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.