internationalregulomeconsortium

1. www.internationalregulomeconsortium.ca

3. Management Structure

4. Phase 1 - Genesis - Jul 03-Jan 04 • Objective • Consult with scientific, management and political resources in order to ascertain viability of large-scale international regulome project • Funding • Nil; travel and workshop expenses (<$10K) absorbed by several existing Rudnicki grants • Accomplishments • Obtained support of Ontario Ministry of Economic Development and Trade (MEDT) and France's Research Directorate • Established international steering committee (initially, Canada and France)

5. Phase 2 - Initiation (Feb-Jul 04) • Objectives • Register project with Genome Canada as an International Consortium Initiative, submission of LOI • Establish International Regulome Consortium • Extend International Steering Committee membership • Publish project whitepaper • Generate interest in the scientific community and general public • Funding • Total of $149,600 in support of 1st International Workshop and follow-up travel and project management, sourced from Genome Canada, Ontario Genomics Institute, CIHR, MEDT, Ottawa Life Sciences Council, Embassies (UK, France, Italy) and private industry (Sun, Invitrogen, Affymetrix)

6. Phase 2 - Initiation (Feb-Jul 04) • Accomplishments • Letter of Intent submitted 18 Feb 04 • Held 1st International Workshop, attended by 75 world-class scientists from six nations, as well as representatives of the private sector, granting agencies, and embassies and officially struck the consortium on 5 May 04 • Expanded Steering Committee membership to include the UK, Netherlands, Singapore and Australia. Other nations expressing interest in scientific participation include the US, Italy, and Germany • Published Ottawa White Paper 5 Jul 04 • Obtained media exposure (12 articles/interviews) for the Consortium and established a public website (http://www.internationalregulomeconsortium.ca)

7. Phase 3 - Ramp-up (Aug 04-Mar 05) • Objectives • Apply for seed funding to support project management, Consortium establishment, proof-of-concept experiments • Draft IRC incorporation documents • Seek industrial partner • Apply for major funding through Genome Canada • Apply to Stem Cell Network for hESC research component • Hold second international workshop • Funding • $400K from CIHR and Government of Ontario

8. Phase 3 - Ramp-up (Aug 04-Mar 05) • Accomplishments • 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 installments of $8.3K posted 31 Mar 05 • $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 Government of Ontario to flow funds submitted 23 Mar 05. • Incorporation documents finalized and forwarded to Steering Committee for signature 24 May 05. • 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. • "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 30-31 May 05 • Participated in French IRC application, review is pending • Submit Notification of Intent letter, CFI International Joint Ventures Fund 2005 • Opened discussions with Sun Microsystems regarding partnership with IRCag • Greenblatt and Rossant labs optimizes tag for tandem affinity purification

9. Phase 3 - Ramp-up (Aug 04-Mar 05) • Accomplishments (continued) • International IRC working groups use multiple tools to identify approximately 2,500 DNA-binding transcription factors from genomic sequence data • Rudnicki lab employs recombineering to engineer tag in multiple gene targeting constructs in BACs • Rudnicki lab introduces tag into endogenous Oct4 gene in mESC by homologous recombination • Rudnicki employs tandem affinity purification to identify protein components of a tagged TF • Singapore group uses di-tag library approach to sequence binding sites for Oct4, Sox2 and Nanog. • First Board of Directors Meeting to occur November, 2005

10. Phase 4 - Establishment (April 05-Mar 06) • Funding • $500K requested from Genome Canada to support this phase • Objectives • Participate in EU IRC applications • Retool/regroup Ontario Genomics Innovation Centre to support IRC activities • Undertake proof-of-concept experiments (scalability of high-throughput techniques) • Apply for Genome Canada International Consortium Initiative funding • Complete IRC incorporation and hold first (interim) Board meeting • Independent scientific review, jointly sponsored by Genome Canada and other partners • Submit application, Ontario Research Fund Research Excellence competition • Submit project outline, CFI International Joint Ventures Fund 2005 • Complete development agreement with Invitrogen • Complete development agreement with Sun Microsystems

11. Phase 5 – Implementation (April 06 – Dec 06) • Funding • Up to $1.5M from Genome Canada to implement the project. • Objectives • Appoint IRC Board of Directors • Staff and equip the team • Implement high throughput approaches • Pursue additional partners • Apply for CFI International Joint Ventures Fund • Apply for Ontario Research Fund and other provinial agencies • Implement high-throughput methodology.

12. Phase 6-10- Execution Y1-Y4 (Jan 07 – Dec 10) • Funding • Up to $5 provided in annual disbursements • (up to 25% of international contributions) • Objectives • Conduct experimental/analytical portion of IRC. • Milestones and deliverables to be negotiated in previous cycle. • Progress will be reviewed annually, six months out of phase with the funding cycle.

