Capturing Chromosome Conformation Across Length Scales

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) ...

University of Massachusetts Medical School

Memory cross-reactive CD8+ T-cell responses may induce protection or immunopathology upon secondary viral challenge. To elucidate the potential role of T cells in sequential flavivirus infection, we characterized cross-reactive CD4+ and CD8+ T-cell responses between attenuated and pathogenic Japanese encephalitis virus (JEV) and pathogenic West Nile virus (WNV). A previously reported WNV NS4b CD8+ T-cell epitope and its JEV variant elicited CD8+ T-cell responses in both JEV- and WNV-infected mice. The peptide variant homologous to the immunizing virus induced greater cytokine secretion and activated higher frequencies of epitope-specific CD8+ T cells. However, there was a virus-dependent, peptide variant-independent pattern of cytokine secretion; the IFNgamma+-to-IFNgamma+TNFalpha+ CD8+ T-cell ratio was greater in JEV- than in WNV-infected mice. Despite similarities in viral burden for pathogenic WNV and JEV viruses, CD8+ T cells from pathogenic JEV-immunized mice exhibited functional and phenotypic profiles similar to those seen for the attenuated JEV strain. Patterns of killer cell lectin-like receptor G1 (KLRG1) and CD127 expression differed by virus type, with a rapid expansion and contraction of short-lived effector cells in JEV infection and persistence of high levels of short-lived effector cells in WNV infection. Such cross-reactive T-cell responses to primary infection may affect the outcomes of sequential flavivirus infections.

Altered effector functions of virus-specific and virus cross-reactive CD8+ T cells in mice immunized with related flaviviruses.

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 ∼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.

Ultrastructural Details of Mammalian Chromosome Architecture.

During mitosis chromosomes reorganise into highly compact, rod-shaped forms, thought to consist of consecutive chromatin loops around a central protein scaffold. Condensin complexes are involved in chromatin compaction, but the contribution of other chromatin proteins, DNA sequence and histone modifications is less understood. A large region of fission yeast DNA inserted into a mouse chromosome was previously observed to adopt a mitotic organisation distinct from that of surrounding mouse DNA. Here, we show that a similar distinct structure is common to a large subset of insertion events in both mouse and human cells and is coincident with the presence of high levels of heterochromatic H3 lysine nine trimethylation (H3K9me3). Hi-C and microscopy indicate that the heterochromatinised fission yeast DNA is organised into smaller chromatin loops than flanking euchromatic mouse chromatin. We conclude that heterochromatin alters chromatin loop size, thus contributing to the distinct appearance of heterochromatin on mitotic chromosomes.

Large domains of heterochromatin direct the formation of short mitotic chromosome loops.

The intricate folding of chromatin enables living organisms to store genomic material in an extremely small volume while facilitating proper cell function. Hi-C is a chromosome conformation capture (3C)-based technology to detect pair-wise chromatin interactions genome-wide, and has become a benchmark tool to study genome organization. In Hi-C, chromatin conformation is first captured by chemical cross-linking of cells. Cells are then lysed and subjected to restriction enzyme digestion, before the ends of the resulting fragments are marked with biotin. Fragments within close 3D proximity are ligated, and the biotin label is used to selectively enrich for ligated junctions. Finally, isolated ligation products are prepared for high-throughput sequencing, which enables the mapping of pair-wise chromatin interactions genome-wide. Over the past decade, "next-generation" sequencing has become cheaper and easier to perform, enabling more interactions to be sampled to obtain higher resolution in chromatin interaction maps. Here, we provide an in-depth guide to performing an up-to-date Hi-C procedure on mammalian cell lines. These protocols include recent improvements that increase the resolution potential of the assay, namely by enhancing cross-linking and using a restriction enzyme cocktail. These improvements result in a versatile Hi-C procedure that enables the detection of genome folding features at a wide range of distances. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fixation of nuclear conformation Basic Protocol 2: Chromosome conformation capture Basic Protocol 3: Hi-C sequencing library preparation.

Hi-C 3.0: Improved Protocol for Genome-Wide Chromosome Conformation Capture.

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.

Liyan Yang

Department of Systems Biology

University of Massachusetts Medical School

Capturing Chromosome Conformation Across Length Scales

Liyan Yang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Systems Biology

University of Massachusetts Medical School

Capturing Chromosome Conformation Across Length Scales

Department of Systems Biology