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.

Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.

SUMMARY The human genome folds to create thousands of intervals, called “contact domains,” that exhibit enhanced contact frequency within themselves. “Loop domains” form because of tethering between two loci – almost always bound by CTCF and cohesin – lying on the same chromosome. “Compartment domains” form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation, but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes, and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although reformation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.

SUMMARY MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here a new map of the transcriptional regulatory circuitry of ES cells that incorporates both protein-coding and miRNA genes, and which is based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for most miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb Group proteins in ES cells and expressed in a tissue-specific fashion in differentiated cells. These data reveal how key ES cell transcription factors promote the miRNA expression program that contributes to normal self-renewal and cellular differentiation, and integrate miRNAs and their targets into an expanded model of the regulatory circuitry controlling ES cell identity.

Genome-wide location analysis, also known as ChIP-Chip, combines chromatin immunoprecipitation and DNA microarray analysis to identify protein-DNA interactions that occur in living cells. Protein-DNA interactions are captured in vivo by chemical crosslinking. Cell lysis, DNA fragmentation and immunoaffinity purification of the desired protein will co-purify DNA fragments that are associated with that protein. The enriched DNA population is then labeled, combined with a differentially labeled reference sample and applied to DNA microarrays to detect enriched signals. Various computational and bioinformatic approaches are then applied to normalize the enriched and reference channels, to connect signals to the portions of the genome that are represented on the DNA microarrays, to provide confidence metrics and to generate maps of protein-genome occupancy. Here, we describe the experimental protocols that we use from crosslinking of cells to hybridization of labeled material, together with insights into the aspects of these protocols that influence the results. These protocols require approximately 1 week to complete once sufficient numbers of cells have been obtained, and have been used to produce robust, high-quality ChIP-chip results in many different cell and tissue types.

Embryonic stem (ES) cells have a unique regulatory circuitry, largely controlled by the transcription factorsOct4, Sox2, and Nanog, which generates a gene expression program necessary for pluripotency and self-renewal. How external signals connect to this regulatory circuitry to influence ES cell fate is not known. We report here that a terminal component of the canonical Wnt pathway in ES cells, the transcription factor T-cell factor-3 (Tcf3), co-occupies promoters throughout the genome in association with the pluripotency regulators Oct4 and Nanog. Thus, Tcf3 is an integral component of the core regulatory circuitry of ES cells, which includes an autoregulatory loop involving the pluripotency regulators. Both Tcf3 depletion and Wnt pathway activation cause increased expression of Oct4, Nanog, and other pluripotency factors and produce ES cells that are refractory to differentiation. Our results suggest that the Wnt pathway, through Tcf3, brings developmental signals directly to the core regulatory circuitry of ES cells to influence the balance between pluripotency and differentiation. Embryonic stem (ES) cells provide a unique opportunity to study early development and hold great promise for regenerative medicine (Thomson et al. 1998;Reubinoff et al. 2000;Pera and Trounson 2004). ES cells are derived from the inner cell mass of the developing blastocyst, and can be propagated in culture in an undifferentiated state while maintaining the capacity to generate any cell type in the body. Discovering how signaling pathways and transcriptional regulatory circuitry contribute to self-renewal and pluripotency is essential for understanding early development and realizing the therapeutic potential of ES cells.A model for the core transcriptional regulatory circuitry of ES cells has emerged from studying the target genes of the ES cell transcription factors Oct4, Sox2, and Nanog (Boyer et al. 2005;Loh et al. 2006). These master regulators occupy the promoters of active genes encoding transcription factors, signal transduction components, and chromatin-modifying enzymes that promote ES cell self-renewal. They also occupy the promoters of a large set of developmental transcription factors that are silent in ES cells, but whose expression is associated with lineage commitment and cellular differentiation. Polycomb Repressive Complexes co-occupy the genes encoding these developmental transcription factors to help maintain a silent transcriptional state in ES cells Lee et al. 2006;Wilkinson et al. 2006;Rajasekhar and Begemann 2007;Stock et al. 2007).External signals can promote ES cell pluripotency or cause these cells to differentiate, but precisely how these pathways are connected to the ES cell regulatory network has not been determined. These signals are produced by the stem cell niche in the developing blastocyst or, for cultured ES cells, can be produced by added factors or serum to maintain stem cell identity or promote differentiation. Recent studies have demonstrated the importance of several signaling pathways in m...

Many DNA-hypermethylated cancer genes are occupied by the Polycomb (PcG) repressor complex in embryonic stem cells (ESCs). Their prevalence in the full spectrum of cancers, the exact context of chromatin involved, and their status in adult cell renewal systems are unknown. Using a genome-wide analysis, we demonstrate that ∼75% of hypermethylated genes are marked by PcG in the context of bivalent chromatin in both ESCs and adult stem/progenitor cells. A large number of these genes are key developmental regulators, and a subset, which we call the “DNA hypermethylation module,” comprises a portion of the PcG target genes that are down-regulated in cancer. Genes with bivalent chromatin have a low, poised gene transcription state that has been shown to maintain stemness and self-renewal in normal stem cells. However, when DNA-hypermethylated in tumors, we find that these genes are further repressed. We also show that the methylation status of these genes can cluster important subtypes of colon and breast cancers. By evaluating the subsets of genes that are methylated in different cancers with consideration of their chromatin status in ESCs, we provide evidence that DNA hypermethylation preferentially targets the subset of PcG genes that are developmental regulators, and this may contribute to the stem-like state of cancer. Additionally, the capacity for global methylation profiling to cluster tumors by phenotype may have important implications for further refining tumor behavior patterns that may ultimately aid therapeutic interventions.

The pollen extracellular matrix contains proteins mediating species specificity and components needed for efficient pollination. We identified all proteins >10 kilodaltons in the Arabidopsis pollen coating and showed that most of the corresponding genes reside in two genomic clusters. One cluster encodes six lipases, whereas the other contains six lipid-binding oleosin genes, including GRP17, a gene that promotes efficient pollination. Individual oleosins exhibit extensive divergence between ecotypes, but the entire cluster remains intact. Analysis of the syntenic region in Brassica oleracea revealed even greater divergence, but a similar clustering of the genes. Such allelic flexibility may promote speciation in plants.