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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

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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 µg of each amplified sample were labeled with Cy5 for IP DNA, and Cy3 for input DNA using CGH labeling kit (Invitrogen). Four µg 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)<0.001, with intra-array median normalization (Boyer et al., 2005). GEO: GSE12482 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE12482). Selected

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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 14×14 correlation matrix. With the matrix, a heatmap reflecting the hierarchical clustering of the correlation coefficients was generated. De novo motif analysis. To determine the binding motif of Sall4, we extracted sequences (+/- 200) for all (132) ChIP-seq peaks in the Sall4 library containing at least 25 overlapping extended tags. An enriched motif (TCGCCATA) was identified in the 132 sequences using the motif discovery algorithm Weeder (Pavesi et al., 2001). Supplemental References Boyer, L. A., Lee, T. I., Cole, M. F., Johnstone, S. E., Levine, S. S., Zucker, J. P., Guenther, M. G., Kumar, R. M., Murray, H. L., Jenner, R. G., et al. (2005). Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947-956. Chen, X., Xu, H., Yuan, P., Fang, F., Huss, M., Vega, V. B., Wong, E., Orlov, Y. L., Zhang, W., Jiang, J., et al. (2008). Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106-1117. Ji, X., Li, W., Song, J., Wei, L., and Liu, X. S. (2006). CEAS: cis-regulatory element annotation system. Nucleic Acids Res 34, W551-554. Pavesi, G., Mauri, G., and Pesole, G. (2001). An algorithm for finding signals of unknown length in DNA sequences. Bioinformatics 17 Suppl 1, S207-214.

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Zhang, J., Tam, W. L., Tong, G. Q., Wu, Q., Chan, H. Y., Soh, B. S., Lou, Y., Yang, J., Ma, Y., Chai, L., et al. (2006). Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1. Nat Cell Biol 8, 1114-1123.

Figure S1. Expression of XEN marker genes in blastocyst-derived XEN cells. Immunofluorescence analysis of Gata6, Gata4, Sox7 and Sox17 expression and nuclear localization in XEN cells. Scale bars = 50 µm.

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Figure S2. Blastocyst-derived XEN-GIS cell line is highly similar to previously characterized XEN cells. (A) Dendrogram showing hierarchical clustering of the transcriptome profile of the XEN-GIS line with that of three characterized XEN lines XEN1-3, IM8A1-I, IM8A-II, four ES cell lines and four mouse embryonic fibroblasts (MEF) lines. (B) Heatmap representation of gene expression profiles in the indicated XEN and ES cell lines. (C) Marker gene expression changes in differentiated XEN-GIS cells analyzed by qRT-PCR. Values are normalized to β-actin and plotted relative to expression levels in undifferentiated XEN-GIS cells, except Afp (*) which was not expressed in the undifferentiated cells. For Afp, an arbitrary CT value at the threshold of real-time PCR detection was used in the calculations. Error bars indicate s.e.m of four replicates.

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Figure S3A. Validation of ES-specific Sall4 ChIP-chip binding sites in ES cells by qPCR. (A) Specific enrichment of Sall4 at 109 ES-specific loci in ES cells was determined by ChIP using anti-Sall4 or anti-GST control antibodies. Fold enrichment was calculated as the ratio of signals at Sall4-bound locus to the mean of three negative control genomic regions. (B) ChIP-qPCR analysis of the ES-specific Sall4 binding sites in XEN cells. Low or no enrichment of Sall4 was observed. Error bars indicate standard deviation of two technical replicates.

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Figure S3B. Validation of XEN-specific Sall4 ChIP-chip binding sites in XEN cells by qPCR. (A) Specific enrichment of Sall4 at 118 XEN-specific loci in ES cells was determined by ChIP using anti-Sall4 or anti-GST control antibodies. Fold enrichment was calculated as the ratio of signals at Sall4-bound locus to the mean of three negative control genomic regions. (B) ChIP-qPCR analysis of the XEN-specific Sall4 binding sites in ES cells. Low or no enrichment of Sall4 was observed. Error bars indicate standard deviation of two technical replicates.

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Figure S3C. Validation of Sall4 ChIP-chip binding sites common to both ES and XEN cells by qPCR. (A) Enrichment of Sall4 at 28 loci in ES cells was determined by ChIP using anti-Sall4 or anti-GST control antibodies. Fold enrichment was calculated as the ratio of signals at Sall4-bound locus to the mean of three negative control genomic regions. (B) ChIP-qPCR analysis of the same 28 Sall4 binding sites in XEN cells. Error bars indicate standard deviation of two technical replicates.

