Role for perinuclear chromosome tethering in maintenance of genome stability

Abstract

Repetitive DNA sequences, which constitute half the genome in some organisms, often undergo homologous recombination. This can instigate genomic instability due to gain or loss of DNA1. Assembly of DNA into silent chromatin is generally thought to serve as a mechanism ensuring repeat stability by limiting access to the recombination machinery2. Consistent with this notion, in the budding yeast Saccharomyces cerevisiae, stability of the highly repetitive ribosomal DNA (rDNA) sequences requires a Sir2-containing chromatin silencing complex that also inhibits transcription from foreign promoters and transposons inserted within the repeats by a process called rDNA silencing2-5. Here, we describe a protein network that stabilizes rDNA repeats of budding yeast via interactions between rDNA-associated silencing proteins and two inner nuclear membrane (INM) proteins. Deletion of either the INM or silencing proteins reduces perinuclear rDNA positioning, disrupts the nucleolus-nucleoplasm boundary, induces the formation of recombination foci, and destabilizes the repeats. In addition, artificial targeting of rDNA repeats to the INM suppresses the instability observed in cells lacking an rDNA-associated silencing protein typically required for peripheral tethering of the repeats. Moreover, in contrast to Sir2 and its associated nucleolar factors, the INM proteins are not required for rDNA silencing, indicating that Sir2-dependent silencing is not sufficient to inhibit recombination within the rDNA locus. These findings demonstrate a role for INM proteins in perinuclear chromosome localization and show that tethering to the nuclear periphery is required for rDNA repeat stability. The INM proteins studied here are conserved and have been implicated in chromosome organization in metazoans6,7. Our results therefore reveal an ancient mechanism in which interactions between INM and chromosomal proteins ensure genome stability.

Perinuclear tethers license telomeric DSBs for a broad kinesin- and NPC-dependent DNA repair process

“…Disruption of the telomere-tethering complex cohibin (composed of Lrs4 and Csm1 proteins) alone or in combination with Esc1 deletion, similarly compromised subtelomeric DSB survival (Fig. 1b) 6,8 . Combining the deletion of Nup84 with the disruption of Esc1 or cohibin did not further lower survival rates indicating that all of these factors likely operate within the same pathway (Fig.…”

“…However, deletion of Lrs4, but not Cik1, compromised silent chromatin as assessed by ChIP analysis using an antibody specific to histone H3 acetylated at lysines 9 and 14 (diAcH3K9/14), a robust mark of open chromatin ( Supplementary Fig. 2) 6,8 . Also, unlike Lrs4 deletion, Cik1 loss did not compromise the localization of a green fluorescent protein (GFP)-tagged TEL-XI-L to the nuclear periphery in either the S or G1 phase of the cell cycle ( Fig.…”

“…Endogenous genes were deleted or modified with C-terminal epitope tags 6,8 . Cells were grown to log phase, 5 ml of cell culture was centrifuged, washed with sterile water and resuspended in 100 ml LiOAc mix (100 mM LiOAc pH 7.3, 10 mM Tris-Cl pH 8.0, 1 mM EDTA).…”

“…In yeast, DSBs within the highly repetitive ribosomal RNA genes (rDNA) can lead to unequal sister chromatid exchange events, compromise chromosome stability and shorten cellular lifespan [4][5][6][7] . DSBs within rDNA repeats were found to transiently exit the Rad52/recombination-repressive environment of the nucleolus in which they typically reside to undergo repair without compromising genome integrity 8,9 . In flies, heterochromatic DSBs exit their Rad51/recombinationrepressive subnuclear domain to undergo repair 10 .…”

Nuclear organization in genome stability: SUMO connections

“…Peripheral tethering of telomeres by an inner nuclear membrane protein Mps3 contributes to the suppression of recombination [65,68]. In the nucleolus (gold) the rDNA repeats are bound to the inner nuclear membrane by the yeast protein Heh1 [69]. Peripheral tethering may limit the access of sites to the recombination machinery that mediates sister chromatid exchange.…”