Core transcriptional regulatory circuitry in human embryonic stem cells

Abstract

The transcription factors OCT4, SOX2, and NANOG have essential roles in early development and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture. To gain insights into transcriptional regulation of human ES cells, we have identified OCT4, SOX2, and NANOG target genes using genome-scale location analysis. We found, surprisingly, that OCT4, SOX2, and NANOG co-occupy a substantial portion of their target genes. These target genes frequently encode transcription factors, many of which are developmentally important homeodomain proteins. Our data also indicate that OCT4, SOX2, and NANOG collaborate to form regulatory circuitry consisting of autoregulatory and feedforward loops. These results provide new insights into the transcriptional regulation of stem cells and reveal how OCT4, SOX2, and NANOG contribute to pluripotency and self-renewal.

Figure 1. Genome-Wide Location Analysis in Human Embryonic Stem Cells

(A) DNA segments bound by transcriptional regulators were identified using chromatin immunoprecipitation (ChIP) and identified with DNA microarrays containing 60-mer oligonucleotide probes covering the region from −8 kb to +2 kb for 17,917 annotated transcription start sites for human genes. ES cell growth and quality control, ChIP protocol, DNA microarray probe design, and data analysis methods are described in detail in Experimental Procedures and Supplemental Data. (B) Examples of OCT4 bound regions. Plots display unprocessed ChIP-enrichment ratios for all probes within a genomic region. Genes are shown to scale below plots (exons and introns are represented by thick vertical and horizontal lines, respectively), and the genomic region represented is indicated beneath the plot. The transcription start site and transcript direction are denoted by arrows.

Figure 2. OCT4, SOX2, and NANOG Target Genes in Human ES Cells

(A) Venn diagram representing the overlap of OCT4, SOX2, and NANOG promoter bound regions. (B) Representative examples of protein-coding genes co-occupied by OCT4, SOX2, and NANOG. Plots display unprocessed ChIP-enrichment ratios for all probes within a genomic region. Genes are shown to scale relative to their chromosomal position. Exons and introns are represented by thick vertical and horizontal lines, respectively. The start and direction of transcription are denoted by arrows. Green, red, and purple lines represent NANOG, SOX2, and OCT4 bound regions, respectively. (C) OCT4, SOX2, and NANOG bind in close proximity. The distances between the midpoint of bound regions for pairs of transcription factors was calculated for the 353 regions bound by all three transcription factors. Negative and positive values indicate whether the first factor is upstream or downstream of the second factor in relation to the gene. The frequency of different distances between the bound regions is plotted as a histogram.

Figure 3. Expression of OCT4, SOX2, and NANOG Co-Occupied Genes

(A) Affymetrix expression data for ES cells were compared to a compendium of expression data from 158 experiments representing 79 other differentiated tissues and cell types (Supplemental data). Ratios were generated by comparing gene expression in ES cells to the median level of gene expression across all datasets for each individual gene. Genes were ordered by relative expression in ES cells, and the results were clustered by expression experiment using hierarchical clustering. Each gene is represented as a separate row and individual expression experiments are in separate columns. Red indicates higher expression in ES cells relative to differentiated cells. Green indicates lower expression in ES cells relative to differentiated cells. Examples of bound genes that are at the top and bottom of the rank order list are shown. (B) Relative levels of gene expression in H9 ES cells compared to differentiated cells were generated and converted to log2 ratios. The distribution of these fold changes was calculated to derive a profile for different sets of genes. Data are shown for the distribution of expression changes between H9 ES cells and differentiated tissues for transcription factor genes that are not occupied by OCT4, SOX2, and NANOG (solid black line) and transcription factor genes occupied by all three (dotted line). The change in relative expression is indicated on the x axis, and the numbers of genes in each bin are indicated on the y axes (left axis for unoccupied genes, right axis for occupied genes). The shift in distribution of expression changes for genes occupied by OCT4, SOX2, and NANOG is significant (p value < 0.001 using a two-sampled Kolmogorov-Smirnov test), consistent with the model that OCT4, SOX2, and NANOG are contributing to the regulation of these genes.

Figure 4. Transcriptional Regulatory Motifs in Human ES Cells

(A) An example of feedforward transcriptional regulatory circuitry in human ES cells. Regulators are represented by blue circles; gene promoters are represented by red rectangles. Binding of a regulator to a promoter is indicated by a solid arrow. Genes encoding regulators are linked to their respective regulators by dashed arrows. (B) The interconnected autoregulatory loop formed by OCT4, SOX2, and NANOG.

Figure 5. Core Transcriptional Regulatory Network in Human ES Cells

A model for the core transcriptional regulatory network was constructed by identifying OCT4, SOX2, and NANOG target genes that encode transcription factors and chromatin regulators and integrating knowledge of the functions of these downstream regulators based on comparison to multiple expression datasets (Supplemental Data) and to the literature. A subset of active and inactive genes co-occupied by the three factors in human ES cells is shown here. Regulators are represented by blue circles; gene promoters are represented by red rectangles; gray boxes represent putative downstream target genes. Positive regulation was assumed if the target gene was expressed whereas negative regulation was assumed if the target gene was not transcribed.