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Figure S4. Sall4 transcriptionally activates the promoters of its target genes. Promoter reporter constructs of Sall4 target genes were co-transfected with Sall4 over-expression or control vector into HEK293T cells. Luciferase reporter gene activities were measured 48hrs post-transfection. Reporter activity with Sall4 over-expression are plotted relative to control vector. The error bars represent standard error of three independent experiments, each performed in triplicates. Primers used in cloning these promoters are available in Supplemental Information.

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Figure S5. Changes in lineage marker gene expression upon knockdown of Sall4 targets in ES cells by RNAi. Quantitative RT-PCR analysis of marker gene expression was performed four days after the cells were transfected with gene-specific shRNA or control shRNA plasmids and cultured under puromycin selection. All values were normalized to levels of β-actin, and plotted relative to expression levels in control shRNA-transfected cells. Error bars indicate standard error of three replicates.

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Figure S6. RNAi knockdown of Slc25a36, Zfp27 and Ezh2 in ES cells leads to the emergence of Cdx2-positive cells. Gene-specific or control shRNA plasmids were transfected into ES cells cultured in trophoblast stem cell medium to promote differentiation into trophoblast stem cells. Cells were fixed and immunostained for Cdx2 three days after transfection. Scale bars = 50 µm.

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Figure S7. Analysis of Sall4 ChIP-seq data. (A) Heatmap showing the cluster analysis of the co-occurrence of Sall4 with other transcription factors within multiple transcription factor-binding loci (MTL) in ES cells. The factors were clustered along both axes based on the degree of colocalization with other factors. Colors reflect the colocalization frequency of each pair of factors in MTL; yellow indicates more frequent colocalization, red indicate less colocalization. (B) Putative Sall4 binding motif identified by de novo sequence analysis of Sall4 ChIP-seq data in ES cells. The TCGCCATA sequence was found to be enriched in 132 high-intensity Sall4 binding peak sequences. Methodology for the analysis is available in Supplemental Information.

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Table S1. Expression of known genes in different XEN cell lines Expression levels (Affymetrix) (Illumina) Gene Symbol Unigene ID XEN1-3 IM8A1-I IM8A1-II XEN-GIS

Correlation between XEN

lines

PE and VE genes Sox7 Sox17 Tcf2 Stra6 Gata6 Gata4 Krt2-8 Dab2 Vegfa Krt1-18 PE genes Pthr1 Sparc Pdgfra Snai1 Plat Thbd Fst VE genes Ihh Acvr1 Furin Cited1 Hnf4a Foxa2 Afp Cyp26a1 Plau ES and TS genes Pou5f1 Nanog Zfp42 Eomes Cdx2

Mm.42162 Mm.279103 Mm.7226 Mm.10801 Mm.329287 Mm.247669 Mm.358618 Mm.240830 Mm.282184 Mm.22479 Mm.3542 Mm.291442 Mm.221403 Mm.2093 Mm.154660 Mm.24096 Mm.4913 Mm.439736 Mm.689 Mm.5241 Mm.2390 Mm.202383 Mm.938 Mm.358570 Mm.42230 Mm.4183 Mm.17031 Mm.6047 Mm.285848 Mm.200692 Mm.20358

981 1024 111 465 2468 1091 1358 206 112 1192

2276 5924 1057 590 1641 219 1869

182 93

450 1208

A 552 A

354 A

A A A A A

1339 1436 189 296 1973 1196 1962 277 263 2104

2128 7284 1487 660 1893 393 3475

63 65

327 1032

A 512 A

1721 A

A A A A A

225 1285 138 85

1086 899 2737 624 158 3834

1384 7376 1056 239 1191 199 2990

156 64

455 1651

A 520 A

1010 A

A A A A A

2344 4528 235 346 3903 1455 ND 1400 1696 4650 538 2134 2739 ND 3873 178 1029 183 519 178 497 1110 ND A 90 A A A A 97 A

Good Good Good Good Good Good N/A

Good Good Good

Good Good Good N/A

Good Good Good

Good No

Good Good No* N/A

Good No

Good

Good Good Good Good Good

Correlation in the expression of a subset of known genes in XEN-GIS and three characterized XEN lines, XEN1-3, IM8A-I, IM8A-II (Kunath et al. 2005). Microarray expression profiling of XEN-GIS was performed on Illumina Beadarrays, while the Affymetrix microarray data of XEN1-3, IM8A1-I and IM8A1-II were obtained from Kunath et al. 2005. “A” indicates absence of gene expression and “ND” indicates genes not detected on the microarray. * Expression of Hnf4a was detected by RT-PCR in XEN1-3, IM8A1-I and IM8A1-II (Kunath et al. 2005).

