Cooperation of Sumoylated Chromosomal Proteins in rDNA Maintenance

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

The histone chaperone DAXX maintains the structural organization of heterochromatin domains

Abstract Background The death domain-associated protein (DAXX) collaborates with accessory proteins to deposit the histone variant H3.3 into mouse telomeric and pericentromeric repeat DNA. Pericentromeric repeats are the main genetic contributor to spatially discrete, compact, constitutive heterochromatic structures called chromocentres. Chromocentres are enriched in the H3K9me3 histone modification and serve as integral, functionally important components of nuclear organization. To date, the role of DAXX as an H3.3-specific histone chaperone has been investigated primarily using biochemical approaches which provide genome-wide views on cell populations and information on changes in local chromatin structures. However, the global chromatin and subnuclear reorganization events that coincide with these changes remain to be investigated. Results Using electron spectroscopic imagine (ESI), a specialized form of energy-filtered transmission electron microscopy that allows us to visualize chromatin domains in situ with high contrast and spatial resolution, we show that in the absence of DAXX, H3K9me3-enriched domains are structurally altered and become uncoupled from major satellite DNA. In addition, the structural integrity of nucleoli and the organization of ribosomal DNA (rDNA) are disrupted. Moreover, the absence of DAXX leads to chromatin that is more sensitive, on a global level, to micrococcal nuclease digestion. Conclusions We identify a novel role of DAXX as a major regulator of subnuclear organization through the maintenance of the global heterochromatin structural landscape. As well, we show, for the first time, that the loss of a histone chaperone can have severe consequences for global nuclear organization

MOESM2 of The histone chaperone DAXX maintains the structural organization of heterochromatin domains

Additional file 2: Peripheral association of H3.3 with H3K9me3-enriched domains