Transcription factors (TFs) and their specific interactions with targets are crucial for specifying gene-expression programs. To gain insights into the transcriptional regulatory networks in embryonic stem (ES) cells, we use chromatin immunoprecipitation coupled with ultra-high-throughput DNA sequencing (ChIP-seq) to map the locations of 13 sequence-specific TFs (Nanog, Oct4, STAT3, Smad1, Sox2, Zfx, c-Myc, n-Myc, Klf4, Esrrb, Tcfcp2l1, E2f1, and CTCF) and 2 transcription regulators (p300 and Suz12). These factors are known to play different roles in ES-cell biology as components of the LIF and BMP signaling pathways, self-renewal regulators, and key reprogramming factors. Our study provides insights into the integration of the signaling pathways into the ES-cell-specific transcription circuitries. Intriguingly, we find specific genomic regions extensively targeted by different TFs. Collectively, the comprehensive mapping of TF-binding sites identifies important features of the transcriptional regulatory networks that define ES-cell identity.

SingaporeDNA methylation is a critical epigenetic regulator in mammalian development. Here, we present a whole-genome comparative view of DNA methylation using bisulfite sequencing of three cultured cell types representing progressive stages of differentiation: human embryonic stem cells (hESCs), a fibroblastic differentiated derivative of the hESCs, and neonatal fibroblasts. As a reference, we compared our maps with a methylome map of a fully differentiated adult cell type, mature peripheral blood mononuclear cells (monocytes). We observed many notable common and cell-type-specific features among all cell types. Promoter hypomethylation (both CG and CA) and higher levels of gene body methylation were positively correlated with transcription in all cell types. Exons were more highly methylated than introns, and sharp transitions of methylation occurred at exon-intron boundaries, suggesting a role for differential methylation in transcript splicing. Developmental stage was reflected in both the level of global methylation and extent of non-CpG methylation, with hESC highest, fibroblasts intermediate, and monocytes lowest. Differentiation-associated differential methylation profiles were observed for developmentally regulated genes, including the HOX clusters, other homeobox transcription factors, and pluripotence-associated genes such as POU5F1, TCF3, and KLF4. Our results highlight the value of high-resolution methylation maps, in conjunction with other systems-level analyses, for investigation of previously undetectable developmental regulatory mechanisms.

Mammalian genomes are viewed as functional organizations that orchestrate spatial and temporal gene regulation. CTCF, the most characterized insulator-binding protein, has been implicated as a key genome organizer. Yet, little is known about CTCF-associated higher order chromatin structures at a global scale. Here, we applied Chromatin Interaction Analysis by Paired-End-Tag sequencing to elucidate the CTCF-chromatin interactome in pluripotent cells. From this analysis, 1,480 cis and 336 trans interacting loci were identified with high reproducibility and precision. Associating these chromatin interaction loci with their underlying epigenetic states, promoter activities, enhancer binding and nuclear lamina occupancy, we uncovered five distinct chromatin domains that suggest potential new models of CTCF function in chromatin organization and transcriptional control. Specifically, CTCF interactions demarcate chromatin-nuclear membrane attachments and influence proper gene expression through extensive crosstalk between promoters and regulatory elements. This highly complex nuclear organization offers insights towards the unifying principles governing genome plasticity and function.

In multicellular organisms, transcription regulation is one of the central mechanisms modelling lineage differentiation and cell-fate determination1. Transcription requires dynamic chromatin configurations between promoters and their corresponding distal regulatory elements2. It is believed that their communication occurs within large discrete foci of aggregated RNA polymerases termed transcription factories in three-dimensional nuclear space3. However, the dynamic nature of chromatin connectivity has not been characterized at the genome-wide level. Here, through a chromatin interaction analysis with paired-end tagging approach3–5 using an antibody that primarily recognizes the pre-initiation complexes of RNA polymerase II6, we explore the transcriptional interactomes of three mouse cells of progressive lineage commitment, including pluripotent embryonic stem cells7, neural stem cells8 and neuro-sphere stem/progenitor cells9. Our global chromatin connectivity maps reveal approximately 40,000 long-range interactions, suggest precise enhancer–promoter associations and delineate cell-type-specific chromatin structures. Analysis of the complex regulatory repertoire shows that there are extensive colocalizations among promoters and distal-acting enhancers. Most of the enhancers associate with promoters located beyond their nearest active genes, indicating that the linear juxtaposition is not the only guiding principle driving enhancer target selection. Although promoter–enhancer interactions exhibit high cell-type specificity, promoters involved in interactions are found to be generally common and mostly active among different cells. Chromatin connectivity networks reveal that the pivotal genes of reprogramming functions are transcribed within physical proximity to each other in embryonic stem cells, linking chromatin architecture to coordinated gene expression. Our study sets the stage for the full-scale dissection of spatial and temporal genome structures and their roles in orchestrating development.