +2kb from the 3’ end of the 2kb from the TSS to +2kb from the 3’ end of the transcripttranscript

• 370 Genes with at least one adjacent Oct4/Sox2 site370 Genes with at least one adjacent Oct4/Sox2 site

• A subset of these sites were evaluated as direct A subset of these sites were evaluated as direct Oct4 transcriptional targets by Chromatin Oct4 transcriptional targets by Chromatin Immunoprecipitation (ChIP) followed by Real-time Immunoprecipitation (ChIP) followed by Real-time PCR for analysis of occupancy at specific lociPCR for analysis of occupancy at specific loci

• Putative binding sites selected by the presence of Putative binding sites selected by the presence of neighboring Oct4 and Sox2 binding sites scanning -neighboring Oct4 and Sox2 binding sites scanning -2kb from the TSS to +2kb from the 3’ end of the 2kb from the TSS to +2kb from the 3’ end of the transcripttranscript

• 370 Genes with at least one adjacent Oct4/Sox2 site370 Genes with at least one adjacent Oct4/Sox2 site

• A subset of these sites were evaluated as direct A subset of these sites were evaluated as direct Oct4 transcriptional targets by Chromatin Oct4 transcriptional targets by Chromatin Immunoprecipitation (ChIP) followed by Real-time Immunoprecipitation (ChIP) followed by Real-time PCR for analysis of occupancy at specific lociPCR for analysis of occupancy at specific loci

Oct4 ChIP Real-time PCR AnalysisOct4 ChIP Real-time PCR AnalysisOct4 ChIP Real-time PCR AnalysisOct4 ChIP Real-time PCR Analysis

• Duplicate ChIP experimetsDuplicate ChIP experimets• Duplicate PCRs from each ChIPDuplicate PCRs from each ChIP• PCR on 10-fold serial dilutions of input DNA were PCR on 10-fold serial dilutions of input DNA were

performed for a subset of amplicons performed for a subset of amplicons • ΔCt values of ~3.3 were obtainedΔCt values of ~3.3 were obtained• A change in Ct of 1 represents an approximate population A change in Ct of 1 represents an approximate population

doublingdoubling• 2-fold enrichment used as cutoff for identification of 2-fold enrichment used as cutoff for identification of

bound targetbound target• Fold enrichment calculated by the ΔΔ Ct method:Fold enrichment calculated by the ΔΔ Ct method:

• Amplicons were sequence validatedAmplicons were sequence validated

• Duplicate ChIP experimetsDuplicate ChIP experimets• Duplicate PCRs from each ChIPDuplicate PCRs from each ChIP• PCR on 10-fold serial dilutions of input DNA were PCR on 10-fold serial dilutions of input DNA were

performed for a subset of amplicons performed for a subset of amplicons • ΔCt values of ~3.3 were obtainedΔCt values of ~3.3 were obtained• A change in Ct of 1 represents an approximate population A change in Ct of 1 represents an approximate population

doublingdoubling• 2-fold enrichment used as cutoff for identification of 2-fold enrichment used as cutoff for identification of

bound targetbound target• Fold enrichment calculated by the ΔΔ Ct method:Fold enrichment calculated by the ΔΔ Ct method:

• Amplicons were sequence validatedAmplicons were sequence validatedFold Enrichment = 2Fold Enrichment = 2 [Ct (–Ab) – Ct (input)] – [Ct (+Ab) – Ct (input)] [Ct (–Ab) – Ct (input)] – [Ct (+Ab) – Ct (input)]Fold Enrichment = 2Fold Enrichment = 2 [Ct (–Ab) – Ct (input)] – [Ct (+Ab) – Ct (input)] [Ct (–Ab) – Ct (input)] – [Ct (+Ab) – Ct (input)]

02468

10121416182022242628

*8L16RikCcne1 Myog

Fold Enrichment

02468

10121416182022242628

*8L16RikCcne1 Myog

Fold Enrichment

02468

10121416182022242628

Phc3Hoxb1Bmi1

Sh3glb1Tdrd7Mef2aCasp6

Fold Enrichment

02468

10121416182022242628

Phc3Hoxb1Bmi1

Sh3glb1Tdrd7Mef2aCasp6

Fold Enrichment

02468

10121416182022242628

Fgf4 O/S

Utf1

Nanog O/S

Phc1Jarid2Hsf2bpParp1

D14Abb1e

Aqr CcnfSall4Igf2bp1

TdhRestTrp53NanogShmt1Ash2l

Rnf134Phb

Brca1Tcf4Rara

Fold Enrichment

02468

10121416182022242628

Fgf4 O/S

Utf1

Nanog O/S

Phc1Jarid2Hsf2bpParp1

D14Abb1e

Aqr CcnfSall4Igf2bp1

TdhRestTrp53NanogShmt1Ash2l

Rnf134Phb

Brca1Tcf4Rara

Fold Enrichment

AA

BB CC

Positively Correlated TargetsPositively Correlated TargetsPositively Correlated TargetsPositively Correlated Targets

