Resolving the 3D landscape of transcription-linked mammalian chromatin folding

Whereas folding of mammalian genomes at the large scale of epigenomic compartments and topologically associating domains (TADs) is now relatively well-understood, how chromatin is folded below this scale remains largely unexplored in mammals. Here, we overcome this limitation using a high-resolution 3C-based method, Micro-C, and probe the links between 3D-genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregate TAD regions into various finer-scale structures with distinct regulatory features (i.e. stripes, dots, and domains linking promoter-promoter (P-P) or enhancer-promoter (E-P), and bundle contacts between Polycomb regions). E-P stripes extending from the edge of domains predominantly link co-expressed loci, often independently of CTCF and cohesin occupancy. Acute inhibition of transcription disrupts the gene-related folding features without altering higher-order chromatin structures. Analysis of ligation events sheds light on both the putative loop extrusion model and the “two-start” zig-zag 30-nanometer model of the chromatin fiber. Our work uncovers the finer-scale genome organization that establishes novel functional links between chromatin folding and gene regulation. We generated 38 replicates of Micro-C in wildtype mouse embryonic stem cells and 3 replicates with RNA polymerase II inhibition. We merged WT data into two different levels of sequencing coverage with 1.3B and 2.6B reads. The Micro-C contact information was then converted to the formats for HiGlass (.mcool) or Juicer browse (.hic). Notes that some replicates are in low read depth or low quality due to the process of optimizing protocol. We also generated ChIP-seq data of Pol II upon transcription inhibition (2 replicates for each).