Supplemental Data Sall4 Regulates Distinct Transcription Circuitries ?· Sall4 Regulates Distinct Transcription…

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    Cell Stem Cell, Volume 3

    Supplemental Data

    Sall4 Regulates Distinct Transcription Circuitries

    in Different Blastocyst-Derived Stem Cell Lineages Chin Yan Lim, Wai-Leong Tam, Jinqiu Zhang, Haw Siang Ang, Hui Jia, Leonard Lipovich, Huck-Hui Ng, Chia-Lin Wei, Wing Kin Sung, Paul Robson, Henry Yang, and Bing Lim Experimental Procedures RNAi design and construction of plasmids for shRNA synthesis. For RNAi validation of gene function, two short hairpin RNA constructs were made for each targeted gene. These oligonucleotides were annealed and cloned into pSuper.puro (Oligoengine). Gata6 shRNA 1 GATCCCCGAGGACCTGTTGCTCTTCAttcaagagaTGAAGAGCAACAGGTCCTCTTTTTA AGCTTAAAAAGAGGACCTGTTGCTCTTCAtctcttgaaTGAAGAGCAACAGGTCCTCGGG Gata 6 shRNA 2 gatccccAtGCAGACAtAACAttCCTttcaagagaAGGAAtGttAtGtCtGCATttttta agcttaaaaaAtGCAGACAtAACAttCCTtctcttgaaAGGAAtGttAtGtCtGCATggg Sox17 shRNA 1 GATCCCCGGACCCGGCTTTCTTTGCAttcaagagaTGCAAAGAAAGCCGGGTCCTTTTTA AGCTTAAAAAGGACCCGGCTTTCTTTGCAtctcttgaaTGCAAAGAAAGCCGGGTCCGGG Sox17 shRNA 2 GATCCCCGCACGGAATTCGAACAGTAttcaagagaTACTGTTCGAATTCCGTGCTTTTTA AGCTTAAAAAGCACGGAATTCGAACAGTAtctcttgaaTACTGTTCGAATTCCGTGCGGG Sox 7 shRNA 1 GATCCCCGAACACGCTGCCTGAGAAAttcaagagaTTTCTCAGGCAGCGTGTTCTTTTTA AGCTTAAAAAGAACACGCTGCCTGAGAAAtctcttgaaTTTCTCAGGCAGCGTGTTCGGG Sox 7 shRNA 2 GATCCCCGTCCTCTGTTTCTGGATGTttcaagagaACATCCAGAAACAGAGGACTTTTTA AGCTTAAAAAGTCCTCTGTTTCTGGATGTtctcttgaaACATCCAGAAACAGAGGACGGG Ezh2 shRNA 1 GATCCCCGGATGGCACTTTCATTGAAttcaagagaTTCAATGAAAGTGCCATCCTTTTTA AGCTTAAAAAGGATGGCACTTTCATTGAAtctcttgaaTTCAATGAAAGTGCCATCCGGG Ezh2 shRNA 3 GATCCCCGCAAATTCTCGGTGTCAAAttcaagagaTTTGACACCGAGAATTTGCTTTTTA AGCTTAAAAAGCAAATTCTCGGTGTCAAAtctcttgaaTTTGACACCGAGAATTTGCGGG Slc25a36 shRNA 1

