Enhancer-promoter interactions and transcription are largely maintained upon acute loss of CTCF, cohesin, WAPL or YY1.

It remains unclear why acute depletion of CTCF (CCCTC-binding factor) and cohesin only marginally affects expression of most genes despite substantially perturbing three-dimensional (3D) genome folding at the level of domains and structural loops. To address this conundrum, we used high-resolution Micro-C and nascent transcript profiling in mouse embryonic stem cells. We find that enhancer-promoter (E-P) interactions are largely insensitive to acute (3-h) depletion of CTCF, cohesin or WAPL.

Mapping Mammalian 3D Genomes by Micro-C.

3D genome mapping aims at connecting the physics of chromatin folding to the underlying biological events, and applications of various chromosomal conformation capture (3C) assays continue to discover critical roles of genome folding in regulating nuclear functions. To interrogate the full spectrum of chromatin folding ranging from the level of nucleosomes to full chromosomes in mammals, we developed an enhanced 3C-based method called Micro-C. The protocol employs Micrococcal nuclease (MNase) to fragment the genome, which overcomes the resolution limit of restriction enzyme-based methods, enabling the estimation of contact frequencies between proximal nucleosomes.

Resolving the 3D Landscape of Transcription-Linked Mammalian Chromatin Folding.

Whereas folding of genomes at the large scale of epigenomic compartments and topologically associating domains (TADs) is now relatively well understood, how chromatin is folded at finer scales remains largely unexplored in mammals. Here, we overcome some limitations of conventional 3C-based methods by using high-resolution Micro-C to probe links between 3D genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregates TAD regions into various finer-scale structures with distinct regulatory features including stripes, dots, and domains linking promoters-to-promoters (P-P) or enhancers-to-promoters (E-P) and bundle contacts between Polycomb regions.