A gene regulatory network in mouse embryonic stem cells

Abstract

We analyze new and existing expression and transcription factor-binding data to characterize gene regulatory relations in mouse ES cells (ESC). In addition to confirming the key roles of Oct4, Sox2, and Nanog, our analysis identifies several genes, such as Esrrb, Stat3, Tcf7, Sall4, and LRH-1, as statistically significant coregulators. The regulatory interactions among 15 core regulators are used to construct a gene regulatory network in ESC. The network encapsulates extensive cross-regulations among the core regulators, highlights how they may control epigenetic processes, and reveals the surprising roles of nuclear receptors. Our analysis also provides information on the regulation of a large number of putative target genes of the network.

Fig. 1.

Overview of the Oct4-sorted series. (A) FACS in an ESC to EB differentiation time course followed by expression profiling. The cells were separated by FACS into undifferentiated (GFP+) and differentiated (GFP−) subpopulations, as indicated by green and white solid circles, respectively. Microarray expression profiling was performed on the sorted subpopulations of cells, and we used the resulting data as the basis for identifying Oct4-sorted± genes. (B) Expression levels of selected marker genes in the Oct4-high and -low samples as measured by the percentage of maximal expression. The error bars indicate the standard errors of the expression levels. (C) Overlaps between the gene sets in the Oct4-sorted, Oct4-RNAi, and RA-induction series. The numbers in the Venn diagrams represent the counts of the contiguous regions (gene sets) where they appear. The P values of the overlaps are calculated by the hypergeometric distribution (SI Text).

Fig. 2.

The overlaps between Oct4-sorted± genes and Oct4-bound (A), Nanog-bound (B), and Phc1-bound (C) genes. The statistical significance (P value) is calculated on the basis of the hypergeometric distribution, and the enrichment level R is defined as the ratio of the number of observed overlaps over that of expected overlaps. The computations of the P value and enrichment level are explained in SI Text.

Fig. 3.

A regulatory network in mouse ESC anchored on the master regulators Oct4, Sox2, and Nanog. The network represents the interactions among the core regulators (pink) and their protein-interaction partners (yellow). Blue and pink arrows indicate regulatory interactions inferred by anchor sites and by sites of coregulators within 150 bp of the anchor sites, respectively. Orange and green lines represent protein interactions identified in ref. and reported in the literature, respectively. Arrows from a dashed ellipse indicate that the targets are regulated by all of the regulators inside the ellipse. Some regulators appear multiple times in the network to reduce the number of intersecting arrows.