    buckleycCross-Out

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    GATCCCCGGCATATCAGAGACTGTTAttcaagagaTAACAGTCTCTGATATGCCTTTTTA AGCTTAAAAAGGCATATCAGAGACTGTTAtctcttgaaTAACAGTCTCTGATATGCCGGG Slc25a36 shRNA 1 GATCCCCGCAGTCTTCTTCTGTGACAttcaagagaTGTCACAGAAGAAGACTGCTTTTTA AGCTTAAAAAGCAGTCTTCTTCTGTGACAtctcttgaaTGTCACAGAAGAAGACTGCGGG Zfp27 shRNA 1 GATCCCCGGACACGTCTTATGTAATTttcaagagaAATTACATAAGACGTGTCCTTTTTA AGCTTAAAAAGGACACGTCTTATGTAATTtctcttgaaAATTACATAAGACGTGTCCGGG Zfp27 shRNA 2 gatccccttACtGtCtCtAtAGGACAttcaagagatGtCCtAtAGAGACAGtAAttttta agcttaaaaattACtGtCtCtAtAGGACAtctcttgaatGtCCtAtAGAGACAGtAAggg Control shRNA GATCCCCGAACGGCATCAAGGTGAACttcaagagaGTTCACCTTGATGCCGTTCTTTTTA AGCTTAAAAAGAACGGCATCAAGGTGAACtctcttgaaGTTCACCTTGATGCCGTTCGGG Chromatin Immunoprecipitation and DNA Microarray Analysis. ChIP assays with feeder-free E14 mouse ESCs were carried out as described previously (Zhang et al., 2006). Briefly, cells were cross-linked with 1 % formaldehyde for 10 min at room temperature and formaldehyde was then inactivated by the addition of 125 mM glycine. Chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using antibodies. Approximately 5x107 cells were used for each ChIP reaction. Amounts of antibodies used for immunoprecipitation were 10 ug for anti-Sall4, 10 ug for anti-H3K4me3 (Abcam) and 15 ug for anti-H3K27me3 (Upstate biotechnology). For all ChIP experiments, quantitative PCR analyses were performed in real-time using the ABI PRISM 7900 Sequence Detection System and SYBR Green Master Mix as described previously (Zhang et al., 2006). Relative occupancy values were calculated by determining the apparent immunoprecipitation efficiency (ratios of the amount of immunoprecipitated DNA to that of the input sample) and normalized to the level observed. ChIP combined with DNA microarray was carried out according to Agilent Mammalian ChIP-on-chip protocol (version 3) (Boyer et al., 2005). Briefly, immunoprecipitated (IP) DNA was blunted with T4 polymerase and ligated to linkers with T4 DNA ligase. For reference, 200 ng of input DNA was used. Ligated DNA was amplified with two rounds of PCR cycles. Two g of each amplified sample were labeled with Cy5 for IP DNA, and Cy3 for input DNA using CGH labeling kit (Invitrogen). Four g of each labeled sample was blocked with mouse Cot-1 DNA (Invitogen) and blocking agent (Agilent), and hybridized to DNA microarrays (G4490A, Agilent) for 40 h at 65 oC. The microarrays were washed and stabilized with acetonitrile (Sigma), followed by stabilization and drying solution (Agilent). The arrays were scanned with Agilent DNA microarray scanner (G2565BA), and probe features were extracted using Feature Extraction Software (G2567AA, version 9.1). The data was then analysed with ChIP Analytics 1.3 (G4477AA), according to the Whitehead Neighbourhood Model, P(Xbar)

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    bound targets, determined by the algorithm, were validated by qPCR to determine the false discovery rate. Quantitative RT-PCR - List of ABI Taqman probes Mapped ID Assay ID Actb Mm00607939_s1 Afp Mm00431715_m1 Bmp4 Mm00432087_m1 Cdx2 Mm00432449_m1 Eomes Mm01351984_m1 Esx1 Mm01297208_m1 Fgf5 Mm00438919_m1 Foxa2 Mm00839704_mH Gata4 Mm00484689_m1 Gata6 Mm00802636_m1 Hand1 Mm00433931_m1 Hnf4a Mm00433964_m1 Ihh Mm00439613_m1 MyoD1 Mm00440387_m1 Nanog Mm02019550_s1 Otx2 Mm00446859_m1 Pax6 Mm00443072_m1 Pdgfra Mm01211694_m1 Pou5f1 Mm00658129_gH Psx1/Rhox6 Mm00655990_g1 Pthr1 Mm00441046_m1 Sall4 Mm00614351_m1 Sox1 Mm00486299_s1 Sox17 Mm00488363_m1 Sox2 Mm00488369_s1 Sox7 Mm00776876_m1 T Mm00436877_m1 Tcf2/Hnf1b Mm00447452_m1 Utf1 Mm00447703_g1 Promoter reporter assays. The promoters of selected genes were PCR amplified from mouse genomic DNA and cloned into pGL4.10 (Promega). Primers used for cloning are listed in table. The Oct4 promoter was previous described in Zhang et al., 2006. To perform the promoter-luciferase assays, HEK293T cells were seeded 24 h before transfection at a density of 2.5 104 cells per well in 96-well culture plates. For testing the effect of Sall4 activity on these promoters, Sall4 over-expressing plasmid, pCAG-Sall4-IRES-GFP, or pCAG-IRES-GFP vector (200 ng) was co-transfected with individual promoter-luciferase (50ng) and pRL-SV40 (1ng) (Promega). Firefly and Renilla luciferase activities were measured with the Dual-Luciferase Reporter system (Promega) 48h post-transfection on Centro LB960 Luminometer (Berthold Technologies). The data were expressed as relative to corresponding control vector transfections, after normalization to Renilla luciferase readings.

