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1 Supplementry Figure 1. Genertion of N- nd C-tgged cyclin D1 knock-in mice., N-tgged cyclin D1 gene trgeting construct, cyclin D1 genomic locus, cyclin D1 locus following homologous recomintion (trgeted DNA) nd the finl cyclin D1 Ntg locus following deletion of the loxp-flnked Neomycin resistnce gene (Neo). Cyclin D1 exons re shown s yellow oxes nd re numered. Flg- (lue ox) nd HA (ornge ox) tgs re lso indicted. Restriction enzyme recognition sites: X, XI; N, Ne; H, HindIII; Pm, PmlI; R, EcoRI; K, KpnI; Hp, HpI; Nd, NdeI; B, BclI., Gene trgeting strtegy to generte C-tgged cyclin D1 llele (s ove). M, MfeI; Nt, NotI. Hygro-TK, Hygromycin resistnce thymidine kinse cssette. Proes A - D used for Southern lotting re mrked. 1
2 D1 Ctg/+ D1 Ctg/+ D1 Ntg/+ HA Cyclin D1 Tgged Endogenous D1 Ctg/+ HA Cyclin D1 c Cyclin D1 CDK4 p27 Ip HA Whole Cell Extrct Ip Cyclin D1 d pr Cyclin D1 Western lot HA CDK4 Ip CDK4 Ip Control IgG Supplementry Figure 2. Anlyses of N- nd C-tgged cyclin D1 lleles., Western lot nlysis of wild-type (), heterozygous cyclin D1 Ctg/+ or cyclin D1 Ntg/+ differentited emryonl stem cells proed with the indicted ntiodies. Non-relevnt lnes in the middle hve een cut out (mrked y gry line)., Western lots of wildtype, heterozygous nd homozygous knock-in retins proed with the indicted ntiodies. c, Cyclin D1 ws immunoprecipitted (Ip) from the retins of wild-type or homozygous knock-in mice using nti-cyclin D1 or nti-ha ntiodies, nd the immunolots were proed with the indicted ntiodies. d, CDK4 ws immunoprecipitted (Ip CDK4) from retins of wild-type or homozygous knock-in mice, nd the immunoprecipittes were used for in vitro kinse rections with recominnt pr s sustrte (upper pnel). Lower pnel: immunoprecipittes were lotted nd proed with the indicted ntiodies. 2
3 D1 -/- D1 Ntg/Ctg D1 -/- D1 Ntg/Ctg c % of tumor free Age (weeks) D1+/+/ /ErB2 ErB2 D1 KI.C.C Ctg/Ctg /ErB2./Neu D1-/- /ErB2 /Neu d D1 -/- /D2 -/- /D2 -/- Supplementry Figure 3. Norml development of cyclin D1 tg/tg nimls., Appernce of retins in dult wild-type (), cyclin D1 -/-, homozygous cyclin nd cyclin s well s in hyrid cyclin D1 Ntg/Ctg knock-in mice. Histologic sections were stined with hemtoxylin nd eosin., Whole mounts of mmmry glnds dissected from femle mice of the indicted genotypes one dy fter the delivery of pups. Mmmry epithelil tree ws stined with crmine red. c, Percentge of tumor-free mice mong cyclin D1 +/+ /MMTV-ErB2, cyclin D1 -/- /MMTV-ErB2 nd cyclin /MMTV-ErB2 femles. Tumor incidence dt for cyclin D1 -/- /MMTV- ErB2 mice ws tken from Yu et l. 1 d, Appernce of cereell in wild-type, cyclin D1 -/- D2 -/- nd cyclin D2 -/- mice. 3
4 Ip FLAG (CycD1) KI Input CDK1/2 CDK5 HCF1 ELL MYST4-Ip JMJD1B-Ip BAF53A-Ip FLAG (CycD1)-Ip Control IgG-Ip ADARB1 HA (CycD1) ZNF423-Ip NF-YA-Ip FLAG (CycD1)-Ip Mouse IgG-Ip Rit IgG-Ip ELK1-Ip STAT3-Ip CUX1-Ip CREB2-Ip FLAG (CycD1)-Ip Control IgG-Ip HA (CycD1) Supplementry Figure 4. Verifiction of selected cyclin D1-interctors., Selected cyclin D1-interctors, identified t high confidence (Mnn-Whitney test p 0.001, frctionl difference 0.8) y mss spectroscopy, were verified using immunoprecipittion Western lotting., Verifiction of the physicl interction etween cyclin D1 nd trnscription fctors whose