Comprehensive gene expression analyses of differentiating ES cells.

ES cells were treated with 10-Seq analyses. (A) RA-responsive genes (those whose expression levels were increased more than 5-fold compared with undifferentiated ES cells: 543 genes out of 14,255 eligible annotated genes) were plotted on the graph using modified fragments/kb of transcript/million fragments mapped (FPKM) values. The x-axis corresponds to expression levels of each gene (shown as log2 transformation of each FPKM value plus 0.1), and the y-axis corresponds to fold change in gene expression levels between ΔSET ES cells and wild-type (shown as Δlog2 transformation). The encircled area was enriched for some Hox and Wnt family genes (arrows, see also Figure S2). (B) Gene ontology analysis of ΔSET-impaired 152 genes. A subset of RA-responsive genes demonstrating a greater-than-2-fold decrease in the modified FPKM values in differentiating ΔSET ES cells was analyzed. Gene enrichment P-values were calculated by Chi-square test. The numbers of genes in each group are shown in parentheses. (C) Bar chart showing relative ratios of the numbers of genes carrying trimethylation of Lys27 [18], a hallmark of the Polycomb-regulated genes. Genes showing a difference greater than 2-fold were analyzed (up- or down-regulated). In addition, RA-responsive genes in the down-regulated genes were further extracted and analyzed [the last group, same as in (B)]. Chi-square testing was used for calculation of P-values against the total gene set, *P<0.001. The numbers of genes in each group are shown in the table below. (D) Radar chart showing relative ratio of status of chromatin signatures for indicated gene group as in (C). (E) Venn diagram showing the relationship between Ash1l-target genes and ΔSET-affected genes. The numbers of genes in each compartment are shown. The total number of annotated genes analyzed was 18,724.