We previously have shown that condensin bound to wild type PolI-silent rDNA repeats () was released from transcriptionally active ErDNA (Wang et al., 2006
). Thus, it is conceivable that this pool of extra condensin in ErDNA cells either remains unbound to chromatin (and/or degraded), or is relocalized to other chromatin sites (). Our observations of condensin relocalization between different chromosomal locations in mitosis (Wang et al., 2005
) and upon modulation of rDNA transcription (Wang et al., 2006
) show that the relocalization is feasible. The fact that both total amount and the fraction of chromatin-bound condensin, as determined by chromatin fractionation (Liang and Stillman, 1997
), are unchanged in ErDNA cells (), argues in favor of the relocalization hypothesis ().
Hypothetical change in condensin function in ErDNA cells
In order to directly test the possibility that condensin localization is altered in ErDNA cells, we analyzed Smc2p distribution by Smc2p-HA ChIP and quantitative real-time PCR (qPCR) for several loci previously tested for condensin binding in wild type (Wang et al., 2005
). A marked increase in Smc2p-HA association in ErDNA cells was observed for most of these loci (). This could indicate that condensin released from nucleolar chromatin in ErDNA cells (Wang et al., 2006
) increases saturation of the existing binding sites.
Additional condensin enrichment at known binding hotspots in ErDNA cells
Extrapolating this finding, there is a possibility that the increased availability of condensin can result in an alteration of its binding genome-wide, e.g. in the emergence of new sites or in changing pattern of condensin enrichment along the chromosomal arms. Thus, we also examined condensin binding in ErDNA cells at the genomic scale, by the combined ChIP and microarray analysis (ChIP-chip approach) and by comparing these results with those from a study of condensin distribution in wild type (Wang et al., 2005
). Two types of microarrays, with open reading frames (ORFs) and intergenic regions (IGRs), were hybridized to Smc2-HA ChIP DNA derived from an asynchronous population of haploid SMC2
ErDNA cells. The resulting median-normalized fluorescence ratio datasets (Supplement 1
) were analyzed for the enrichment of condensin at genomic loci. Microarray elements that showed a two-fold or greater enrichment in binding over the median dataset value were considered condensin-binding.
It was evident that in the ErDNA strain some shift has occurred in the PeakFinder-defined peaks position at the condensin occupied zones (). However, these differences in the PeakFinder output are somewhat exaggerated, because only peak maxima are shown and no widths (). Thorough examination of the condensin-bound zones in ErDNA strain showed that most of ErDNA peaks were still adjacent to the wild type peaks of condensin binding: more than 50% of PeakFinder-defined condensin peaks in ErDNA stain were within 2kb from the nearest peak location in wild type, an equivalent of an average single S. cerevisiae
gene (including regulatory sequences). Without peak-cutting filtering, about 800 newly enriched microarray elements emerged in the ErDNA dataset (including only data with at least two replicas, Supplement 1
). However the basic rules of condensin distribution: site spacing, IGR/ORF ratio and pericentromeric enrichment did not change significantly. Thus, we conducted a statistical test on even more rigorously limited sample (data points with three replicas), comparing the present study data with the similarly filtered data from wild type (Wang et al., 2005
). As a result, we revealed a trend: there was a two-fold increase in sites that emerged in ErDNA cells over sites that disappeared. Moreover, ORF loci were strongly prevalent among the newly occupied sites (Supplements 2
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 (more ...)
We also paid particular attention to the possible change in condensin occupancy that may have reflected nucleolus-dependent changes in nuclear organization. Namely, tRNA genes play an important role in genomic organization, as they are recruited to the nucleolus in wild type cells (Thompson et al., 2003
) and thus should become more dispersed in ErDNA nuclei. Probably due to this recruitment (Thompson et al., 2003
), tRNA genes showed increased condensin occupancy (as compared to other IGRs) in wild type: 20% of 241 tRNA genes analyzed in (Wang et al., 2005
) coincided with condensin peaks. However, in ErDNA strain ChIP-chip analysis 15% of 262 analyzed tRNA genes were still condensin-enriched (Supplement 1
), indicating that this aspect of intranuclear chromosome packaging did not change significantly in ErDNA cells.
The new pattern of condensin binding in ErDNA cells, including the novel sites as well as expansions of the pre-existing peaks, could potentially be an example of a more universal eukaryotic pattern of condensin distribution, which is not biased by the substantial condensin recruitment to the segregation of actively transcribed nucleoli in S. cerevisiae
with wild type rDNA. The newly emerged peaks, however, are especially important, as they may uncover previously obscured functions of condensin. PeakFinder analysis of condensin distribution in the ErDNA strain has indicated that for most chromosomes, if their subtelomeric regions were present on the array, a notable increase in peak frequency occurred at the telomere-proximal regions (). Thus, we investigated this phenomenon in greater detail (and without PeakFinder transformation) by plotting both relative enrichment values and numbers of new condensin-positive array elements (ORFs or IGRs), as functions of their distance from the centromere. For ORF arrays, newly-found elements were spread uniformly along the chromosomal arms, both in their enrichment values () and distribution (), similarly to wild type (Wang et al., 2005
). However, for IGR arrays, a notable preference for subtelomeric regions was apparent in the ErDNA strain (). The most telomere-proximal 10% of chromosome arms contained more than three times as many condensin-enriched IGRs, a feature not seen in wild type strains (Wang et al., 2005
). This subtelomeric enrichment was confirmed in an independent ChIP-chip experiment, which directly compared microarray hybridization of alternatively labeled Smc2-HA-bound (ChIP) DNA from wild type and ErDNA strains (data not shown).
Subtelomeric region bias in condensin relocalization in ErDNA cells
Because telomeres are highly repeated sequences, and thus were largely excluded from the arrays we used, the ChIP-chip data did not allow us to determine to what degree telomeres themselves bind condensin. Moreover, as telomeric repeats are quite short, the standard ChIP quantification method by qPCR was not feasible. However it is important to understand whether telomeric repeats themselves are the hotspots of condensin binding. In this case one would observe a peak of binding intensity immediately proximal to the telomere with accompanying decay of binding as distance to the telomere increases. Thus we carried out a higher-resolution ChIP/qPCR scanning of the telomere-proximal region of the left arm of chromosome IX in ErDNA cells. This analysis revealed that condensin enrichment spans an extensive area (exceeding 10 kb) next to the telomere, but no condensin binding was observed immediately proximal to telomeric repeats (). This finding indicates that ends of chromosomes are enriched in condensin in ErDNA cells without enrichment of the telomeric repeats themselves. The mechanism of this enrichment remains to be elucidated.