Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization.

Several general principles of global 3D genome organization have recently been established, including non-random positioning of chromosomes and genes in the cell nucleus, distinct chromatin compartments, and topologically associating domains (TADs). However, the extent and nature of cell-to-cell and cell-intrinsic variability in genome architecture are still poorly characterized. Here, we systematically probe heterogeneity in genome organization. High-throughput optical mapping of several hundred intra-chromosomal interactions in individual human fibroblasts demonstrates low association frequencies, which are determined by genomic distance, higher-order chromatin architecture, and chromatin environment. The structure of TADs is variable between individual cells, and inter-TAD associations are common. Furthermore, single-cell analysis reveals independent behavior of individual alleles in single nuclei. Our observations reveal extensive variability and heterogeneity in genome organization at the level of individual alleles and demonstrate the coexistence of a broad spectrum of genome configurations in a cell population.

Jump-starting life: balancing transposable element co-option and genome integrity in the developing mammalian embryo.

Remnants of transposable elements (TEs) are widely expressed throughout mammalian embryo development. Originally infesting our genomes as selfish elements and acting as a source of genome instability, several of these elements have been co-opted as part of a complex system of genome regulation. Many TEs have lost transposition ability and their transcriptional potential has been tampered as a result of interactions with the host throughout evolutionary time. It has been proposed that TEs have been ultimately repurposed to function as gene regulatory hubs scattered throughout our genomes. In the early embryo in particular, TEs find a perfect environment of naïve chromatin to escape transcriptional repression by the host. As a consequence, it is thought that hosts found ways to co-opt TE sequences to regulate large-scale changes in chromatin and transcription state of their genomes. In this review, we discuss several examples of TEs expressed during embryo development, their potential for co-option in genome regulation and the evolutionary pressures on TEs and on our genomes.

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.

Detecting chromatin interactions between and along sister chromatids with SisterC.

Chromosome segregation requires both compaction and disentanglement of sister chromatids. We describe SisterC, a chromosome conformation capture assay that distinguishes interactions between and along identical sister chromatids. SisterC employs 5-bromo-2'-deoxyuridine (BrdU) incorporation during S-phase to label newly replicated strands, followed by Hi-C and then the destruction of 5-bromodeoxyuridine-containing strands via Hoechst/ultraviolet treatment. After sequencing of the remaining intact strands, this allows assignment of Hi-C products as inter- and intra-sister interactions based on the strands that reads are mapped to. We performed SisterC on mitotic Saccharomyces cerevisiae cells. We find precise alignment of sister chromatids at centromeres. Along arms, sister chromatids are less precisely aligned, with inter-sister connections every ~35 kilobase (kb). Inter-sister interactions occur between cohesin binding sites that are often offset by 5 to 25 kb. Along sister chromatids, cohesin results in the formation of loops of up to 50 kb. SisterC allows study of the complex interplay between sister chromatid compaction and their segregation during mitosis.

CTCF sites display cell cycle-dependent dynamics in factor binding and nucleosome positioning.

CCCTC-binding factor (CTCF) plays a key role in the formation of topologically associating domains (TADs) and loops in interphase. During mitosis TADs are absent, but how TAD formation is dynamically controlled during the cell cycle is not known. Several contradicting observations have been made regarding CTCF binding to mitotic chromatin using both genomics- and microscopy-based techniques. Here, we have used four different assays to address this debate. First, using 5C, we confirmed that TADs and CTCF loops are readily detected in interphase, but absent during prometaphase. Second, ATAC-seq analysis showed that CTCF sites display greatly reduced accessibility and lose the CTCF footprint in prometaphase, suggesting loss of CTCF binding and rearrangement of the nucleosomal array around the binding motif. In contrast, transcription start sites remain accessible in prometaphase, although adjacent nucleosomes can also become repositioned and occupy at least a subset of start sites during mitosis. Third, loss of site-specific CTCF binding was directly demonstrated using CUT&RUN. Histone modifications and histone variants are maintained in mitosis, suggesting a role in bookmarking of active CTCF sites. Finally, live-cell imaging, fluorescence recovery after photobleaching, and single molecule tracking showed that almost all CTCF chromatin binding is lost in prometaphase. Combined, our results demonstrate loss of CTCF binding to CTCF sites during prometaphase and rearrangement of the chromatin landscape around CTCF motifs. This, combined with loss of cohesin, would contribute to the observed loss of TADs and CTCF loops during mitosis and reveals that CTCF sites, key architectural cis-elements, display cell cycle stage-dependent dynamics in factor binding and nucleosome positioning.

Epigenetic characteristics of the mitotic chromosome in 1D and 3D.

While chromatin characteristics in interphase are widely studied, characteristics of mitotic chromatin and their inheritance through mitosis are still poorly understood. During mitosis, chromatin undergoes dramatic changes: transcription stalls, chromatin-binding factors leave the chromatin, histone modifications change and chromatin becomes highly condensed. Many key insights into mitotic chromosome state and conformation have come from extensive microscopy studies over the last century. Over the last decade, the development of 3C-based techniques has enabled the study of higher order chromosome organization during mitosis in a genome-wide manner. During mitosis, chromosomes lose their cell type-specific and locus-dependent chromatin organization that characterizes interphase chromatin and fold into randomly positioned loop arrays. Upon exit of mitosis, cells are capable of quickly rearranging the chromosome conformation to form the cell type-specific interphase organization again. The information that enables this rearrangement after mitotic exit is thought to be encoded at least in part in mitotic bookmarks, e.g. histone modifications and variants, histone remodelers, chromatin factors, and non-coding RNA. Here we give an overview of the chromosomal organization and epigenetic characteristics of interphase and mitotic chromatin in vertebrates. Second, we describe different ways in which mitotic bookmarking enables epigenetic memory of the features of interphase chromatin through mitosis. And third, we explore the role of epigenetic modifications and mitotic bookmarking in cell differentiation.

Histone Purification Combined with High-Resolution Mass Spectrometry to Examine Histone Post-Translational Modifications and Histone Variants in Caenorhabditis elegans.

Histones are the major proteinaceous component of chromatin in eukaryotic cells and an important part of the epigenome, affecting most DNA-related events, including transcription, DNA replication, and chromosome segregation. The properties of histones are greatly influenced by their post-translational modifications (PTMs), over 200 of which are known today. Given this large number, researchers need sophisticated methods to study histone PTMs comprehensively. In particular, mass spectrometry (MS)-based approaches have gained popularity, allowing for the quantification of dozens of histone PTMs at once. Using these approaches, even the study of co-occurring PTMs and the discovery of novel PTMs become feasible. The success of MS-based approaches relies substantially on obtaining pure and well-preserved histones for analysis, which can be difficult depending on the source material. Caenorhabditis elegans has been a popular model organism to study the epigenome, but isolation of pure histones from these animals has been challenging. Here, we address this issue, presenting a method for efficient isolation of pure histone proteins from C. elegans at good yield. Further, we describe an MS pipeline optimized for accurate relative quantification of histone PTMs from C. elegans. We alkylate and tryptically digest the histones, analyze them by bottom-up MS, and then evaluate the resulting data by a C. elegans-adapted version of the software EpiProfile 2.0. Finally, we show the utility of this pipeline by determining differences in histone PTMs between C. elegans strains that age at different rates and thereby achieve very different lifespans. © 2020 The Authors. Basic Protocol 1: Large-scale growth and harvesting of synchronized C. elegans Basic Protocol 2: Nuclear preparation, histone extraction, and histone purification Basic Protocol 3: Bottom-up mass spectrometry analysis of histone PTMs and histone variants.