VIR telomere. YFR057w ORF codes for a protein of unknown function and is located within 1 kb from the end of chromosome. As Sir2p spreads as far as 3 kb length in the chromosome VIR [33], its ORF is repressed under normal conditions but de- repressed in Dsir2 strain (Figure S2). We quantified the steady state level of YFR057w transcript in iG170Dhsp82 at both 25uC and 37uC and compared with ACT1 transcript. The semi quantitative RT-PCR showed significant increase in YFR057w transcript at 37uC compared to 25uC, whereas the level of ACT1 transcript remained the same at both temperatures (Figure 1B). In order to rule out the possibility that the loss of silencing is not a mere effect due to shift in temperature we measured the YFR057w transcript level of wild type strain (SLY20) at both 25uC and 37u (Figure 1B). No loss of silencing was observed in wild type strain at higher temperature. This result is consistent with the previous finding that higher temperature causes more silencing [34]. The above two experiments conclusively prove that Hsp82 has a role in regulating telomere silencing as well as mating type silencing. Since, Sir2p is the key protein involved in both of these silencing mechanisms; we wanted to see whether there is any change in the level of Sir2p in iGD170hsp82 strain at different temperatures by Western blot analysis. When monitored at non permissive temperature (37uC) the level of Sir2p was found to be substantially diminished (Figure 1C), whereas the level of Hsp82 was comparable at both the temperatures, Act1p being a loading control. The experiment was repeated four times and the mean value of the quantification of the band intensity on Western blot showed that there was at least 50% reduction in the level of Sir2p at 37uC compared to 25uC (Figure 1D). We hereby speculate that Sir2p may be a putative client of Hsp82, whose activity inside the cell is directly or indirectly regulated by Hsp82. At restrictive temperature when Hsp82 is nonfunctional, Sir2p level is reduced by 50% and thus at lower levels it is unable to provide mating type silencing and telomere silencing activity. Thus for transcriptional silencing at telomere and at mating type loci, the budding yeast requires native Hsp82 protein. Effect of Hsp82 over-expression on mating type silencing and transcriptional silencing at telomere Next, we wanted to determine whether single knockout mutant allele Dhsp82 or Dhsc82 has any effect on Sir2p function. We generated two deletion mutants, hsp82 and hsc82 in SLY20 strain, which had ADE2 gene next to telomere at chromosome VIIL. Under normal condition, when SLY20 was allowed to grow on low adenine medium at 30uC, they developed mostly as red colored colonies due to the lack of ADE2 expression, whereas Dsir2 strain SLY12 developed as white colored colonies owing to the expression of ADE2. Our result showed that deletion mutants SLY4 (Dhsc82), SLY5 (Dhsp82) exhibited wild type phenotype, i.e. they all produced pink colored colony (Figure 2Ai) due to transcriptional silencing of ADE2 gene, suggesting that Hsp82 and Hsc82 are redundant to each other. Similarly when we checked YFR057w transcript level of the single deletion mutants, we found YFR057w was silenced in all of them as expected from earlier experiment (Figure 2Aii). Thus single knockout alleles do not show any change in transcriptional silencing at subtelomeric locus and are redundant to each other. As Hsp82 level is increased within the cells whenever cells are exposed to any kind of stressed conditions, we aimed to determine the effect of elevated amount of Hsp82 on Sir2p level and examined whether the silencing function of Sir2p was altered in Hsp82 over-expressed condition. We mimicked the Hsp82 over- expressed condition within the cell by transforming a centromeric plasmid pHCA/hsp82 in SLY20 to generate SLY13 strain, where Hsp82 Is Involved in Sir2p Abundance Hsp82 was expressed under a strong promoter GPD. Interestingly, we found SLY13 cells resulted mostly in white colored colonies indicating a decrease in TPE (Figure 2Ai), however SLY46 strain containing the blank vector showed pink color colonies. In order to test whether the loss of TPE, due to over-expression of HSP82, is restricted to chromosome VII alone or other subtelomeric genes are also de-repressed, we monitored the mRNA level of YFR057w gene. We quantified the steady state level of YFR057w transcript in Hsp82 over-expressed cell (SLY13) and compared with wild type cell (SLY20). The semi quantitative RT-PCR showed significant increase in YFR057w transcript in Hsp82 over-expressed cells compared to the wild type, whereas ACT1 transcript remained same in both the genotype (Figure 2B). This leads us to speculate that over-expression of Hsp82 acts as a negative regulator of transcriptional silencing at telomeres. In order to find out the effect of single knock out (Dhsp82 or Dhsc82) strains or Hsp82 over-expression on mating type silencing, we measured the mating ability of SLY4, SLY5, SLY10, SLY13, and SLY32 and compared with wild type and Dsir2 strains. It was observed that all of them exhibited wild type mating phenotype when mated with the tester strain YDS32 (MATa) (Figure 3A). The mating type frequency for each of the deletion mutants (Dhsp82 and Dhsc82) were plotted (Figure S3) and it was found to be comparable with that of the wild type. We also determined the level of MATa2 transcript in HSP82 overexpressing cell (SLY13) and compared its level with wild type (SLY20) and Dsir2 (SLY12) strains. While Dsir2 strain showed abundant levels of MATa2 as expected, SLY13 showed negligible amount of MATa2 transcript comparable to that of the wild type (Figure 3B). We, therefore, conclude that neither the single knockout mutants nor the over- expression of Hsp82 affect mating type silencing. We were also interested to explore whether Sba1 (human ortholog of p23), which is a cochaperone of Hsp82 has any role in Sir2p maturation. To this end we created Dsba1 deletion strain SLY6. We had also over-expressed Hsp82 in sba1 background (SLY31). While doing TPE assay for both SLY6 and SLY31, we found that SLY6 mostly developed as pink colored colonies whereas SLY31 showed white colored colonies (Figure 2Ai). The Dsba1 deletion strain could also silence the subtelomeric gene YFR057w (Figure 2Aii) like wild type. The mating type silencing function of both SLY6 and SLY31 remained unperturbed (Figure 3A). These results indicate that Hsp82 mediated homeostasis of Sir2p is independent of Sba1. Over-expression of Hsp82 reduces the cellular pool of Sir2p in a dose dependent manner As over-expression of Hsp82 leads to down regulation of transcriptional silencing at telomeres, we monitored whether there was any change in Sir2p level as a function of Hsp82 expression. Equal amount of proteins isolated from different strains was loaded and they were probed with anti-Hsp90 antibody as well as anti-Sir2 antibody. Our result showed that in Hsp82 over- expressing cells (SLY10 and SLY13 strains) the level of Sir2p was considerable diminished (Figure 4A). The quantification of the bands from at least three independent experiments showed that in SLY13 strain while Hsp82 amount was increased by 50% compared to the wild type, the level of Sir2 was reduced by more than 50% in some cases (Figure 4B). In all other strain background such as SLY4 (Dhsc82), SLY5 (Dhsp82), SLY6 (Dsba1) the level of Sir2p remained unaltered. These results also suggest that Hsc82 (which is poorly detected by the antibody) compensates for the lack of Hsp82 (in SLY5) and thus the level of Sir2p is not affected. Since cellular level of Hsp82 changes in response to different stressed condition, it is important to know whether Sir2p level is PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23406