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Table S2. Expression of genes in XEN cells upon Sall4 knockdown Log2 Fold difference

(compared to control KD)

Gene Symbol Unigene ID TargetID Sall4 KD 3 Days

Sall4 KD 4 Days

Sall4 KD 5 Days

PE and VE genes Sox7 Sox17 Tcf2 Stra6 Gata6 Gata4 Krt2-8 Dab2 Vegfa Krt1-18 PE genes Pthr1 Sparc Pdgfra Snai1 Plat Thbd Fst VE genes Ihh Acvr1 Furin Cited1 Hnf4a Foxa2 Afp Cyp26a1 Plau Top 40 XEN expressed genes (compared to expression in ES) Glipr1 Foxq1 Clic6 Ctsh Hs3st1 Plat Gata6 Hprt Pdgfra Ankrd1 Sox17

Mm.42162 Mm.279103 Mm.7226 Mm.10801 Mm.329287 Mm.247669 Mm.358618 Mm.240830 Mm.282184 Mm.22479 Mm.3542 Mm.291442 Mm.221403 Mm.2093 Mm.154660 Mm.24096 Mm.4913 Mm.439736 Mm.689 Mm.5241 Mm.2390 Mm.202383 Mm.938 Mm.358570 Mm.42230 Mm.4183 Mm.173790 Mm.44235 Mm.44747 Mm.2277 Mm.12559 Mm.154660 Mm.329287 Mm.299381 Mm.221403 Mm.10279 Mm.279103

scl46189.2_223-S scl17017.6_157-S scl41128.11_74-S scl36890.23.1_31-S GI_46909570-S scl0014463.2_238-S scl016691.2_87-S scl013132.15_5-S scl49877.10_136-S scl016668.7_96-S scl35310.8_24-S scl40203.12_25-S scl27703.26_20-S scl19886.3.1_22-S scl33993.13_72-S scl18545.1_131-S scl014313.3_29-S scl16606.3_174-S scl0011477.2_228-S scl018550.2_67-S scl53978.5.1_19-S scl19959.12_665-S scl18548.4_142-S scl27608.15.10_44-S scl53164.6.1_158-S scl46587.11.1_61-S scl37497.8.1_151-S scl44946.1_175-S scl0209195.6_234-S scl36630.14.1_50-S scl26655.5_57-S scl33993.13_72-S GI_46909570-S scl54930.9.1_1-S scl27703.26_20-S scl52528.9.1_3-S scl17017.6_157-S

-0.52 -0.72 -0.28 -0.04 -0.35 -0.01 0.20

-0.07 0.08 0.09

-0.60 -0.24 -0.09 0.44 0.41 0.67 0.75

-1.50 -0.14 0.15

-0.41 -0.76 -0.20

ND ND ND

0.17 0.44

-0.48 0.03 0.13 0.41

-0.35 -0.12 -0.09 0.73

-0.72

-0.97 -1.10 -0.50 -0.80 -0.69 -0.23 -0.02 -0.10 -0.12 0.10

-0.95 -0.74 -0.25 0.23 0.27 0.20 0.51

-1.90 -0.81 0.12

-0.61 -0.83 -0.17

ND ND ND

0.14 0.09

-0.76 -0.21 0.01 0.27

-0.69 -0.23 -0.25 0.74

-1.10

-1.24-1.11-0.83-0.80-0.60-0.27-0.010.110.160.17

-1.35-0.64-0.41-0.110.000.260.38

-1.64-0.64-0.59-0.50-0.46-0.15

NDNDND

0.05-0.03-0.54-0.230.070.00

-0.60-0.05-0.410.82

-1.11

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Drp2 Ccnd2 Loxl2 Tnnc1 Emb Gata4 Sox7 Kdelr3 9130005N14Rik Lamb1-1 S100a1 Flrt3 Lmna Chst1 Fxyd3 Gdf9 1200002N14Rik F3 Ncoa4 Dab2 Klb (AV071179) Sparc Myl3 Lgmn 1810045K07Rik St3gal1 (Siat4a) BC022687 Dhrs7 Sphk1

Mm.121662 Mm.333406 Mm.116714 Mm.439921 Mm.274926 Mm.247669 Mm.42162 Mm.298199 Mm.258545 Mm.172674 Mm.24662 Mm.426781 Mm.466615 Mm.38021 Mm.263847 Mm.9714 Mm.35450 Mm.273188 Mm.390717 Mm.240830 Mm.45274 Mm.291442 Mm.7353 Mm.17185 Mm.290353 Mm.248334 Mm.186678 Mm.289653 Mm.20944

scl54634.28.1_195-S scl28384.6_282-S scl0094352.2_42-S scl46496.6.1_3-S scl013723.9_229-S scl0014463.2_238-S scl46189.2_223-S scl0002476.1_437-S scl068908.1_119-S scl016777.14_23-S scl21916.1_21-S scl0071436.2_277-S scl0016905.1_7-S scl076969.2_187-S scl017178.4_5-S scl41569.3_290-S scl37649.7_307-S scl22672.6.1_17-S scl000311.1_2-S scl013132.15_5-S scl27771.5.1_89-S scl40203.12_25-S scl36423.7.11_132-S scl42127.12.1_16-S scl000679.1_109-S scl0020442.2_266-S scl42724.5.1_68-S scl066375.2_40-S scl0020698.1_299-S

-0.26 0.33 0.32

-0.63 -1.05 -0.01 -0.52 -0.38 0.02

-0.26 -0.07 -0.61 -0.31 -0.56 -0.30 0.17

-0.28 -0.11 0.25

-0.07 -0.56 -0.24 -0.96 -0.30 -0.21 0.46 0.00

-0.02 -0.68

-0.56 0.15 0.22

-0.70 -1.19 -0.23 -0.97 -0.42 0.08

-0.53 -0.26 -1.27 -0.27 -1.13 -0.56 -0.08 -0.56 -0.28 0.39

-0.10 -1.08 -0.74 -1.95 -0.60 0.05 0.40 0.12

-0.08 -1.20

-0.730.130.18

-0.27-0.96-0.27-1.24-0.680.23

-0.650.04

-0.65-0.39-1.50-0.87-0.78-0.64-0.190.610.11

-1.16-0.64-2.18-0.76-0.100.470.22

-0.27-1.39

Changes in expression of a subset of genes after RNAi-mediated Sall4 knockdown in XEN cells. The log2 fold difference in gene expression between Sall4 knockdown and control knockdown XEN cells after RNAi treatment for 3, 4, and 5 days are shown for genes known to be expressed in the extra-embroynic endoderm lineages, and for genes with the highest differential expression in XEN compared to ES cells. The log2 fold difference in gene expression is represented as ND (not determined) for genes that are not expressed. The log2 fold difference for each time point was obtained from the average of triplicate experiments. Tables S3 and S4 are available as Excel files.

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Table S5. Sall4 target genes with highly differential expression in Sall4 knockdown ES cells.

The list of Sall4-bound genes that exhibited >1.75 fold change in expression upon RNAi-mediated Sall4 knockdown in ES cells. Expression microarray profiling was performed on triplicate sets of ES cells transfected with Sall4- or control- shRNA and cultured in puromycin drug selection for four days. Average fold changes in gene expression between Sall4 knockdown and control knockdown cells were determined for each Sall4-bound gene.

Gene Symbol Fold Change (Downregulated) Gene Symbol Fold Change

(Upregulated)

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Liph Eras Pla2g10 Jam2 Ephx2 Trh Akp2 Tdgf1 Fgf15 Sfrp1 Slc7a3 Camk2b Twistnb Porcn Ldhb Kit Fblim1 D11Ertd636e Jakmip1 Acp6 Slc25a36 Msh6 Tcl1 Gbx2 Sox2 Upp1 Ezh2 Got1l1 Stat4 Calca Dido1 Lefty1 Mybl2 Sox2 Zfp57

