Subnuclear relocalization and silencing of a chromosomal region by an ectopic ribosomal DNA repeat

Abstract

Our research addresses the relationship between subnuclear localization and gene expression in fission yeast. We observed the relocalization of a heterochromatic region, the mating-type region, from its natural location at the spindle-pole body to the immediate vicinity of the nucleolus. Relocalization occurred in response to a DNA rearrangement replacing a boundary element (IR-R) with a ribosomal DNA repeat (rDNA-R). Gene expression was strongly silenced in the relocalized mating-type region through mechanisms that differ from those operating in wild type. Also different from the wild-type situation, programmed recombination events failed to take place in the rDNA-R mutant. Increased silencing and perinucleolar localization depended on Reb1, a DNAbinding protein with cognate sites in the rDNA. Reb1 was recently shown to mediate long-range interchromosomal interactions in the nucleus through dimerization, providing a mechanism for the observed relocalization. Replacing the full rDNA repeat with Reb1-binding sites, and using mutants lacking the histone H3K9 methyltransferase Clr4, indicated that the relocalized region was silenced redundantly by heterochromatin and another mechanism, plausibly antisense transcription, achieving a high degree of repression in the rDNA-R strain.nuclear organization | gene silencing | chromatin | boundary elements | antisense transcription E ukaryotic genomes are spatially organized within the nucleus (reviewed in refs. 1-5). In interphase, chromatin domains and entire chromosomes tend to occupy defined positions relative to each other or to nuclear landmarks. Chromosomal regions capable of associating with the nuclear periphery, with nuclear substructures, or with each other have been proposed to function as anchors underlying this organization (4-7). Coupled with a nonuniform distribution of proteins in the nucleus, the dynamic, directed localization of chromosomal regions to subnuclear compartments might influence and perhaps regulate gene expression and recombination. Both stable associations and transient contacts might impose lasting marks on the regions that participate in them (8, 9). Despite our increasingly detailed view of subnuclear organization, however, the two fundamental questions of how the organization is achieved and of its physiological significance remain largely open.In the fission yeast Schizosaccharomyces pombe, extended silent heterochromatic domains are found at the three centromeres, at the telomeres of chromosome 1 and 2, and in the right arm of chromosome 2, in the mating-type region (10-16). Large clusters of rDNA repeats at both ends of chromosome 3 are also partially heterochromatic (13,16). In addition to their distinctive chromatin structures, these regions display distinctive subnuclear localizations. The fission yeast nucleus is structured (17)(18)(19)(20)(21)(22)(23), and the nuclear proteome is not uniformally distributed (24). One pole of the interphase nucleus is defined by the microtubule organizing center or spindle-pole body (SPB) spanning ...

Establishment of expression-state boundaries by Rif1 and Taz1 in fission yeast

“…Thus, the control of replication might be required for boundary positioning by regulating the spreading of CLRC. Consistently, our large-scale screens identified factors involved in DNA replication control in addition to Taz1, Rif1, and Mcm4, including the replication fork protection complex and, in the case of the rDNA, the replication blocker Reb1 (27). We speculate that delayed replication at the edge of the domain following boundary deletion (Fig.…”

“…We performed genome-wide screens for genomic elements that, when inserted in the place of IR-R, permit heterochromatic repression of ade6 + while preserving expression of a LEU2 gene in the adjacent euchromatin. In addition to the rDNA repeats that we described previously (27), the boundaries of centromere 1 and centromere 3 and the endogenous boundaries of the mating-type region, six unique chromosomal elements originating from the arms of chromosome 1 and 2 were repeatedly isolated, in a total of 35 independent integration events for which representative clones are shown in Fig. 1.…”

“…The previously described S. pombe strain PG2897 and plasmid pGT299 (27) were used throughout the study to integrate DNA constructs at the edge of the mating-type region as described in detail in Supporting Information. This included the boundary-trap screen, deletion analysis of trapped elements, and insertion of Gal4 binding sites.…”

“…Table S3 shows the chromosomal coordinates of the trapped boundary elements and Table S4 provides an overview of the primers, plasmids, and strains for the deletion analysis of trapped elements. Expression of the (EcoRV)::ade6 + and (BlpI)::LEU2 reporter genes was monitored as described previously (27).…”

A Duplicated NUCLEOLIN Gene with Antagonistic Activity Is Required for Chromatin Organization of Silent 45S rDNA in Arabidopsis

“…Derepression of VAR1 rRNA genes in nuc2-2 mutant plants could be explained by the observed reorganization of rDNA from inactive to active NOR chromatin (Jakociunas et al, 2013) but also by possible rDNA copy number variation. This phenomenon has previously been observed in fasciata (fas) mutant plants ( Figure 5A), a subunit of the H3/H4 histone chaperone Chromatin Associated Factor1 complex (Mozgová et al, 2010).…”