Structural variants (SVs) can result in changes in gene expression due to abnormal chromatin folding and cause disease. However, the prediction of such effects remains a challenge. Here we present a polymer-physics-based approach (PRISMR) to model 3D chromatin folding and to predict enhancer-promoter contacts. PRISMR predicts higher-order chromatin structure from genome-wide chromosome conformation capture (Hi-C) data. Using the EPHA4 locus as a model, the effects of pathogenic SVs are predicted in silico and compared to Hi-C data generated from mouse limb buds and patient-derived fibroblasts. PRISMR deconvolves the folding complexity of the EPHA4 locus and identifies SV-induced ectopic contacts and alterations of 3D genome organization in homozygous or heterozygous states. We show that SVs can reconfigure topologically associating domains, thereby producing extensive rewiring of regulatory interactions and causing disease by gene misexpression. PRISMR can be used to predict interactions in silico, thereby providing a tool for analyzing the disease-causing potential of SVs.

Chromosomes have a complex architecture in the cell nucleus, which serves vital functional purposes, yet its structure and folding mechanisms remain still incompletely understood. Here we show that genome-wide chromatin architecture data, as mapped by Hi-C methods across mammalian cell types and chromosomes, are well described by classical scaling concepts of polymer physics, from the sub-Mb to chromosomal scales. Chromatin is a complex mixture of different regions, folded in the conformational classes predicted by polymer thermodynamics. The contact matrix of the Sox9 locus, a region linked to severe human congenital diseases, is derived with high accuracy in mESCs and its molecular determinants identified by the theory; Sox9 self-assembles hierarchically in higher-order domains, involving abundant many-body contacts. Our approach is also applied to the Bmp7 locus. Finally, the model predictions on the effects of mutations on folding are tested against available data on a deletion in the Xist locus. Our results can help progressing new diagnostic tools for diseases linked to chromatin misfolding.

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The CCCTC-binding factor (CTCF) works together with the cohesin complex to drives the formation of chromatin loops and topologically associating domains, but its role in gene regulation has not been fully defined. Here, we investigated the effects of acute CTCF loss on chromatin architecture and transcriptional programs in mouse embryonic stem cells undergoing differentiation to neural precursor cells. We identified CTCF-dependent enhancer-promoter contacts genome-wide and found that they disproportionally affect genes that are bound by CTCF at promoter and dependent on long-distance enhancers. Disruption of promoter-proximal CTCF binding reduced both long-range enhancer-promoter contacts and transcription, which are restored by artificial tethering of CTCF to the promoter. Promoter-proximal CTCF binding is correlated with transcription of over 2,000 genes, across a diverse set of adult tissues. Taken together, our study shows that CTCF binding to promoters may promote long-distance-enhancer dependent transcription at specific genes in diverse cell types.

The spatial organization of chromosomes has key functional roles, yet how chromosomes fold remains poorly understood at the single-molecule level. Here, we employ models of polymer physics to investigate DNA loci in human HCT116 and IMR90 wild-type and cohesin depleted cells. Model predictions on single-molecule structures are validated against singlecell imaging data, providing evidence that chromosomal architecture is controlled by a thermodynamics mechanism of polymer phase separation whereby chromatin self-assembles in segregated globules by combinatorial interactions of chromatin factors that include CTCF and cohesin. The thermodynamics degeneracy of single-molecule conformations results in broad structural and temporal variability of TAD-like contact patterns. Globules establish stable environments where specific contacts are highly favored over stochastic encounters. Cohesin depletion reverses phase separation into randomly folded states, erasing average interaction patterns. Overall, globule phase separation appears to be a robust yet reversible mechanism of chromatin organization where stochasticity and specificity coexist.