Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin

Abstract

Background The cohesin complex organizes the genome-forming dynamic chromatin loops that impact on all DNA-mediated processes. There are two different cohesin complexes in vertebrate somatic cells, carrying the STAG1 or STAG2 subunit, and two versions of the regulatory subunit PDS5, PDS5A and PDS5B. Mice deficient for any of the variant subunits are embryonic lethal, which indicates that they are not functionally redundant. However, their specific behavior at the molecular level is not fully understood. Results The genome-wide distribution of cohesin provides important information with functional consequences. Here, we have characterized the distribution of cohesin subunits and regulators in mouse embryo fibroblasts (MEFs) either wild type or deficient for cohesin subunits and regulators by chromatin immunoprecipitation and deep sequencing. We identify non-CTCF cohesin-binding sites in addition to the commonly detected CTCF cohesin sites and show that cohesin-STAG2 is the preferred variant at these positions. Moreover, this complex has a more dynamic association with chromatin as judged by fluorescence recovery after photobleaching (FRAP), associates preferentially with WAPL and is more easily extracted from chromatin with salt than cohesin-STAG1. We observe that both PDS5A and PDS5B are exclusively located at cohesin-CTCF positions and that ablation of a single paralog has no noticeable consequences for cohesin distribution while double knocked out cells show decreased accumulation of cohesin at all its binding sites. With the exception of a fraction of cohesin positions in which we find binding of all regulators, including CTCF and WAPL, the presence of NIPBL and PDS5 is mutually exclusive, consistent with our immunoprecipitation analyses in mammalian cell extracts and previous results in yeast. Conclusion Our findings support the idea that non-CTCF cohesin-binding sites represent sites of cohesin loading or pausing and are preferentially occupied by the more dynamic cohesin-STAG2. PDS5 proteins redundantly contribute to arrest cohesin at CTCF sites, possibly by preventing binding of NIPBL, but are not essential for this arrest. These results add important insights towards understanding how cohesin regulates genome folding and the specific contributions of the different variants that coexist in the cell.

Shared and distinct molecular effects of regulatory genetic variants provide insight into mechanisms of distal enhancer-promoter communication

“…Which molecular mechanisms could link enhancer activity and connectivity? The cohesin complex, and particularly its isoform containing the STAG2 subinit, is known to be recruited to active enhancers, including those that do not bind CTCF 85,86 . This recruitment could be directly impacted by genetic aberrations in enhancer activity.…”

The consequences of cohesin mutations in myeloid malignancies

“…Mouse models harbouring mutations in both the genes have shown a lower leucocyte count and an increased granulocyte/monocyte fraction along with a decrease in hemoglobin levels. It was observed that there was an increase in their hematopoietic stem and progenitor cells thus indicating a higher self-renewal capacity in them ( Cuadrado et al, 2022) . This study highlights the importance of mouse models to study clonal hierarchy and their co-operativity in MDS and its aetiology ( Figure 2 ).…”

“…Cohesin mutations have also been shown to reduce overall chromatin accessibility while increasing accessibility for specific transcription factor motifs related to HSC self-renewal, such as ERG1, GATA2, and RUNX1 ( Mazumdar et al, 2015 ). Studies which aimed to study how STAG1 and STAG2 cohesin subunits are distributed throughout the genome also showed that non-CTCF cohesin binding sites are bound by cohesin-STAG2 ( Cuadrado et al, 2022 ). This is further proof of STAG2 having a specialised function in myeloid malignancies.…”

Different NIPBL requirements of cohesin-STAG1 and cohesin-STAG2

“…In vitro, NIPBL is also required both for topological entrapment of plasmid DNA and for loop extrusion by cohesin [20][21][22] . PDS5 proteins compete with NIPBL for binding cohesin and are, together with WAPL, required for cohesin unloading [23][24][25][26] . Thus, NIPBL-bound, presumably loop extruding cohesin, cannot be unloaded.…”