Goel, V. Y. | bioRxiv Science

bioRxiv2023

Integrative PTEN Enhancer Discovery Reveals a New Model of Enhancer Organization

Enhancers possess both structural elements mediating promoter looping and functional elements mediating gene expression. Traditional models of enhancer-mediated gene regulation imply genomic overlap or immediate adjacency of these elements. We test this model by combining densely-tiled CRISPRa screening with nucleosome-resolution Region Capture Micro-C topology analysis. Using this integrated approach, we comprehensively define the cis-regulatory landscape for the tumor suppressor PTEN, identifying and validating 10 distinct enhancers and defining their 3D spatial organization. Unexpectedly, we identify several long-range functional enhancers whose promoter proximity is facilitated by chromatin loop anchors several kilobases away, and demonstrate that accounting for this spatial separation improves the computational prediction of validated enhancers. Thus, we propose a new model of enhancer organization incorporating spatial separation of essential functional and structural components.

genomics↗

bioRxiv2022

Region Capture Micro-C reveals coalescence of enhancers and promoters into nested microcompartments

Although enhancers are central to the regulation of mammalian gene expression, the mechanisms underlying Enhancer-Promoter (E-P) interactions remain unclear. Chromosome conformation capture (3C) methods effectively capture large-scale 3D genome structure but struggle to achieve the depth necessary to resolve fine-scale E-P interactions. Here, we develop Region Capture Micro-C (RCMC) by combining MNase-based 3C with a tiling region-capture approach and generate the deepest 3D genome maps reported thus far with only modest sequencing. By applying RCMC in mouse embryonic stem cells and reaching the genome-wide equivalent of [~]200 billion unique contacts, RCMC reveals previously unresolvable patterns of highly nested and focal 3D interactions, which we term microcompartments. Microcompartments frequently connect enhancers and promoters and are largely robust to loss of loop extrusion and inhibition of transcription. We therefore propose that many E-P interactions form through a compartmentalization mechanism, which may explain why acute cohesin depletion only modestly affects global gene expression.

genomics↗