Figure 5.

Otx2 and Oct4 Coregulate Target Gene Expression

(A) Volcano plot of microarray data showing gene expression changes in mESCs (Rex1GFPd2) cells 12 hr after “2i” withdrawal following Otx2 depletion or treatment with control siRNAs (top). The table depicts the intersection between expression microarray and Otx2 ChIP-seq data sets (bottom).

(B) Pie charts showing the proportion of genes up or downregulated upon withdrawal of 2i from ESCs for 12 hr, for subcategories of genes that are either activated (top) or repressed (bottom) by Otx2.

(C) Summary of gene expression changes affected by Otx2 and Oct4 depletion observed 12 hr after 2i release. p Values are calculated from a hypergeometric distribution.

(D) UCSC genome browser view of Otx2 binding profiles and RNA-seq tags associated with the Slc16a3 locus. Data are shown for the indicated times following release from 2i and for the RNA-seq data, treatment with control (ctrl) or the indicated RNAi (i) duplexes. RPKM values for each time point are shown on the right.

(E and F) Heatmap (E) and selected examples (F) of qRT-PCR analysis with the Fluidigm system of mRNA expression of the indicated genes over 24 hr following 2i withdrawal. Cells were treated with control, Otx2, or Oct4 siRNAs where indicated. Data in (E) are row Z score normalized and are presented in (F) as means ± SD (n ≥ 3). Genes associated with Oct4 binding regions are indicated by purple dots.

(G) Expression analysis of the genes in the “primary neural tube formation” gene ontology category that are direct targets of Otx2 and Oct4, either in 2i conditions or upon withdrawal from 2i for 12 hr in the presence of control (ctrl) or Otx2 siRNAs. Data are from RNA-seq analysis and are shown as a heatmap (row Z-normalized; left) and the individual profiles of the top six genes in the heatmap are shown graphically (middle). A UCSC genome browser view of the ChIP profiles across the Enah locus is shown on the right.

See also Figure S5.