SummaryTopological domains are key architectural building blocks of chromosomes, but their functional importance and evolutionary dynamics are not well defined. We performed comparative high-throughput chromosome conformation capture (Hi-C) in four mammals and characterized the conservation and divergence of chromosomal contact insulation and the resulting domain architectures within distantly related genomes. We show that the modular organization of chromosomes is robustly conserved in syntenic regions and that this is compatible with conservation of the binding landscape of the insulator protein CTCF. Specifically, conserved CTCF sites are co-localized with cohesin, are enriched at strong topological domain borders, and bind to DNA motifs with orientations that define the directionality of CTCF’s long-range interactions. Conversely, divergent CTCF binding between species is correlated with divergence of internal domain structure, likely driven by local CTCF binding sequence changes, demonstrating how genome evolution can be linked to a continuous flux of local conformation changes. We also show that large-scale domains are reorganized during genome evolution as intact modules.

Structural variants (SVs) can contribute to oncogenesis through a variety of mechanisms. Despite their importance, the identification of SVs in cancer genomes remains challenging. Here, we present a framework that integrates optical mapping, high-throughput chromosome conformation capture (Hi-C), and whole-genome sequencing to systematically detect SVs in a variety of normal or cancer samples and cell lines. We identify the unique strengths of each method and demonstrate that only integrative approaches can comprehensively identify SVs in the genome. By combining Hi-C and optical mapping, we resolve complex SVs and phase multiple SV events to a single haplotype. Furthermore, we observe widespread structural variation events affecting the functions of noncoding sequences, including the deletion of distal regulatory sequences, alteration of DNA replication timing, and the creation of novel three-dimensional chromatin structural domains. Our results indicate that noncoding SVs may be underappreciated mutational drivers in cancer genomes.

Male gametes are generated through a specialised differentiation pathway involving a series of developmental transitions that are poorly characterised at the molecular level. Here, we use droplet-based single-cell RNA-Sequencing to profile spermatogenesis in adult animals and at multiple stages during juvenile development. By exploiting the first wave of spermatogenesis, we both precisely stage germ cell development and enrich for rare somatic cell-types and spermatogonia. To capture the full complexity of spermatogenesis including cells that have low transcriptional activity, we apply a statistical tool that identifies previously uncharacterised populations of leptotene and zygotene spermatocytes. Focusing on post-meiotic events, we characterise the temporal dynamics of X chromosome re-activation and profile the associated chromatin state using CUT&RUN. This identifies a set of genes strongly repressed by H3K9me3 in spermatocytes, which then undergo extensive chromatin remodelling post-meiosis, thus acquiring an active chromatin state and spermatid-specific expression.