HSP90 Controls SIR2 Mediated Gene Silencing Shyamasree Laskar1, Mrinal K. Bhattacharyya2, Rama Shankar1, Sunanda Bhattacharyya1* 1 Department of Biotechnology, School of Life Sciences, University of Hyderabad, Hyderabad, Andhra Pradesh, India, 2 Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Andhra Pradesh, India Abstract In recent years, Hsp90 is found to interact with several telomeric proteins at various phases of cell cycle. The Hsp90 chaperone system controls assembly and disassembly of telomere structures and thus maintains the dynamic state of telomere. Here, for the first time we report that the activity of another telomeric protein Sir2p is modulated by Hsp82, the ortholog of Hsp90 from budding yeast (Saccharomyces cerevisiae). In a temperature sensitive Hsp90 deficient yeast strain (iG170Dhsp82), less abundant Sir2p is observed, resulting in de-repression of telomere silencing and a complete loss of mating type silencing. Intriguingly, over expression of Hsp90, either by exposing cells to heat shock or by introducing HSP82 overexpression plasmid also yields reduced level of Sir2p, with a consequential loss of telomere silencing. Thus, Hsp90 homeostasis maintains the cellular pool of Sir2p and thereby controls the reversible nature of telomere silencing. Interestingly, such regulation is independent of one of its major co-chaperones Sba1 (human ortholog of p23). Citation: Laskar S, Bhattacharyya MK, Shankar R, Bhattacharyya S (2011) HSP90 Controls SIR2 Mediated Gene Silencing. PLoS ONE 6(8): e23406. doi:10.1371/ journal.pone.0023406 Editor: Mary Bryk, Texas A&M University, United States of America Received January 20, 2011; Accepted July 16, 2011; Published August 4, 2011 Copyright: 2011 Laskar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from CSIR (Council for Scientific and Industrial Research), India to MKB (Grant number 37/1343/08/EMR-II); and Purse grant from University of Hyderabad to SB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sbtsl@uohyd.ernet.in Introduction Hsp90 is a highly abundant eukaryotic protein, which is involved in maturation and folding of some special class of proteins, which are primarily involved in signal transduction [1,2,3]. It dimerises in an ATP dependent manner with several cochaperones and provides the maturation of the target protein at a near native state [4]. In budding yeast there are two isoforms of Hsp90; Hsc82 (human ortholog of Hsp90b), which is constitutively expressed in the cell and Hsp82 (human ortholog of Hsp90a), which is induced whenever cells are exposed to any kind of stressed condition. It is known that expression of either one of the two isoforms of Hsp90 is required for yeast viability [5]. The two isoforms share 97% sequence identity and they comprise (1–2) % of the total cytosolic proteins. Hsp90 level is significantly increased in the cell upon exposure to stress, including temperature, nonphysiological pH, nutrient deprivation and malignancy [6]. Recent studies have unraveled novel roles of Hsp90, where Hsp90 and its cochaperone p23 are involved in stabilization of different protein DNA complexes during RNA transcription, telomere maintenance, DNA replication etc. [7,8,9,10]. Hsp90 and p23 also influence in vitro and in vivo telomerase activity [11]. It is also known that other cochaperones, for example, Hsp70, Hsp40 and Hop are also needed for proper maturation of telomerase [12]. However, unlike the steroid hormone receptor maturation, where Hsp90-p23 complex is only transiently attached to the target protein, in the case of telomerase activation, Hsp90 and p23 are found to be associated with functional telomerase complex even after the completion of telomere addition. In yeast, Hsp82 (the ortholog of Hsp90) helps in maintaining a dynamic equilibrium between the extendable and unextendable state of telomere through active interaction with Cdc13 [13]. In recent years, a plethora of proteomic, bioinformatics, and genetic approaches unravel many more telomeric proteins as potential clients of Hsp82. Examples include Cdc13, Stn1, [14,15], Sir2 [16], Mre11 [17], Ku80, Mec1 and Est1 [15]. Some of these gene products are involved in telomere position effect (TPE) [18], a phenomenon where transcription of gene is repressed by its close proximity to telomere. In budding yeast, telomere silencing is initiated by recruitment of Sir2p-Sir4p complex at subtelomeric regions by telomere bound Rap1p and Ku70/Ku80 heterodimer. Sir2p mediated histone deacetylation helps Sir3p binding, which triggers the spreading of the Sir complex along the subtelomeric regions, causing heterochromatinisation. Sir2p, which is the key member of sirtuins, is conserved across the evolution [19]. It is a NAD+ dependent histone deacetylase and is involved in transcriptional silencing of the silent mating type loci, HML and HMR, as well as genes near telomeres [20,21]. A high throughput study aims at identifying S. cerevisiae deletion mutants affecting telomere length reveal slightly short telomere phenotype in hsc82 deletion mutant [22]. However contradictory results from other studies show no apparent change in telomere length in hsp82 or hsc82 deletion mutants [14]. It is well established that length of telomere positively regulates the efficiency of silencing, i.e., the longer the telomere, greater is the silencing [23]. However, the effect of Hsp82 deficiency on TPE has not been explored. Moreover, why over-expression of Hsp82 leads to telomere length shortening is also poorly understood, except for the fact that HSP82 and HSC82 are the high copy suppressors for the stn1-1 and cdc13-1 mutants [14]. In this paper, we show that Hsp82 homeostasis plays a critical role in maintaining cellular pools of enzymatically active Sir2p. In Dhsp82, Dhsc82 double knock out background, which is maintained PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23406