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. 2008 Jan;59(1):45-53.
doi: 10.1016/j.plasmid.2007.09.003. Epub 2007 Nov 19.

Transcriptional homogenization of rDNA repeats in the episome-based nucleolus induces genome-wide changes in the chromosomal distribution of condensin

Bi-Dar Wang  1 , Alexander Strunnikov
Affiliations

Transcriptional homogenization of rDNA repeats in the episome-based nucleolus induces genome-wide changes in the chromosomal distribution of condensin

Bi-Dar Wang et al. Plasmid. 2008 Jan.

Abstract

Condensin activity establishes and maintains mitotic chromosome condensation, however the mechanisms of condensin recognition of specific chromosomal sites remain unknown. rDNA is the chief condensin binding locus in Saccharomyces cerevisiae, and the level of nucleolar transcription is one of the key factors determining condensin loading to the nucleolar organizer. A new aspect of this transcriptional control is demonstrated in cells with a diffuse (episomal) nucleolar organizer, where active transcription excludes condensin from the transcribed regions of rDNA. Genome-wide ChIP-chip analysis showed that these cells acquire an altered and a more robust pattern of chromosomal condensin distribution, with increased enrichment of wild-type hotspots and with emergence of new sites, most notably in the subtelomeric regions. This genome-wide condensin relocalization induced by the increase in rDNA transcription and, possibly, nucleolar architecture uncovers a novel potential role of the nucleolus in the general chromosome organization.

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Figures

Figure 1
Figure 1. Hypothetical change in condensin function in ErDNA cells
(A) Putative pattern of condensin binding within wild type rDNA array. Larger box – 35S RNA gene, smaller box – 5S rRNA gene, open circle – rDNA origin of replication. Repeats are shown either specialized for rRNA production (active Pol I transcription) or nucleolar segregation (silent for Pol I transcription) according to (Wang et al., 2006). (B) Release and hypothetical relocalization of condensin in ErDNA cells. Condensin loss from transcribed rDNA sites was documented in (Wang et al., 2006). (C) (D) Condensin remains fully chromatin-bound in ErDNA cells. (C) Western-blotting analysis of chromatin fractionation according to (Liang and Stillman, 1997) shows that Smc4p is fully associated with chromatin (Chr) in ErDNA cells (ΔΔ rDNA). Sup – chromatin-unbound protein fraction (1/3 of chromatin equivalent). (*) - proteins cross-reacting with anti-Smc4p antibodies, (•) – Smc4p breakdown products resulting from chromatin extraction. Chromatin extraction was done with nocodazole-arrested cells. (D) Analysis of other condensin subunits as in (C). Only chromatin fractions are shown. Hmo1p – a control chromatin protein. Wild type rDNA strain - 532-W303, ErDNA strain - 532-NOY891/pRDNwt. Specific antibodies to condensin subunit have been described (Freeman et al., 2000).
Figure 2
Figure 2. Additional condensin enrichment at known binding hotspots in ErDNA cells
The wild type no-tag strain W303 (negative control), Smc2p-HA WT rDNA (532-W303) and Smc2p-HA ErDNA (532-NOY891/pRDNwt) strains were used for ChIP/qPCR. Sites were selected from (Wang et al., 2005). TUB2 was used throughout as a negative control.
Figure 3
Figure 3
Genome view of ChIP-chip data for Smc2p-HA binding in wild type and episomal rDNA strains. Only peaks based on at least two replicas were included. The graphs were produced using the PeakFinder application (Glynn et al., 2004). The PeakFinder output only shows the calculated maximum position (i.e. a single array element) for the condensin occupied region, but does not reflect the span of the bound regions. Exanimation of the contunuous datasets shows that condensin-bound regions are usually wider than a single array element (see Supplement 1 and Results). Chromosomal coordinates are as in Supplement 1.
Figure 4
Figure 4. Subtelomeric region bias in condensin relocalization in ErDNA cells
(A) (B) Distribution of condensin occupancy along chromosome arms for new condensin-binding sites in ErDNA cells. Sites plotted were identified by ChIP-chip analysis as enriched in condensin in Smc2p-HA ErDNA strain (532-NOY891/pRDNwt) but not in strains with a wild type NOR (Wang et al., 2005). Condensin enrichment values (fold increase over median) are plotted as a function of relative position along the chromosome arm (in %), where the CEN locus is 0 and the telomere position is 100. The ORF array analysis (A) shows no polarity in distribution of novel sites, while IGR array (B) demonstrates distinct subtelomeric enrichment. (C) (D) Distribution of new condensin-occupied sites in ErDNA cells along the chromosome arms. ChIP-chip data were plotted to show the number of new condensin-enriched elements along the lengths of chromosome arms, sorted into 10% bins, for ORF (C) and IGR (D) arrays. The increase in the number of subtelomeric condensin sites is evident in IGR ChIP-chip analysis (E). ChIP/qPCR validation of subtelomeric condensin enrichment in ErDNA cells. The wild type (532-W303) and ErDNA (532-NOY891/pRDNwt) strains were used for ChIP/qPCR. The test sites were selected from the left arm of chromosome IX in arbitrary (mostly 1-kb) intervals with 300-bp probe span. TUB2 was used as a negative control (Wang et al., 2005).
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