Tracking mitotic chromosome formation: How cells pack DNA into fully compact, rod-shaped chromosomes during mitosis has fascinated cell biologists for more than a century. Gibcuset al.delineated the conformational transition trajectory from interphase chromatin to mitotic chromosomes minute by minute during the cell cycle. The mitotic chromosome is organized in a spiral staircase architecture in which chromatin loops emanate radially from a centrally located helical scaffold. The molecular machines condensin I and II play distinct roles in these processes: Condensin II is essential for helical winding, whereas condensin I modulates the organization within each helical turn.Science, this issue p.eaao6135

Chromosome conformation capture-based methods such as Hi-C have become mainstream techniques for the study of the 3D organization of genomes. These methods convert chromatin interactions reflecting topological chromatin structures into digital information (counts of pair-wise interactions). Here, we describe an updated protocol for Hi-C (Hi-C 2.0) that integrates recent improvements into a single protocol for efficient and high-resolution capture of chromatin interactions. This protocol combines chromatin digestion and frequently cutting enzymes to obtain kilobase (kb) resolution. It also includes steps to reduce random ligation and the generation of uninformative molecules, such as unligated ends, to improve the amount of valid intra-chromosomal read pairs. This protocol allows for obtaining information on conformational structures such as compartment and topologically associating domains, as well as high-resolution conformational features such as DNA loops.

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.

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability^1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)^3–6, subTADs⁷ and loops⁸ in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.

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 conformationBasic Protocol 2: Chromosome conformation captureBasic Protocol 3: Hi‐C sequencing library preparation

Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments^1,2. However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skateLeucoraja erinaceaas a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins—including gene expression, chromatin occupancy and three-dimensional conformation—we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with severalhoxagenes, consistent with the redeployment ofhoxgene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.