Negatively Correlated TargetsNegatively Correlated TargetsNegatively Correlated TargetsNegatively Correlated Targets Negative ControlsNegative ControlsNegative ControlsNegative Controls

Known Oct TargetsKnown Oct TargetsKnown Oct TargetsKnown Oct Targets

Polycomb andPolycomb and

Trithorax GroupTrithorax Group

GenesGenes

Polycomb andPolycomb and

Trithorax GroupTrithorax Group

GenesGenes

DNA RepairDNA RepairDNA RepairDNA Repair

Cell CycleCell CycleCell CycleCell Cycle

ApoptosisApoptosisApoptosisApoptosis

Oct4 ChIP Real-time PCR AnalysisOct4 ChIP Real-time PCR AnalysisOct4 ChIP Real-time PCR AnalysisOct4 ChIP Real-time PCR Analysis

Oct4

Self-renewal and Pluripotency

Polycomb Group

Ctbp2

Phc1

Rnf134

Suz12Phc3

Bmi1

Sh3glb1

Nr3c1

Casp6Pdx41

Pdcd7 Siva

Thap1

Api5Caipin1

Bag4

Eif4bp1

Aatf1

Cadh1

Anti-Apoptosis

Trithorax Group

Hcfc1

Ash2lAsh1l

Smarcc1

Whsc2

Chromatin Domainsand

Nuclear ArchitecturePml

CoilNcl

Gemin4

Pum1

Gemin5

Mep50

Nup54

Ccnf nMyc1

Igf2bp1 Ccne1

Cell Cycle

Nipp1 Ccnb1

Ccnb2

Ccna2

Cdc25a

D14abb1e

Jarid1b

Fancd2

DNA Repair

Parp1

Trp53Brca1

Mre11a

Rad51

Chek1 Blm

Msh2

Tdrd7

Bmi1Bmi1Bmi1Bmi1Phc3Phc3Phc3Phc3

Igf2bp1Igf2bp1Igf2bp1Igf2bp1

pRbpRbpRbpRb Ccnf Ccnf Ccnf Ccnf

Jarid1b Jarid1b Jarid1b Jarid1b

D14Abb1eD14Abb1eD14Abb1eD14Abb1e

Ash2lAsh2lAsh2lAsh2l Phc1Phc1Phc1Phc1

Mef2a Mef2a Mef2a Mef2a

Jarid2Jarid2Jarid2Jarid2 Sall4Sall4Sall4Sall4

Rnf134Rnf134Rnf134Rnf134

RestRestRestRest

Brca1Brca1Brca1Brca1Tdrd7Tdrd7Tdrd7Tdrd7

Parp1Parp1Parp1Parp1

PhbPhbPhbPhb

Trp53Trp53Trp53Trp53

Rara Rara Rara Rara

Tdh Tdh Tdh Tdh

Sh3glb1Sh3glb1Sh3glb1Sh3glb1

Casp6Casp6Casp6Casp6

Hoxb1Hoxb1Hoxb1Hoxb1

Nipp1 Nipp1 Nipp1 Nipp1

MesodermMesoderm MesodermMesoderm Ectoderm Ectoderm Ectoderm Ectoderm

Aqr Aqr Aqr Aqr

Chromatin StructureChromatin StructureChromatin StructureChromatin Structure

ApoptosisApoptosisApoptosisApoptosis

DNA RepairDNA RepairDNA RepairDNA Repair

Cell Cycle Cell Cycle Cell Cycle Cell Cycle

Oct4 Oct4 Oct4 Oct4

repressiverepressivechromatinchromatin


permissivepermissivechromatinchromatin


anti-anti-apoptoticapoptotic

anti-anti-apoptoticapoptotic

pro-pro- apoptoticapoptotic


checkpointcheckpointcontrolcontrol


DNADNArepairrepairDNADNA

repairrepair

anti-anti-differentiationdifferentiation


pro-pro-differentiationdifferentiation


inactive-inactive-pRbpRb

inactive-inactive-pRbpRb

active-active-pRbpRb


BBBB





anti-anti- apoptoticapoptotic

anti-anti- apoptoticapoptotic







DNADNArereppairairDNADNA

rereppairair


checkpointcheckpointcontrolcontrol inactive-inactive-

pRbpRbinactive-inactive-

pRbpRb



CCCC

Self-Renewal and PluripotencySelf-Renewal and PluripotencySelf-Renewal and PluripotencySelf-Renewal and Pluripotency

DifferentiationDifferentiationDifferentiationDifferentiation

Core Transcriptional Regulatory CircuitryCore Transcriptional Regulatory Circuitryin Human Embryonic Stem Cellsin Human Embryonic Stem CellsBoyer et al. Cell 122, 1 (2005)Boyer et al. Cell 122, 1 (2005)

