internationalregulomeconsortium
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Transcript of internationalregulomeconsortium
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
-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-
48kD-48kD-48kD-48kD-
35kD-35kD-35kD-35kD-
10kD-10kD-10kD-10kD-
19kD-19kD-19kD-19kD-
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
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
AAAA
protein modification
11
intracellular signalling
4 transcriptional regulation
9
transport7
unknown16
apoptosis4
chromatin3
protein modification
11
intracellular signalling
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
Transcriptional regulationTranscriptional regulation
Intracellular signalingIntracellular signaling
mRNA splicingmRNA splicing
Cell cycleCell cycle
ChromatinChromatin
UnknownUnknown
Transcriptional regulationTranscriptional regulation
Protein modificationProtein modification
TransportTransport
Intracellular signalingIntracellular signaling
ApoptosisApoptosis
UnknownUnknown
Transcriptional regulationTranscriptional regulation
Protein modificationProtein modification
TransportTransport
Intracellular signalingIntracellular signaling
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
repressiverepressivechromatinchromatin
permissivepermissivechromatinchromatin
permissivepermissivechromatinchromatin
anti-anti-apoptoticapoptotic
anti-anti-apoptoticapoptotic
pro-pro- apoptoticapoptotic
pro-pro- apoptoticapoptotic
checkpointcheckpointcontrolcontrol
checkpointcheckpointcontrolcontrol
DNADNArepairrepairDNADNA
repairrepair
anti-anti-differentiationdifferentiation
anti-anti-differentiationdifferentiation
pro-pro-differentiationdifferentiation
pro-pro-differentiationdifferentiation
inactive-inactive-pRbpRb
inactive-inactive-pRbpRb
active-active-pRbpRb
active-active-pRbpRb
BBBB
repressiverepressivechromatinchromatin
repressiverepressivechromatinchromatin
permissivepermissivechromatinchromatin
permissivepermissivechromatinchromatin
anti-anti- apoptoticapoptotic
anti-anti- apoptoticapoptotic
pro-pro- apoptoticapoptotic
pro-pro- apoptoticapoptotic
anti-anti-differentiationdifferentiation
anti-anti-differentiationdifferentiation
pro-pro-differentiationdifferentiation
pro-pro-differentiationdifferentiation
DNADNArereppairairDNADNA
rereppairair
checkpointcheckpointcontrolcontrol
checkpointcheckpointcontrolcontrol inactive-inactive-
pRbpRbinactive-inactive-
pRbpRb
active-active-pRbpRb
active-active-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