Cohesin plays an important role in chromatid cohesion and has additional functions in higher-order chromatin organization and in transcriptional regulation. The binding of cohesin to euchromatic regions is largely mediated by CTCF or the mediator complex. However, it is currently unknown how cohesin is recruited to pericentric heterochromatin in mammalian cells. Here we define the histone methyltransferase Suv4-20h2 as a major structural constituent of heterochromatin that mediates chromatin compaction and cohesin recruitment. Suv4-20h2 stably associates with pericentric heterochromatin through synergistic interactions with multiple heterochromatin protein 1 (HP1) molecules, resulting in compaction of heterochromatic regions. Suv4-20h mutant cells display an overall reduced chromatin compaction and an altered chromocenter organization in interphase referred to as ''chromocenter scattering.'' We found that Suv4-20h-deficient cells display chromosome segregation defects during mitosis that coincide with reduced sister chromatid cohesion. Notably, cohesin subunits interact with Suv4-20h2 both in vitro and in vivo. This interaction is necessary for cohesin binding to heterochromatin, as Suv4-20h mutant cells display substantially reduced cohesin levels at pericentric heterochromatin. This defect is most prominent in G0-phase cells, where cohesin is virtually lost from heterochromatin, suggesting that Suv4-20h2 is involved in the initial loading or maintenance of cohesion subunits. In summary, our data provide the first compelling evidence that Suv4-20h2 plays essential roles in regulating nuclear architecture and ensuring proper chromosome segregation.

SUMO is a posttranslational modifier that can modulate protein activities, interactions, and localizations. As the GFP-Smt3p fusion protein has a preference for subnucleolar localization, especially when deconjugation is impaired, the nucleolar role of SUMO can be the key to its biological functions. Using conditional triple SUMO E3 mutants, we show that defects in sumoylation impair rDNA maintenance, i.e., the rDNA segregation is defective and the rDNA copy number decreases in these mutants. Upon characterization of sumoylated proteins involved in rDNA maintenance, we established that Top1p and Top2p, which are sumoylated by Siz1p/Siz2p, most likely collaborate with substrates of Mms21p to maintain rDNA integrity. Cohesin and condensin subunits, which both play important roles in rDNA stability and structures, are potential substrates of Mms21, as their sumoylation depends on Mms21p, but not Siz1p and Siz2p. In addition, binding of cohesin and condensin to rDNA is altered in the mms21-CH E3-deficient mutant.

DNA double-strand breaks (DSBs) activate a signaling pathway known as the DNA damage response (DDR) which via protein–protein interactions and post-translational modifications recruit signaling proteins, such as 53BP1, to chromatin flanking the lesion. Depletion of the SET8 methyltransferase prevents accumulation of 53BP1 at DSBs; however, this phenotype has been attributed to the role of SET8 in generating H4K20 methylation across the genome, which is required for 53BP1 binding to chromatin, prior to DNA damage. Here, we report that SET8 acts directly at DSBs during the DNA damage response (DDR). SET8 accumulates at DSBs and is enzymatically active at DSBs. Depletion of SET8 just prior to the induction of DNA damage abrogates 53BP1’s accumulation at DSBs, suggesting that SET8 acts during DDR. SET8’s occupancy at DSBs is regulated by histone deacetylases (HDACs). Finally, SET8 is functionally required for efficient repair of DSBs specifically via the non-homologous end-joining pathway (NHEJ). Our findings reveal that SET8’s active role during DDR at DSBs is required for 53BP1’s accumulation.