DNA recognition sequences were enriched mong cyclin D1-ound genomic frgments (Fig. 2d in the min text). Grey line mrks plce where irrelevnt lnes hve een cut out. In, right pnel nd in, ntiodies for the indicted trnscription fctors were conjugted nd crosslinked using DMP (Pierce #21667) to Sephrose G eds nd used for immunoprecipittions from knock-in orgns, followed y Western lotting with nti-ha ntiody (to detect tgged cyclin D1). In, left hnd pnel, cyclin D1 ws immunoprecipitted from orgns of tgged knock-in mice (KI), or for control, from corresponding orgns of wild-type mice (, not expressing tgged cyclin D1), nd the immunolots were proed with the indicted ntiodies. 4
5 KNOCK-IN KNOCK-IN nti-flag Ip (log2) nti-flag Ip (log2) (KI) nti-flag Ip (log2) nti-igg Ip (log2) WCE (log2) WCE (log2) WILD-TYPE KNOCK-IN vs. WCE (log2) Anti-FLAG Ip (log2) () FLAG No enrichment 2 fold enrichment 3 fold enrichment 4 fold enrichment 5 fold enrichment or more c FLAG IgG Supplementry Figure 5. ChIP-chip nd trgeted ChIP nlyses., Control ChIPchip nlyses using Mouse PromoterChip rry. Sctterplots of chromtin immunoprecipittion (Ip) performed on knock-in or wild-type emryos using nti-flag ntiody (M2, Sigm), or IgG control. Log 2 intensity vlues of nti-flag Ip mteril (Cy5 lel) re plotted ginst log 2 intensity vlues of whole cell extrct DNA (WCE, Cy3 lel). Enrichment of specific DNA trgets ws oserved in nti-flag immunoprecipittes from knock-in emryos (top row, left pnel). Note, tht no enrichment ws oserved when isotype-mtched IgG ws used for immunoprecipittion in the knock-in emryos (top row, right pnel). Also, no enrichment ws found when nti-flag immunoprecipittion ws performed in wild-type emryos (which do not express tgged cyclin D1) (ottom row, left pnel). Bottom row, right pnel shows log 2 intensity vlues of nti-flag Ip from knock-in emryos (KI, Cy5 lel) plotted ginst log 2 intensity vlues of nti-flag Ip from wild-type emryos (, Cy3 lel). DNA ws hyridized to Mouse PromoterChip BCBC 5A rrys (from Bet Cell Biology Consortium)., c, Site-specific ChIP. Confirmtion of selected cyclin D1 trgets, identified in whole-emryo ChIP-chip, using site-specific quntittive PCR on ChIP DNA from () E14.5 emryos or (c) P0 retins. Retins were microdissected from cyclin D1 knock-in neontes. The fold enrichment in nti-flag ChIP ws clculted reltive to the PCR of the sme mount of whole cell DNA. The color intensity reflects the fold chnge, s shown in the legend. The dt re the verge of three experiments. 5
6 D1-ound promoters D1-not ound promoters Numer of promoters Numer of promoters CpG proportion CpG proportion Fold enrichment Fold chnge tgged knock-in mice (KI), or for control, from corresponding orgns of wild-type mice (, not expressing tgged cyclin D1), nd the immunolots were proed with the indicted ntiodies. Supplementry Figure 5. ChIP-chip nd trgeted ChIP nlyses., Control ChIPchip nlyses using Mouse PromoterChip rry. Sctterplots of chromtin immunoprecipittion (Ip) performed on knock-in or wild-type emryos using nti-flag ntiody (M2, Sigm), or IgG control. Log 2 intensity vlues of nti-flag Ip mteril (Cy5 lel) re plotted ginst log Cyclin D1 2 intensity vlues of whole cell extrct DNA (WCE, Cy3 lel). Enrichment of specific DNA trgets ws oserved in nti-flag immunoprecipittes from knock-in emryos (top row, left pnel). Note, tht no enrichment ws oserved when Cumultive isotype-mtched numer of genes IgG ws used for immunoprecipittion in the knock-in emryos (top row, right pnel). Also, no enrichment ws found when nti-flag immunoprecipittion ws performed in wild-type emryos (which do not express tgged c cyclin D1) (ottom row, left pnel). Bottom row, right pnel shows log 4 2 intensity vlues 3of nti-flag Ip from knock-in emryos (KI, Cy5 lel) plotted ginst log 2 intensity vlues 2 of nti-flag Ip from wild-type emryos (, Cy3 lel). DNA ws hyridized 1to Mouse PromoterChip BCBC 5A rrys (from Bet Cell Biology Consortium)., 0 c, Site-specific ChIP. Confirmtion of selected cyclin D1 trgets, identified in whole-emryo -1 ChIP-chip, using site-specific quntittive PCR on ChIP -2 DNA from () E14.5 emryos or (c) P0 retins. Retins were microdissected from cyclin -3 D1 knock-in neontes. The fold enrichment in nti-flag ChIP ws clculted reltive to -4 the PCR of the sme mount of whole cell DNA. The color intensity reflects the fold Gpdh Px6 Chx10 Dkk3 chnge, s shown in the legend. The dt re the verge of three experiments. Supplementry Figure 6. Anlyses of cyclin D1 trnscriptionl function., CpG content in promoter regions. Shown is distriution of CpG dinucleotide content per sepir within the 500p promoter regions of cyclin D1-ound promoters, nd promoters not ound y cyclin D1., Comprison of gene expression etween retins of wild-type nd cyclin D1 -/- mice. Trnscripts were ordered from the lowest to the highest rtio etween cyclin D1 -/- vs. retins. c, Levels of trnscripts for Px6, Chx10 nd Dkk3, mrkers of retinl progenitor cells 2 were not chnged in cyclin D1-null retins (s judged y RT- PCR), indicting tht reduced expression of Notch1 in cyclin D1 -/- retins ws not cused y decresed numers of progenitor cells. Supplementry Figure 7. Reduced recruitment of cyclin D1 to NF-Y trget promoters following knock-down of NF-YA in R28 cells. Rt retinl precursor R28 cells were infected with viruses encoding nti-nf-ya shrna, or control shrna. The recruitment of cyclin D1 (or NF-YA) to selected NF-Y trget promoters ws quntified using chromtin immunoprecipittion followed y rel-time PCR. For control, we guged cyclin D1-recruitment to non-nf-y trget promoters (devoid of NF-Y DNA recognition sequences) in the presence or sence of nti-nf-ya shrna. Trgets contining NF-Y DNA recognition sequences within cyclin D1-ound promoter regions were selected s descried in Full Methods. The dt is presented s percentge of recruitment oserved in NF-YA shrna smples s compred to control shrna smples. Shown re men vlues 6
7 Non NF-Y trgets (controls) NF-Y trgets 120 Percent of enrichment compred to control shrna CycD1 NF-YA NF-YA Tuulin Control shrna NF-YA shrna 0 Supplementry Figure 7. Reduced recruitment of cyclin D1 to NF-Y trget promoters following knock-down of NF-YA in R28 cells. Rt retinl precursor R28 cells were infected with viruses encoding nti-nf-ya shrna, or control shrna. The recruitment of cyclin D1 (or NF-YA) to selected NF-Y trget promoters ws quntified using chromtin immunoprecipittion followed y rel-time PCR. For control, we guged cyclin D1-recruitment to non-nf-y trget promoters (devoid of NF-Y DNA recognition sequences) in the presence or sence of nti-nf-ya shrna. Trgets contining NF-Y DNA recognition sequences within cyclin D1-ound promoter regions were selected s descried in Full Methods. The dt is presented s percentge of recruitment oserved in NF-YA shrna smples s compred to control shrna smples. Shown re men vlues +SD of three independent experiments. Right pnel shows Western lotting with nti- NF-YA ntiodies. 7
