Spatial Chromosome Folding and Active Transcription Drive DNA Fragility and Formation of Oncogenic MLL Translocations.

How spatial chromosome organization influences genome integrity is still poorly understood. Here, we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CCCTC-binding factor (CTCF) and cohesin-bound sites at the bases of chromatin loops, and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hotspots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability and are key contributors to the occurrence of genome rearrangements that drive cancer.

HMGB2 Loss upon Senescence Entry Disrupts Genomic Organization and Induces CTCF Clustering across Cell Types.

Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.

Dynamics of Double-Strand Breaks: Implications for the Formation of Chromosome Translocations.

Illegitimate joining of chromosome breaks can lead to the formation of chromosome translocations, a catastrophic type of genome rearrangements that often plays key roles in tumorigenesis. Emerging evidence suggests that the mobility of broken DNA loci can be an important determinant in partner search and clustering of individual breaks, events that can influence translocation frequency. We summarize here the recent literature on the mechanisms that regulate chromatin movement, focusing on studies exploring the motion properties of double-strand breaks in the context of chromatin, the functional consequences for DNA repair, and the formation of chromosome fusions.

RNA polymerase II is required for spatial chromatin reorganization following exit from mitosis.

Mammalian chromosomes are three-dimensional entities shaped by converging and opposing forces. Mitotic cell division induces marked chromosome condensation, but following reentry into the G1 phase of the cell cycle, chromosomes reestablish their interphase organization. Here, we tested the role of RNA polymerase II (RNAPII) in this transition using a cell line that allows its auxin-mediated degradation. In situ Hi-C showed that RNAPII is required for both compartment and loop establishment following mitosis. RNAPs often counteract loop extrusion, and in their absence, longer and more prominent loops arose. Evidence from chromatin binding, super-resolution imaging, and in silico modeling allude to these effects being a result of RNAPII-mediated cohesin loading upon G1 reentry. Our findings reconcile the role of RNAPII in gene expression with that in chromatin architecture.

Genome-wide detection of DNA double-strand breaks by in-suspension BLISS.

sBLISS (in-suspension breaks labeling in situ and sequencing) is a versatile and widely applicable method for identification of endogenous and induced DNA double-strand breaks (DSBs) in any cell type that can be brought into suspension. sBLISS provides genome-wide profiles of the most consequential DNA lesion implicated in a variety of pathological, but also physiological, processes. In sBLISS, after in situ labeling, DSB ends are linearly amplified, followed by next-generation sequencing and DSB landscape analysis. Here, we present a step-by-step experimental protocol for sBLISS, as well as a basic computational analysis. The main advantages of sBLISS are (i) the suspension setup, which renders the protocol user-friendly and easily scalable; (ii) the possibility of adapting it to a high-throughput or single-cell workflow; and (iii) its flexibility and its applicability to virtually every cell type, including patient-derived cells, organoids, and isolated nuclei. The wet-lab protocol can be completed in 1.5 weeks and is suitable for researchers with intermediate expertise in molecular biology and genomics. For the computational analyses, basic-to-intermediate bioinformatics expertise is required.

Operons are a conserved feature of nematode genomes.

The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla. Operons are present in the class Chromadorea, one of the two main nematode classes, but their distribution in the other class, the Enoplea, is not known. We have surveyed the genomes of Trichinella spiralis, Trichuris muris, and Romanomermis culicivorax and identified the first putative operons in members of the Enoplea. Consistent with the mechanism of polycistronic RNA resolution in other nematodes, the mRNAs produced by genes downstream of the first gene in the T. spiralis and T. muris operons are trans-spliced to spliced leader RNAs, and we are able to detect polycistronic RNAs derived from these operons. Importantly, a putative intercistronic region from one of these potential enoplean operons confers polycistronic processing activity when expressed as part of a chimeric operon in Caenorhabditis elegans. We find that T. spiralis genes located in operons have an increased likelihood of having operonic C. elegans homologs. However, operon structure in terms of synteny and gene content is not tightly conserved between the two taxa, consistent with models of operon evolution. We have nevertheless identified putative operons conserved between Enoplea and Chromadorea. Our data suggest that operons and "spliced leader" (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing.