PMC

(A) Immunoblots for proteins from mouse embryonic fibroblasts (MEFs) and embryonic stem cells (ESCs) as indicated on top. Antibodies were indicated on left. (B) Real-time PCR and (C) Immunoblot analyses for RA-induced ESC differentiation. (D) Real-time PCR and (E) Immunoblot analyses for ESC differentiation during EB formation. In (B, D), fold changes of each transcript relative to its expression in day 0 EB formation were presented. For C, E, β-actin was used as the loading control.

(A) Left, schematic for wild type and Mof knockout alleles. Genotyping primers (red arrows) were indicated. Right, genotyping results for wild type, floxed Mof alleles as well as Cre-ER^™ by PCR. (B) Immunoblots for Mof and H4 K16ac in Mof ^+/+, Mof ^flox/+ and Mof ^flox/flox cells after 4-OHT treatment. Immunoblot for β-actin was used as the loading control. (C) Electron microscopy images of wild type (left) and Mof knockout nuclei (right). Densely stained heterochromatin was indicated by arrow. Scale bars, 2 micron. (D) Alkaline phosphatase staining of Mof ^+/+, Mof ^+/− and Mof ^−/− ESCs. (E) Light microscopy images of day 4 EB for Mof ^+/+, Mof ^+/− and Mof ^−/− ESCs. Scale bars, 0.5mm. Also see .

(A) Chromosome-distribution of Mof binding peaks in mouse ESCs. Y-axis, count of ChIP-seq reads per kilo base. X-axis, chromosome name. (B) Distribution of Mof binding sites relative to nearest Refseq genes. Top, schematic for eight counting categories. Bottom, pie chart for percentage distribution of Mof peaks in each category. (C) Distribution of Mof peaks in a 12kb region from −2kb to +10kb around TSS (indicated by red arrow). Y-axis, percentage of Mof peaks relative to total Mof peaks within the defined region. X-axis, bin numbers, with each represents a 500bp region. Mof peaks were indicated as class I and class II peaks on bottom. (D) Comparison of Mof distribution within the defined 12kb region in mESCs (blue) and human CD4⁺ cells (red). TSS and Class I and II sites were indicated on bottom. Also see

(A) Immunoblots for Nanog, Mof and H4 K16ac in WT or Mof ^−/− ESCs rescued with control vector, wild type Mof, mutant Mof or Nanog. Antibodies used in the experiments were indicated on right. Both exogenous wild type and mutant Mof were Myc-tagged. (B) AP staining of wild type ESCs and Mof ^−/− ESCs expressing exogenous wild type Mof, Mof mutant or Nanog as indicated. Top, percentage of AP positive clones of Mof ^−/− and three rescue ESCs relative to wild type ESCs was presented. Means and standard deviations (as error bars) from two independent experiments were presented. Bottom, images (20×) for each cell lines as indicated on bottom. (C) Real-time PCR analyses for pluripotency (left) and differentiation genes (right) in Mof ^−/− and three rescue cell lines as indicated. All mRNA levels were normalized against β-actin and were presented as relative fold changes to wild type ESCs. Means and standard deviations (as error bars) from at least three independent experiments were presented. Also see .

(A) Heat map of expression of conserved Nanog and Oct4 joint targets () in WT and Mof ^−/− ESCs. Fold change of gene expression relative to WT ESCs was indicated at bottom. (B, C) Real-time PCR analyses for pluripotency (B) and differentiation genes (C) in Mof ^−/− and Mof ^+/+ ESCs as indicated. All mRNA levels were normalized against β-actin and were presented as relative expression in Mof null versus wild type ESCs. (D) ChIP for pluripotency genes that were down regulated in Mof ^−/− ESCs. (E) ChIP for differentiation genes that were up regulated in Mof ^−/− ESCs. For (D–E), primer sets were designed corresponding to Mof binding peaks identified by ChIP-seq (indicated in ). The antibody was indicated on top. Signals for each experiment were normalized to 5% input. For (B–E), Means and standard deviations (as error bars) from at least three independent experiments were presented. Also see .

(A) Venn diagram for overlap of Mof bound genes (yellow) and genes that were either up regulated (blue) or down regulated (orange) in Mof ^−/− ESCs. Fisher’s exact test (p< 2.2×10⁻¹⁶) was performed to test for statistical significance of enrichment of up or down regulated genes with direct Mof binding. (B) GO term analyses for Mof down regulated genes (top) and Mof up regulated genes (bottom). Selected developmental pathways were presented and log p-value was used to rank the enrichment. (C) Top, Venn diagram for overlap of Mof targets with class I (green) or class II (yellow) binding sites. Bottom, a table for number of genes that were up or down regulated in each category. (D, E) GSEA of Mof targets with class I binding sites (D) or class II binding sites (E) and Nanog (left) or Oct4 (right) transcriptome (). NES, normalized enrichment score; FDR (p value), false discovery rate. Also see .

(A) Top, Venn diagram for direct physical overlap of binding peaks for Mof (blue), Wdr5 (pink) and H3 K4me3 (orange) (). Total number and percentage of overlapping peaks relative to Wdr5, or H3 K4me3 peaks were summarized in the table below. (B) Distribution of Mof and Mof/Wdr5 joint peaks (top) or Mof/H3K4me3 joint peaks (bottom) as class I or class II peaks. Red arrow, TSS. Y-axis, % peaks relative to total peaks within the defined region. X-axis, each bin represents a 500bp region. (C) The box plots for fold changes in expression of total (white), Mof/Wdr5 (pink) and Mof only (blue) target genes. (D) The box plots for fold changes in expression of total (white), Mof/H3K4me3 (orange) and Mof only (blue) target genes. For (C, D), bottom and top of the boxes correspond to the 25th and 75th percentiles and the internal band is the 50^th percentile (median). The plot whiskers extending outside the boxes correspond to the lowest and highest datum within 1.5 interquartile ranges. p-values were calculated using non-paired Wilcoxon tests as indicated. The number of genes in each category was indicated on bottom. Left, down regulated gene set. Right, up regulated gene set. (E) ChIP experiments for Wdr5 (top) and H3 K4me3 (bottom) at selected joint target genes in WT and Mof ^−/− ESCs. The antibodies used for ChIP were indicated on top. Signals for each experiment were normalized to 5% input. Means and standard deviations (as error bars) from at least three independent experiments were presented. Also see .