current
TCPA: Current C strategies measure 2-way contacts between pairs of sites. The results demonstrate that, in a population of cells, individual genomic sites often contact more than one other distal site. For example, many genes appear in contact with multiple dispersed regulatory DNA elements and vice versa, many regulatory DNA elements appear to contact each other as well as multiple genes. It is often proposed that such contacts occur simultaneously in so-called ‘hubs’. Yet, cell population-based pair-wise contact matrices do not allow making statements about cooperative interactions versus mutually-exclusive interactions. To understand the nature (and mere existence) of chromatin hubs, we need to know how the multitude of genes and regulatory sequences coordinate their action in 3D space. To do this in a meaningful manner, high-throughput strategies must be developed alongside with robust statistical methods and intuitive visualization tools for the detection, analysis and interpretation of multi-way DNA contacts. For this, we have invested in and propose to further develop and apply Multi-Contact Chromatin Capture that exploits Oxford Nanopore Technologies (ONT) long single molecule sequencing technology. Using Multi-Contact HiC (MC-HiC) we will generate genome-wide multi-way contact maps of >100x coverage for at least two of the 4DN reference cell lines (GM12878 and Castx129 ESCs). In addition, we will employ our targeted strategy, Multi-Contact 4C (MC-4C), for in depth analysis of the most intricate spatial regulatory networks. Our work is expected to greatly enhance our understanding of how the multitude of regulatory sequences and genes spatially coordinate their action on individual alleles. It should also deliver valuable new molecular biology and computational tools for studying these under-explored dimensions of genome folding and functioning.