13. High Throughput Gene Targeting • Recombinational vector generation in BACs • TAP-tag and flox up to 2000 genes in mice • ES/embryo aggregation

14. Wild-type Oct4 Allele 5.16 kb Targeted Oct4 Allele 6His-TEV-3FLAG Neo Targeted Oct4 Allele (after FLP Recombinase) 6His-TEV-3FLAG FRT Site

15. Oct4CHF#1 Oct4CHF#1 C2C12 C2C12 J1 J1 64- 64- -Oct4-cTAG 49- 49- -Oct4 -Flag -Tubulin -Tubulin C-TAP Tagging of Oct4

16. Mass Spectroscopy • Tandem affinity purification of complexes • LC MS MS Transcription Complex

17. a-FLAG M2 Associated proteins Ni+ resin Pax7 Pax7 Pax7 Pax7 TAP Tag Methodology A – Build construct C– Purify complex C-terminal TAP tag N- -C 6xHis TEV 3xFLAG TEV Protease 3xFLAG Peptide B – Express construct in cells EDTA

18. Mass Spec of Pax7-cTAP Complex mPax7d-CTAP- final eluate His-FLAG tag – final eluate A B mPax7d-CTAP-TEV-IP Ni+resin mPax7d-CTAP-IPaFLAG mPax7d-CTAP-lysate mPax7d-CTAP-TEV 181kD- RNA splicing factor 115kD- 82kD- Octamer binding protein 64kD- PTB-assoc. splicing factor 48kD- Putative CDK 35kD- Wdr5 protein Western blot a-Pax7 Putative KH-domain containing protein 19kD- 10kD- Silver stain

19. HisFLAG tag mPax7d-CTAP NTAP-mPax7d Fold Activation Pax7 Lix1 Rnf30 Rik221 Normal Activity of Pax7-CTAP Real Time PCR

20. 1 2 3 4 Pax7 Western Blot Purification of mPax7 CTAP • Lysate • Lysate IP -FLAG • Eluate from -FLAG beads (3xFLAG peptide+ TEV) • Eluate from Ni+ resin

21. PTB-assoc. splicing factor (PSF) HSP 70 Pax7 and NonO/p54nrb Putative CDK domain protein Wdr5 Putative KH-domain containing protein Mass Spec of Pax7-cTAP Complex mPax7d-CTAP His-FLAG tag 181kD- 115kD- 82kD- 64kD- 48kD- 35kD- 19kD- 10kD- Silver Stain

22. Pax7-CTAP Protein phosphatase 1cgamma Gelsolin Tropomodulin Gamma-actin GAPDH gi|51829802 PSF Molecular chaperone HSP70.2/HSP70 NonO/P54nrb Unnamed Putative CDK domain protein Hypothetical protein MGC34648 Wdr5 protein Solute carrier family 25, member 5 Thioredoxin domain containing 4 Pax7 gi|12847921 unnamed protein product Molecular chaperone grp78 precursor His-FLAG tag Protein phosphatase 1cgamma Gelsolin Tropomodulin Gamma-actin GAPDH gi|51829802 Ribosomal protein L27a Nucleoside-diphosphate kinase ADP-ribosylation factor Fscn1 protein Actin Coronin-3 Hnrp-L Ckap4 protein Pax7 Interacting Proteins

23. IP -Pax7 -Wdr5 -NonO -Ash2L Western -Pax7 1 2 3 1 2 3 1 2 3 1 2 3 -Wdr5 1 2 3 1 2 3 1 2 3 -NonO 1 2 3 1 2 3 1 2 3 -Ash2L 1 2 3 1 2 3 1 2 3 -Erk1/2 1 2 3 Validation of Pax7 Interactions Lysate IP Protein G IP Antibody

24. The Pax7 Transcriptional Complex • PSF + p54nrb/NonO + HSP 70 • Multi-functional interacting nuclear proteins • Involved in DNA binding and transcription as heterodimer • NonO enhances association of DBD to cognate sites • Ishitani et al., (2003) • Emili et al., (2002) • Otto et al., (2001) • Wdr5 & Ash2L • Components of histone methyltransferase complex with Menin, MLL, RbBP5, HCF1/2 • Directs H3K4 di- and tri-methylation • Marks sites of gene activation (Hox in DM ) • Yokoyama et al., (2004) • Milne et al., (2005)

25. Identification of Target Genes • Tandem Affinity Purification of TF-cTAP bound chromatin • Di-Tag DNA sequencing of purified TF-binding sites

