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Background: Cohesin is an important structural regulator of the genome, regulating both three-dimensional genome organization and gene expression. The core cohesin trimer interacts with various HEAT repeat accessory subunits, yielding cohesin complexes of distinct compositions and potentially distinct functions. The roles of the two mutually exclusive HEAT repeat subunits PDS5A and PDS5B are not well understood., Results: Here, we determine that PDS5A and PDS5B have highly similar localization patterns across the mouse embryonic stem cell (mESC) genome and they show a strong overlap with other cohesin HEAT repeat accessory subunits, STAG1 and STAG2. Using CRISPR/Cas9 genome editing to generate individual stable knockout lines for PDS5A and PDS5B, we find that loss of one PDS5 subunit does not alter the distribution of the other PDS5 subunit, nor the core cohesin complex. Both PDS5A and PDS5B are required for proper gene expression, yet they display only partially overlapping effects on gene targets. Remarkably, gene expression following dual depletion of the PDS5 HEAT repeat proteins does not completely overlap the gene expression changes caused by dual depletion of the STAG HEAT repeat proteins, despite the overlapping genomic distribution of all four proteins. Furthermore, dual loss of PDS5A and PDS5B decreases cohesin association with NIPBL and WAPL, reduces SMC3 acetylation, and does not alter overall levels of cohesin on the genome., Conclusions: This work reveals the importance of PDS5A and PDS5B for proper cohesin function. Loss of either subunit has little effect on cohesin localization across the genome yet PDS5A and PDS5B are differentially required for gene expression., (© 2022. The Author(s).)

Transcription factors control cell-specific gene expression programs by binding regulatory elements and recruiting cofactors and the transcription apparatus to the initiation sites of active genes. One of these cofactors is cohesin, a structural maintenance of chromosomes (SMC) complex that is necessary for proper gene expression. We report that a second SMC complex, condensin II, is also present at transcriptional regulatory elements of active genes during interphase and is necessary for normal gene activity. Both cohesin and condensin II are associated with genes in euchromatin and not heterochromatin. The two SMC complexes and the SMC loading factor NIPBL are particularly enriched at super-enhancers, and the genes associated with these regulatory elements are especially sensitive to reduced levels of these complexes. Thus, in addition to their well-established functions in chromosome maintenance during mitosis, both cohesin and condensin II make important contributions to the functions of the key transcriptional regulatory elements during interphase.

The Msh2-Msh3 heterodimer recognizes various DNA mispairs, including loops of DNA ranging from 1 to 14 nucleotides and some base-base mispairs. Homology modeling of the mispair-binding domain (MBD) of Msh3 using the related Msh6 MBD revealed that mismatch recognition must be different, even though the MBD folds must be similar. Model-based point mutation alleles of Saccharomyces cerevisiae msh3 designed to disrupt mispair recognition fell into two classes. One class caused defects in repair of both small and large insertion/deletion mispairs, whereas the second class caused defects only in the repair of small insertion/deletion mispairs; mutations of the first class also caused defects in the removal of nonhomologous tails present at the ends of double-strand breaks (DSBs) during DSB repair, whereas mutations of the second class did not cause defects in the removal of nonhomologous tails during DSB repair. Thus, recognition of small insertion/deletion mispairs by Msh3 appears to require a greater degree of interactions with the DNA conformations induced by small insertion/deletion mispairs than with those induced by large insertion/deletions that are intrinsically bent and strand separated. Mapping of the two classes of mutations onto the Msh3 MBD model appears to distinguish mispair recognition regions from DNA stabilization regions.