4.80 3.38 3.26 3.20 3.14 2.81 2.80 2.73 2.67 2.60 2.42 2.41 2.37 2.35 2.28 2.27 2.22 2.08 2.07 2.05 2.03 1.98 1.94 1.93 1.92 1.90 1.90 1.90 1.79 1.79 1.78 1.77 1.76 1.75 1.75

Wisp1 Msx1 Fgfbp1 Cdc42ep5 Cyr61 Endod1 Cited2 Ckmt1 Bhlhb2 Fgf13 Gjb3 Dmrta2 Cldn4 Amotl2 Ifngr2 Col4a1 Kif13a Sox4 Edn1 Spsb2 Htra1 Rtn1 Gpc4 Rhob Sftpd Hmox1 Rhou Fem1b Ssbp3 Pawr D4Bwg0951e Srxn1 S100a10 Ctgf

4.21 3.91 3.62 3.45 3.17 3.01 2.89 2.70 2.64 2.63 2.44 2.43 2.32 2.30 2.20 2.15 2.14 2.11 2.10 2.08 2.05 2.05 2.04 1.93 1.89 1.85 1.83 1.83 1.82 1.82 1.82 1.80 1.80 1.79

19

Table S6. Sall4 target genes with highly differential expression in Sall4 knockdown XEN cells.

The list of Sall4-bound genes that exhibited >1.75 fold change in expression upon RNAi-mediated Sall4 knockdown in XEN cells. Expression microarray profiling was performed on triplicate sets of XEN cells transfected with Sall4- or control- shRNA and cultured under puromycin drug selection for five days. Average fold changes in gene expression between Sall4 knockdown and control knockdown cells were determined for each Sall4-bound gene.

Gene Symbol Fold Change (Downregulated)

Gene Symbol

Fold Change (Upregulated)

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

Enpep Sall4 Car4 Msx2 4632428N05Rik Podxl Pthr1 Mybl2 2310057J16Rik Sox7 Pthr1 Lama1 Tbx3 Klb Dkkl1 Sox17 Kcnk13 Clcn5 Mttp Emb Slc39a14 Spink3 Adam19 Ptp4a1 Ripk2 Plscr1 Eml4 Lin28 Spsb1 Nudt4 Dkkl1 Eva1

8.32 5.51 4.33 3.14 3.05 2.69 2.56 2.49 2.39 2.36 2.36 2.33 2.24 2.23 2.16 2.16 2.12 2.03 2.01 1.95 1.89 1.88 1.86 1.84 1.84 1.82 1.81 1.77 1.76 1.76 1.75 1.75

Lgals3 Krt1-13 Apobec3 Car2 Efna1 Vit Adprhl1 Aldh1a1 Sox5 Prss23 Sox5 Spon2 Nfib Ugt2b34 Slc44a1 Lta4h Ptplad2 Idh1 Lta4h Gsta4 Spnb2 Socs3 Gipc2 Ghr Nfib Tspan8 D8Ertd82e Ppp1r9a Oxr1 Spnb2 Rarb Lrrc39 Serpinb6c Ankrd1 C9 Unc5b Bmp2k

4.73 4.66 4.48 3.49 3.42 3.15 3.13 3.09 2.62 2.47 2.47 2.42 2.34 2.21 2.14 2.13 2.09 2.06 2.06 2.05 2.04 2.00 1.99 1.98 1.94 1.89 1.88 1.87 1.86 1.82 1.79 1.78 1.77 1.77 1.75 1.75 1.75

20

Table S7. Gene ontology classification of Sall4 target genes

Tables S8 and S9 are available as Excel files.

Sall4 Target Genes Over-represented GO categories % p-value

ES -specific (552)

Developmental process Transcription Organ development Embryonic development Pattern specification Cell differentiation Organ morphogenesis

31.7 24.6 17.8 8.3 5.4

19.6 8.3

4.23 X 10-14 1.10 X 10-12 1.31 X 10-11 3.79 X 10-11 3.52 X 10-09 4.40 X 10-09 1.35 X 10-08

XEN -specific (516)

Transcription Developmental process Localization Embryonic development

20.9 26.0 25.6 5.8

2.37 X 10-06

2.03 X 10-05 2.07 X 10-05 1.52 X 10-04

ES and XEN (174)

Organ development Developmental process Transcription Cell differentiation Cell cycle

22.4 35.1 27.6 23.6 12.1

4.44 X 10-08 1.94 X 10-07 8.55 X 10-07 2.98 X 10-06 6.86 X 10-05