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 approach3,4,5 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 neurosphere 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.
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The authors thank J. Mariani for the preparation of RNA from NPC; K. Murphy and A. Ku for their assistance with zebrafish enhancer assays; and A. Visel and A. Nord for discussion and their comments on the manuscript. S.N. and R.F. were supported by grants from ASTIL Regione Lombardia (SAL-19 ref. no. 16874), Telethon (GGP12152), Cariplo (Rif. 2010-0673) and AIRC (IG-5801). N.A. is supported by NINDS grant number R01NS079231, NICHD grant number R01HD059862, NHGRI grant numbers R01HG005058 and R01HG006768, NIDDK award number R01DK090382, NIGMS award number GM61390 and Simons Foundation SFARI no. 256769. R.Y.B. is supported by NINDS grant number R01NS079231 and the UCSF Program for Biomedical Breakthrough Research (PBBR). This work was supported by Agency for Science, Technology and Research (A*STAR), Singapore, the Office of Science of the U.S. Department of Energy under contract no. DE-AC02-05CH11231 and National Institutes of Health ENCODE grants (R01 HG004456-01, R01HG003521-01 and 1U54HG004557-01) to Y.R. and C.-L.W.
The authors declare no competing financial interests.
Extended data figures and tables
a–c, Fold enrichments (y axes) of RNAPII ChIP in selected regions (x axes) from three different cell lines (mouse ESCs (a), NSCs (b) and NPCs (c)) are shown. Two replicates of ChIP were tested via ChIP-qPCR and are represented as different colours (red and blue).
RNAPII binding sites, intra- and interchromosomal interactions identified from each cell type are shown.
Extended Data Figure 3 Promoter-mediated interactions and associated gene expression levels in NSCs and NPCs.
a, Distribution of defined interaction between promoters, inter- and intragenic regions in NSCs (top) and NPCs (bottom). b, Boxplots of the expression level (RPKM, y axes) between genes tethered by RNAPII and genes without tethered interactions (x axes) in NSCs (top) and NPCs (bottom).
Enhancers for Nanog (top left), Phc1 (top right), Lefty1 (bottom right) and Oct4 (bottom left) uncovered through 3C analysis in mESC V6.5 (middle black track) and ChIA-PET analysis in mESC E14 (bottom red track).
Phylogenetic conservation represented by PhastCon scores30 of the putative enhancer regions in comparison with other types of genomic regions.
NSC- and NPC-specific interactions detected from promoters of early developmental genes (left) Adam12 (top; chromosome 7:141165832—141495831), Vav3 (middle; chromosome 3:108932769—109282768) and Hoxa (bottom; chromosome 6:51730841—52230840) as well as key telencephalic homeobox transcription factors (right) Otx1 (top, chromosome 11:21878211—21998210) and Meis2 (bottom, chromosome 2:115603679—116003678). Dotted connecting lines depict the defined interactions with the distances labelled. The RNAPII binding peaks are shown in the middle track, followed by PET mapping in NSCs and NPCs, respectively.
Extended Data Figure 7 Examples for interacting promoter nodes with their cell specificity and enhancer connectivity found in all three cell types.
a, ESC-specific promoter nodes (Fzd7) with E:P (2:1). b, NSC- and NPC-specific promoter nodes (Fabp7) with E:P (1:1 and M:1 M: multiple, ≥2). c, Promoter nodes (Sox2) found in three different cell lines with dynamic E:P interactions.
Extended Data Figure 8 Connectivity constructed from the one-hop interactions mediated from reprogramming factor genes in ESCs.
Different colours represent different categories of cell specificity; the different sizes of the nodes represent non-promoter, promoter and iPS (induced pluripotent stem cell) factor nodes.
All the interaction nodes directly connecting Sox2 are highlighted and gene names are labelled. The connectivities between Sox2, Myc and Pou3f2 are highlighted by thick grey lines.
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Zhang, Y., Wong, CH., Birnbaum, R. et al. Chromatin connectivity maps reveal dynamic promoter–enhancer long-range associations. Nature 504, 306–310 (2013). https://doi.org/10.1038/nature12716
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