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    Primers for promoter reporter constructs Gene Promoter

    Region Size Primer Name Sequence

    Aldh1a1 chr19:20301400-20302583

    1184bp Aldh1a1-Forward aagctagcTTGCCTGAGGCGAATTTCCAGC

    Aldh1a1pr-Reverse

    aaggatccTGGTTTGGCTCCTGGAACACAGG

    Dag1 chr9:107996961-107997804

    844bp Dag1-Forward ggcgctagcAAGAATGTGGGCCACCGGGG

    Dag1-Reverse ggcagatctAGGGACACCGCCGCCACTCC

    Dhrs8 chr5:103211036-103212099

    1064bp Dhrs8-Forward ggcgctagcGAGCACTGTCCATTCTTCCT

    Dhrs8-Reverse ggcagatctACTTCATCCTTTTGGAGGCC

    Elf3 chr1:135147614-135150772

    3159bp Elf3-Forward aaggtaccTCTGAAGATGAAGAGTGGAGCC

    Elf3-Reverse aaggatccTCCATTTCTGTCTGGCAAAGCC

    Ezh2 chr6: 47631667-47636793

    5127 bp Ezh2 Forward acggatccACTCCACTGCCTTCGATGTCCCACT

    Ezh2 Reverse acctcgagAGGCTAGGGTAAGCCAGAAGGCAC

    Gata6 chr18:11258066-11259121

    1056bp Gata6-Forward actcgagATAACCATTTGGAGGGAGCGAC

    Gata6-Reverse aaagcttTCCTGATTGGACTCACCGAGCC

    Kcnk13 chr12:97458855-97461236

    2382bp Kcnk13-Forward aagctagcTGGCTGGCTCTGTCAACAGTCTATAG

    Kcnk13-Reverse aaggatccATGTGCTCTGCCGACCTTAGCC

    Khdrbs1 chr4:129186876-129187529

    654bp Khdrbs1-Forward ggcgctagcTCCCTTTCGCACACTCATCA

    Khdrbs1-Reverse ggcagatctTTGGCGGGGGTGGAAGAACA

    Lin28 chr4:133397168-133398046

    879bp Lin28-Forward ggcgctagcCCGAGTTTGAGAGGCAGCAC

    Lin28-Reverse ggcagatctAGCCCATGGTCGTCTGCTGA

    Mybl2 chr2:162792720-162795598

    2879bp Mybl2-Forward aggtaccAGCACCTGTGTGATCAGTGC

    Mybl2-Reverse actcgagCGTCAGCGTGTCTGCAGGTC

    Pla2g10 chr16:13277131-13277933

    803bp Pla2g10-Forward ggcgctagcAGGGGGGGTGGTTCCAAGGTTT

    Pla2g10-Reverse ggcagatctTCACCTTCTTTCCGTGGGGC

    Sall4 chr2:168559732-168562542

    2811bp Sall4 -FL Forward

    aaggatccTGGAGAGACTGAGAACCATACATCGCAC

    Sall4 -FL Reverse

    acaagcttTCCGCCACCAATTCCTGGAGTTG

    Sox7 chr14:58804421-58807629

    3209bp Sox7 Forward acagatctAAGTAGAAGGATAGAGGGAGAAGGACTG

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    Sox7 Reverse acaagcttAACGCGGGTCACTTGGCTTC

    Sulf1 chr1:12863144-12863717

    574bp Sulf1-Forward aagctagcTTCCTCATCCATTCAGTCCTGCC

    Sulf1-Reverse aaggatccAGGAGCAGAGATTAGGTGGCAGC

    Wisp1 chr15:66973748-66975241

    1494bp Wisp1-Forward aaggtaccTCTCAGTTACTGGACCACCAACC

    Wisp1-Reverse acaagcttTGGAAGTCAGCGTCACAGGAGC

    Zfp42 chr8:42091901-42094490

    2590bp Zfp42-Forward aagctagcTCAGGGTCCTGGGAATCAAACC

    Zfp42-Reverse aaggatccAGGTAGGTGCCCTGTTACCTCGC

    Intersection of Sall4 ChIP-Seq data with MTL in ESCs. To determine the common target (co-regulated) genes of Oct4, Sox2, Nanog and Sall4, co-localization of the binding loci in the neighborhood of a target gene was analyzed. Firstly, the co-localized binding loci of all these four factors were extracted and mapped to genes using CEAS (Ji et al., 2006) with the mouse genome assembly version Build 36 (mm8). Sall4 binding loci generated from Sall4 ChIP-seq data with enrichment 9 intensity reads were used to intersect with the 3582 MTL obtained from Chen et al., 2008. Amongst these 3582 MTL, clustering of 14 transcription factors (13 TFs + Sall4) was performed based on co-occurrences of the binding loci to evaluate the similarity of the TF targeting. The co-occurrences between 14 TFs within the MTL were first computed and the correlation coefficients between each pair of co-occurrence vector were then calculated and modeled to a 1414 correlation matrix. With the matrix, a heatmap reflecting the hierarchical clustering of

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