26. Pax7 Pax7 Pax7 Pax7 -FLAG Chromatin Tandem Affinity Purification A – Express construct in cells C – Purify complex Associated proteins TEV Protease 3xFLAG peptide Bound genomic DNA Ni+ resin B – Crosslink proteins to DNA (1% formaldehyde) EDTA DNA Purification DNA fragmented by sonication TF Binding sites

27. Cells fixed with 1% formaldehyde for 10’ @ r.t. Lysis in SDS buffer Sonication time: 10s First IP: -FLAG (poly) Ch-TAP: Standard ChIP Conditions • Pax7-TAP not recovered following binding to -FLAG beads! • Residual detection of complex on protein-A beads (not shown) • Formaldehyde interactions with His-tag interfering with antibody recognition of FLAG epitope? • Metz et al, 2004 - Identification of formaldehyde-induced modifications in proteins: reactions with model peptides

28. Standard ChIP: -MyoD Antibody Lane Formaldehyde ChIP 1. 1%  2. 0.1%  3. 0.01%  4. 0.001%  5. 0.0001%  Myogenin Promoter (150bp PCR product) 1 2 3 4 5

29. ChIP of MyoD-cTAP with -FLAG • MyoD-6His-TEV-3FLAG • Crosslink 0.1% formaldehyde • ChIP with -FLAG • Detect E-box in myogenin promoter Myogenin Promoter (150bp PCR product)

30. Ch-TAP: Formaldehyde Titration • Titration of formaldehyde concentration reveals ability to IP Pax7-cTAP • Pax7 complex is recoverable through TEV cleavage and second affinity purification

31. Ch-TAP: DNA Recovery and Analysis WGA • whole genome amplification • PCR library Ch-TAP • 0.1% formaldehyde • PCR detection of binding site Input Input Input Ch-TAP/WGA Ch-TAP/WGA Ch-TAP/WGA

32. Transcriptional Regulation in mESCs Differentiation pluripotency Pluripotency What are the transcriptional regulatory networks that guide self-renewal, pluripotency and early lineage commitment?

33. Oct4 Correlation Analysis-Rationale • Diverse set of data from StemBase • http://www.scgp.ca:8080/StemBase • 45 Stem Cell, Putative Stem Cell, and Differentiated Derivatives were collected in biological triplicate and analyzed as part of the Stem Cell Genomics Project • Affymetrix MOE430 GeneChip Set • Oct4 used as a ‘marker’ for self-renewal and pluripotency • Genes highly correlated to Oct4 may lead to the identification of • Genes that play an important role in maintaining ‘ES’ • Oct4 target genes • Genes that regulate Oct4 expression

34. Oct4 Correlation Analysis-Method • Method to assign statistical significance to genes that cluster together • Pearson Correlation Coefficient (rho) calculated for all probesets to Oct4 probeset (|rho| ≥ 0.75 cutoff) • 10,000 iterative random resamplings including 65-70% of the data were performed • Probesets included on the list pass the rho cutoff in at least 40% of the trials • Computation time is about 3 days depending on computer

35. Correlation Analysis-Results and Validation • Results • 74 probesets negatively correlated • 1225 probesets positively correlated • Correlations of known Oct4 target genes • UTF1 100% • FGF4 99% • Nanog 97% • Sox2 49% • Sox2 correlation is lower because expression is not restricted to ES • Absence of lineage committed genes • Limitation of Method • Relevant genes that have non-ES restricted expression may be excluded due to stringency of cutoffs

36. Myog Tdrd7 Nanog Mef2a Sox2 100000 GeneCorrelation Nanog +99% Sox2 +49% Tdrd7 -95% Mef2a -49% Myog 0% 10000 1000 Signal Intensity 100 10 Oct4 (P/A) 1 J1_0h R1_0h Neural Dermis Adipose V6.5_0h Myoblasts Myospheres Bone_marrow Bone_marrow Bone_marrow Bone_marrow Bone_marrow Bone_marrow Bone_marrow Bone_marrow Bone_marrow Neurospheres Neurospheres Embryonic_J1 R1_Serum6473 R1_Serum6999 Mammospheres Retinal_primary Embryonic_D4E Embryonic_D4D Embryonic_C2D Embryonic_C2A Embryonic_D4A Embryonic_C2E Embryoid_bodies Embryoid_bodies Embryoid_bodies Cancer_embryonic Undiff_Mammosphere Retinal_first_passage Muscle_derived_stem_cell Muscle_derived_stem_cell D3 Osteoblast Differentiation Bone_marrow_Mast_precursors Bone_marrow_Mast_precursors EC mESC Muscle Retinal Differentiated Adipose Neuronal mESC Hematopoietic Epithelial