8 Decresed in D1 -/- Incresed in D1 -/- Not chnged in D1 -/- Frction of sites Distnce from trnscription strt site Supplementry Figure 8. Distriution of cyclin D1 inding sites reltive to trnscription strt sites of RefSeq genes. Green rs: genes with decresed expression in cyclin D1 -/- retins (s determined y SAM using locl FDR cutoff of 0.2). Red rs: genes upregulted in cyclin D1 -/-. Grey rs: genes which did not show ltered expression in cyclin D1-null retins. 8
9 Notch1 gene TSS Exon 1 Intron 1 Exon 2 Intron 2 Ac H4K5 Ac H3K9, 14 Cyclin D1 CBP Figure modified from Enseml genome uilt Supplementry Figure 9. Schemtic representtion of the murine Notch1 gene. Coding exons re depicted s filled oxes, 3 nd 5 untrnslted regions s open oxes. The gene spns 45,760 p of genomic DNA. Note tht cyclin D1 nd CBP ind the Notch1 gene within intron II, pprox. 2.2 k downstrem from the min trnscription strt site, nd the PCR primers for nti-flag nd nti-cbp trgeted ChIP were designed round this site (see Supplementry Tle 8). Histone cetyltion (H4K5 nd H3K9,14) of the Notch1 promoter region ws detected using PCR primers spnning the trnscription strt site (TSS). 9
10 NIN virus ires LTR nlcz LTR NIN-NICD virus ires LTR NICD nlcz LTR Supplementry Figure 10. Digrm of retrovirl constructs used for retinl infections. NICD, Notch intrcellulr domin; nlcz, nucler et glctosidse; LTR, long terminl repet. 10
11 Fold chnge DMSO PD Roscovitine R-S780-P DMSO PD Cyclin D1 CBP Ac H3K9,14 Actin 40 Fold chnge CycD1 +CycD1-KE Cyclin D1 CBP Ac H3K9,14 Supplementry Figure 11. Anlyses of cyclin D function in CBP recruitment., Rt retinl precursor R28 cells were cultured for 12 hours in the presence of PD ( specific inhiitor of cyclin D-Cdk4/6 kinse) 3 or Roscovitine (inhiitor of Cdk1, Cdk2 nd Cdk5), or solvent (DMSO), nd the recruitment of cyclin D1 or CBP to the Notch1 upstrem regultory region, nd cetyltion of H3K9,14 within the Notch1 promoter were guged y chromtin-immunoprecipittion followed y quntittive rel-time PCR. Shown re men vlues + SD of three independent experiments using 0.2 um PD nd 10 um Roscovitine. Similr results were otined with 1.0 um PD nd 50 um Roscovitine (dt not shown). Right pnel shows immunolotting of R28 cell lystes with n ntiody recognizing retinolstom protein phosphorylted on Serine 780 residue ( trget for cyclin D-Cdk4/6 kinses). Note reduced phosphoryltion of this residue in PD treted smple, s expected., R28 cells were engineered to stly overexpress wild-type cyclin D1 (+CycD1), or cyclin D1 K112E (+CycD1-KE). The ltter is cyclin D1 point-mutnt which is unle to ctivte Cdk kinse ctivity in vitro 4 nd in vivo 5. Recruitment of cyclin D1 nd CBP to the Notch1 regultory region nd cetyltion of H3K9,14 ws guged y chromtin immunoprecipittion rel-time PCR nd expressed s fold chnge compred to cells not overexpressing cyclin D1. Shown re men vlues of three independent experiments +SD. 11
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