Additional Supporting Information may be found in the online version of this article.

STEM_1410_sm_SuppFigure1.tif1495KFig. S1. Inducible ESC lines for Geminin over-expression and knockdown. (A) A2lox ESCs were used to obtain stable, clonal ESC lines for Dox-inducible over-expression (Flag-GemOE) or knockdown (GFP-Gem shRNAmir #7 or #11; KD). Expression cassette sequences used for GemKD are shown, including the Geminin sense (highlighted in blue) and antisense (highlighted in green) sequences. (B) Gem and control Actin protein levels from one GemKD and one GemOE ES line were evaluated by Western blot at days 3, 4, and 5, following Dox addition on day(D) 0 or 3, with comparison to the no Dox (-) control. (C) Day 4 GemKD EBs without (- Dox) or with (+Dox) Gem knockdown were evaluated by FACS for GFP expression.
STEM_1410_sm_SuppFigure2.tif967KFig. S2. Gem down-regulated genes are highly expressed in EBs, while Gem up-regulated genes are highly expressed in ESCs. (A) Examples of mesendoderm genes that are downregulated by Geminin, graphed to show their expression profile in days 0−5 EBs. (B) Aggregate microarray data for GemKD or GemOE was compared with aggregate microarray data for ESEB expression changes. Genes regulated by GemKD or OE are represented graphically to show percentages of Gem-regulated genes that either increase or decrease in expression during days 1- 7 of EB formation. (C) GemKD EBs grown in the absence (-Dox) or presence (+Dox) of Gem knockdown were assayed for ability to form beating cardiomyocytes. Gem knockdown significantly promoted the percentage of spontaneously beating EBs compared to control. Students t-test, p<.05, n=3.
STEM_1410_sm_SuppFigure3.pdf930KFig. S3. Gem knockdown inhibits cell migration. Day 3 EBs, without (-Dox) or with (+Dox) Gem knockdown, were dissociated and plated on adherent plates overnight. On day 4, the circular hydrogel spot (.68mm in diameter) was dissolved (0 hours), and subsequently photographed at 10× magnification at the time points shown. Gem knockdown cells were slower to migrate into the spot.
STEM_1410_sm_SuppFigure4.tif1110KFig. S4. Gem knockdown does not affect cell cycle progression. Cell cycle analysis was performed on day 4 EBs, with or without Gem knockdown by Dox addition from day 0. (A) DNA content using propidium iodide and (B) expression of a mitotic marker (phosphorylated histone H3; pH3) were determined by FACS analysis. Representative data is shown from one of three independent experiments.
STEM_1410_sm_SuppFigure5.tif2706KFig. S5. Gem knockdown does not affect the activation of intracellular effectors of Bmp, Activin/Nodal, or Fgf signaling. EBs were generated for 3, 4, or 5 days, with or without GemOE or GemKD induction by Dox addition. Levels of activated, phospho-specific Smad1, Smad2, and Erk1/Erk2 were detected by western blot, and were compared to total Smad1, Smad2, and Erk1/2 protein levels. Densitometry analyses of the ratio of phosphorylated to total protein levels are represented graphically at right.
STEM_1410_sm_SuppFigure6.tif1427KFig. S6. Conditioned media from Gem knockdown EBs is sufficient to increase mesendodermal gene expression. Day 4 EBs generated from the parental A2lox line were treated for 3 hours with conditioned media (CM) from day 4 GemKD EBs, generated with or without Dox-mediated GemKD. Mesendodermal gene expression was analyzed by qRTPCR in untreated (no CM) and in -DoxCM and +DoxCM treated samples.
STEM_1410_sm_SuppFigure7.pdf723KFig. S7. Gem knockdown does not promote mesendoderm formation by affecting cell nonautonomous Bmp, Activin, or Fgf signaling. Day 4 EBs from the parental A2lox line were treated for 24 hours with conditioned media (CM) collected from GemKD EBs, generated with (+DoxCM) or without (-DoxCM) Gem knockdown. Some EBs were treated with CM and recombinant Noggin (Nog, A), SB431542 (SB; B), or SU5402 (SU; C). Percentages of cells expressing Brachyury, Mixl1, or Eomes were defined by FACS. Error bars represent S.D. from the average of three experiments.
STEM_1410_sm_SuppFigure8.tif1334KFig. S8. Gem down-regulated genes are enriched for Polycomb binding and a ‘bivalent’ histone modification signature. Genes up- or down-regulated upon GemOE or KD (defined by microarray) were compared to ChIP enrichment profiles for (A) Polycomb binding1 and (B) bivalent (H3K4me3 and H3K27me3) and H3K4me3-only histone modification status2 in ESCs. All genes represented on the microarrays were compared with all genes present in each dataset used for comparison (array). This full ‘array’ comparison defines baseline frequencies of data overlap expected to be observed by chance when subsets of each dataset are compared. **pvalue< 1e−5. *p-value<.001. (C) Polycomb binding and histone modification status1,2 for a group of Gem down-regulated genes with roles in development/mesendoderm formation.
STEM_1410_sm_SuppFigure9.tif1119KFig. S9. ChIP analysis following Gem knockdown. Day 4 EBs, without (-Dox) or with (+Dox) GemKD, were analyzed by ChIP with Smad2, H3K9ac, H3K27me3, and IgG control antibodies. Binding at Mixl1, Brachyury and Goosecoid promoter regions at −500, +1, and +500 relative to transcription start site was determined by single qPCR at each position indicated and enrichment determined as described. A representative experiment (of 3) is shown.
STEM_1410_sm_SuppTable1.pdf138KTable S1. Microarray analysis of GemKD up- and down-regulated and GemOE up- and down-regulated genes. Probesets, gene symbols, and average fold change and differences for the three experiments (with versus without Dox) are shown.
STEM_1410_sm_SuppTable2.pdf43KTable S2. Antibodies, qRT-PCR primers, and ChIP primers. For antibodies, commercial sources and catalog numbers are indicated. For ChIP primer pairs, name indicates approximate location of amplicon relative to the transcription start site.
STEM_1410_sm_SuppTable3.pdf42KTable S3. Embryonic genes involved in mesoderm and endoderm formation are overrepresented among Geminin down-regulated genes obtained from microarray analysis. The Metacore/GeneGO Analysis suite was used to define top biological functions among the genes that were up- or down-regulated upon Geminin knockdown or up- or down-regulated upon Geminin over-expression.
STEM_1410_sm_SuppInfo.pdf23KSupporting Information

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