Hsp82 Is Involved in Sir2p Abundance Figure 5. Heat shock induces derepression of transcriptional silencing at telomere. (Ai) The Western blot shows the relative levels of Hsp82p, Sir2 and Act1 in wild type strain at normal vs. heat shock condition. (Aii) Three independent experimental data are normalized with respect to Actin and plotted. Each bar represents mean density 6 SD. (Bi) Heat shock induces the derepression of subtelomeric gene YFR057w, ACT1 being the loading control. (Bii) Three independent experiments are performed and the data are plotted after normalizing with ACT1. Each bar represents mean density 6 SD. doi:10.1371/journal.pone.0023406.g005 activity is due to the reduced cellular pool of Sir2p; on the other hand in Dhsp82Dhsc82 double mutant cell (iG170Dhsp82) the derepression of silencing is not only due to reduced level of Sir2p but could also be due to the presence of functionally inactive Sir2p. It appears that even the low abundance of active Sir2p (as low as 1/4th of the usual amount) is good enough to ensure mating type silencing (as observed in SLY32 cells). The absence of MATa2 transcript in HSP82 overexpressing cells also confirms their mating ability. We reason that these cells are capable of mating type silencing, because they still possess wild type HSC82 and HSP82 genes due to which Sir2p maturation and functional activation are perhaps unaltered. Taking all these together, it is very tempting to propose that loss of TPE under overexpression condition and knockout condition could be mechanistically distinct. At present we do not know whether Hsp82 is directly controlling the cellular pool of Sir2p or it does so through a mediator. Thirdly, our result suggests that Hsp82 over-expression leads to euchromatinisation of chromosome ends. The transitions between the two states of chromatin are very well studied in TPE model in budding yeast S. cerevisiae [18]. However, the reversible nature of such heterochromatin-euchromatin transition and vice versa is poorly understood. Although, the epigenetic marks (especially H3 and H4 modifications) associated with such changes are well established [37], the regulation at the DNA-protein interface is ill understood. It is known to a greater extent that how do the silencing proteins nucleate and then spread along the heterochro- matin [38]. However, the molecular events occurring during the exit of the silencing complex from the heterochromatin giving rise to euchromatin formation is largely unknown. Findings from this work leads us to speculate that may be under in vivo condition Hsp82 is required to maintain a dynamic equilibrium of total amount of silencing proteins. This idea has been proven under stressed conditions such as heat shock that alter Hsp82 levels in the cells. We have exposed the cells to heat shock and under that conditions observed that total Sir2p abundance is also reduced by more than 50% which is associated with complete lose of silencing at telomere. Thus the adaptation in Sir2p abundance in response to the Hsp82 homeostasis could be responsible for the reversible nature of transcriptional silencing at telomere. We speculate that Hsp82 modulates interaction of silencing complex (Sir2-Sir3-Sir4) with DNA in such a way that it facilitates the removal (or degradation) of the major silencing proteins, once the chromatin does not need to be maintained at the heterochromatin state. Similar effect is also observed at telomere end, where Hsp82 acts as a releasing factor for Cdc13 to control the telomere protein dynamics, and maintains the extendable and unextentable states of telomere [13]. Finally, our genetic analysis shows that Hsp82 mediated maturation of Sir2 is independent of its cochaperone Sba1. This observation does not fit with classical steroid hormone receptor chaperone model, where Sba1 is found to be required at the last stages during client release. Thus, apart from traditional chaperonic role, newer roles of Hsp82 are emerging, where it acts as a regulator of various DNA protein interactions occurring inside the cell. Our report also provides evidence along the same line. Future studies need to be done to understand how Hsp82 actually regulates Sir2 level. PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23406