Genome information processing by the INO80 chromatin remodeler positions nucleosomes [preprint]

The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones. INO80 processes such information through an allosteric interplay between its core- and Arp8-modules that probes mechanical properties of nucleosomal and linker DNA. At promoters, INO80 integrates this readout of DNA shape/mechanics with a readout of co-evolved sequence motifs via interaction with general regulatory factors bound to these motifs. Our findings establish a molecular mechanism for robust and yet adjustable +1 nucleosome positioning and, more generally, remodelers as information processing hubs that enable active organization and allosteric regulation of the first level of chromatin

Systematic evaluation of chromosome conformation capture assays [preprint]

Chromosome conformation capture (3C)-based assays are used to map chromatin interactions genome-wide. Quantitative analyses of chromatin interaction maps can lead to insights into the spatial organization of chromosomes and the mechanisms by which they fold. A number of protocols such as in situ Hi-C and Micro-C are now widely used and these differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of experimental parameters of 3C-based protocols. We find that different protocols capture different 3D genome features with different efficiencies. First, the use of cross-linkers such as DSG in addition to formaldehyde improves signal-to-noise allowing detection of thousands of additional loops and strengthens the compartment signal. Second, fragmenting chromatin to the level of nucleosomes using MNase allows detection of more loops. On the other hand, protocols that generate larger multi-kb fragments produce stronger compartmentalization signals. We confirmed our results for multiple cell types and cell cycle stages. We find that cell type-specific quantitative differences in chromosome folding are not detected or underestimated by some protocols. Based on these insights we developed Hi-C 3.0, a single protocol that can be used to both efficiently detect chromatin loops and to quantify compartmentalization. Finally, this study produced ultra-deeply sequenced reference interaction maps using conventional Hi-C, Micro-C and Hi-C 3.0 for commonly used cell lines in the 4D Nucleome Project

Ultrastructural Details of Mammalian Chromosome Architecture

Over the past decade, 3C-related methods have provided remarkable insights into chromosome folding in vivo. To overcome the limited resolution of prior studies, we extend a recently developed Hi-C variant, Micro-C, to map chromosome architecture at nucleosome resolution in human ESCs and fibroblasts. Micro-C robustly captures known features of chromosome folding including compartment organization, topologically associating domains, and interactions between CTCF binding sites. In addition, Micro-C provides a detailed map of nucleosome positions and localizes contact domain boundaries with nucleosomal precision. Compared to Hi-C, Micro-C exhibits an order of magnitude greater dynamic range, allowing the identification of approximately 20,000 additional loops in each cell type. Many newly identified peaks are localized along extrusion stripes and form transitive grids, consistent with their anchors being pause sites impeding cohesin-dependent loop extrusion. Our analyses comprise the highest-resolution maps of chromosome folding in human cells to date, providing a valuable resource for studies of chromosome organization

Systematic evaluation of chromosome conformation capture assays

Chromosome conformation capture (3C) assays are used to map chromatin interactions genome-wide. Chromatin interaction maps provide insights into the spatial organization of chromosomes and the mechanisms by which they fold. Hi-C and Micro-C are widely used 3C protocols that differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of 3C experimental parameters. We identified optimal protocol variants for either loop or compartment detection, optimizing fragment size and cross-linking chemistry. We used this knowledge to develop a greatly improved Hi-C protocol (Hi-C 3.0) that can detect both loops and compartments relatively effectively. In addition to providing benchmarked protocols, this work produced ultra-deep chromatin interaction maps using Micro-C, conventional Hi-C and Hi-C 3.0 for key cell lines used by the 4D Nucleome project

A Role for the Mre11-Rad50-Xrs2 Complex in Gene Expression and Chromosome Organization

International audienc