37. B Highly Represented Gene Ontology Categories A Positively Correlated Genes Negatively CorrelatedGenes Unknown Transcriptional regulation Intracellular signaling mRNA splicing Cell cycle Chromatin Unknown Transcriptional regulation Protein modification Transport Intracellular signaling Apoptosis

38. Binding Site Analysis of Oct4 Correlated Genes • Putative binding sites selected by the presence of neighboring Oct4 and Sox2 binding sites scanning -2kb from the TSS to +2kb from the 3’ end of the transcript • 370 Genes with at least one adjacent Oct4/Sox2 site • A subset of these sites were evaluated as direct Oct4 transcriptional targets by Chromatin Immunoprecipitation (ChIP) followed by Real-time PCR for analysis of occupancy at specific loci

39. Oct4 ChIP Real-time PCR Analysis • Duplicate ChIP experimets • Duplicate PCRs from each ChIP • PCR on 10-fold serial dilutions of input DNA were performed for a subset of amplicons • ΔCt values of ~3.3 were obtained • A change in Ct of 1 represents an approximate population doubling • 2-fold enrichment used as cutoff for identification of bound target • Fold enrichment calculated by the ΔΔ Ct method: • Amplicons were sequence validated Fold Enrichment = 2 [Ct (–Ab) – Ct (input)] – [Ct (+Ab) – Ct (input)]

40. Oct4 ChIP Real-time PCR Analysis A DNA Repair Polycomb and Trithorax Group Genes Known Oct Targets Positively Correlated Targets B C Cell Cycle Apoptosis Negatively Correlated Targets Negative Controls

41. Chromatin Domains and Nuclear Architecture Trithorax Group Polycomb Group Phc1 Pml Gemin5 Nup54 Bmi1 Ash1l Ash2l Mep50 Coil Gemin4 Smarcc1 Rnf134 Hcfc1 Ncl Phc3 Pum1 Whsc2 Suz12 Ctbp2 Cell Cycle Anti-Apoptosis Igf2bp1 Ccne1 Ccna2 Siva Ccnf nMyc1 Cdc25a Pdcd7 Sh3glb1 Thap1 Pdx41 D14abb1e Nipp1 Ccnb1 Api5 Bag4 Casp6 Jarid1b Ccnb2 Aatf1 Caipin1 Nr3c1 Eif4bp1 Cadh1 DNA Repair Parp1 Mre11a Fancd2 Brca1 Rad51 Msh2 Trp53 Chek1 Blm Tdrd7 Oct4 Self-renewal and Pluripotency

42. Chromatin Structure Ash2l Phc1 Rnf134 Cell Cycle Apoptosis Phc3 Bmi1 Casp6 Tdh Igf2bp1 Sh3glb1 Phb Nipp1 Oct4 DNA Repair D14Abb1e Trp53 pRb Tdrd7 Ccnf Brca1 Jarid1b Parp1 Aqr Rara Sall4 Rest Jarid2 Hoxb1 Mef2a Mesoderm Ectoderm

43. anti- differentiation anti- apoptotic checkpoint control repressive chromatin pro- differentiation pro- apoptotic permissive chromatin DNA repair checkpoint control anti- apoptotic repressive chromatin inactive- pRb anti- differentiation inactive- pRb pro- apoptotic permissive chromatin active- pRb DNA repair pro- differentiation active- pRb B Self-Renewal and Pluripotency C Differentiation

44. 623 1687 1271 Core Transcriptional Regulatory Circuitryin Human Embryonic Stem CellsBoyer et al. Cell 122, 1 (2005) • Identification of Oct4, Sox2, and Nanog transcriptional targets by location analysis • -8 kb to +2 kb from TSS of 17,917 annotated genes • No binding site analysis (direct or indirect binding?) • Substantial overlap with Oct4 Correlated genelist • Hoxb1, Tcf4, Jarid2, Rest, Bmi1 confirmed targets • No insight into mechanisms of gene activation or repression

45. Oct4 Nanog Oct4 Sox2 Nanog Oct4 Sox2 Sox2 Nanog Key Regulators are able to Both Activate and Repress Gene Expression Oct4 Sox2 Nanog Trp53 Phc3 Mynn Six2 Crsp3 Barx2 Pak1 Ncoa1 Cadh1 Etv3 Bclaf1 Etv5 Transcript expressed Transcript not expressed Oct4 Isl1, Myf5 What factors are responsible for mediating gene activation/repression? Cofactor Recruitment? Cis-regulatory Elements? Chromatin Structure? Combination of the above?