Core Transcriptional Regulatory CircuitryCore Transcriptional Regulatory Circuitryin Human Embryonic Stem Cellsin Human Embryonic Stem CellsBoyer et al. Cell 122, 1 (2005)Boyer et al. Cell 122, 1 (2005)

• Identification of Oct4, Sox2, and Nanog Identification of Oct4, Sox2, and Nanog transcriptional targets by location analysistranscriptional targets by location analysis

• -8 kb to +2 kb from TSS of 17,917 annotated genes-8 kb to +2 kb from TSS of 17,917 annotated genes

• No binding site analysis (direct or indirect binding?)No binding site analysis (direct or indirect binding?)

• Substantial overlap with Oct4 Correlated genelistSubstantial overlap with Oct4 Correlated genelist

• Hoxb1, Tcf4, Jarid2, Rest, Bmi1 confirmed targetsHoxb1, Tcf4, Jarid2, Rest, Bmi1 confirmed targets

• No insight into mechanisms of gene activation or No insight into mechanisms of gene activation or repressionrepression

• Identification of Oct4, Sox2, and Nanog Identification of Oct4, Sox2, and Nanog transcriptional targets by location analysistranscriptional targets by location analysis

• -8 kb to +2 kb from TSS of 17,917 annotated genes-8 kb to +2 kb from TSS of 17,917 annotated genes

• No binding site analysis (direct or indirect binding?)No binding site analysis (direct or indirect binding?)

• Substantial overlap with Oct4 Correlated genelistSubstantial overlap with Oct4 Correlated genelist

• Hoxb1, Tcf4, Jarid2, Rest, Bmi1 confirmed targetsHoxb1, Tcf4, Jarid2, Rest, Bmi1 confirmed targets

• No insight into mechanisms of gene activation or No insight into mechanisms of gene activation or repressionrepression

623 623

12711271

16871687

Key Regulators are able to Both Activate and Key Regulators are able to Both Activate and Repress Gene ExpressionRepress Gene ExpressionKey Regulators are able to Both Activate and Key Regulators are able to Both Activate and Repress Gene ExpressionRepress Gene Expression

What factors are responsible for mediating gene activation/repression?What factors are responsible for mediating gene activation/repression?Cofactor Recruitment?Cofactor Recruitment?

Cis-regulatory Elements?Cis-regulatory Elements?Chromatin Structure?Chromatin Structure?

Combination of the above?Combination of the above?

What factors are responsible for mediating gene activation/repression?What factors are responsible for mediating gene activation/repression?Cofactor Recruitment?Cofactor Recruitment?

Cis-regulatory Elements?Cis-regulatory Elements?Chromatin Structure?Chromatin Structure?

Combination of the above?Combination of the above?

Oct4Oct4 Sox2Sox2 NanogNanog

Oct4Oct4 NanogNanog Sox2Sox2 NanogNanog

NanogNanogSox2Sox2Oct4Oct4

Oct4 Oct4 Sox2Sox2

Phc3Phc3Trp53Trp53 MynnMynn Six2Six2 Barx2Barx2Crsp3Crsp3

Ncoa1Ncoa1Pak1Pak1 Cadh1Cadh1 Etv3Etv3 Bclaf1Bclaf1 Etv5Etv5

Isl1, Myf5Isl1, Myf5Oct4Oct4

Transcript not expressed

Transcript not expressed

Transcript expressedTranscript expressed

Documents

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