The Sir2-Sum1 Complex Represses Transcription Using Both Promoter-Specific and Long-Range Mechanisms to Regulate Cell Identity and Sexual Cycle in the Yeast Kluyveromyces lactis Meleah A. Hickman 1,2 , Laura N. Rusche 1,3 * 1 Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of America, 2 University Program in Genetics and Genomics, Duke University, Durham, North Carolina, United States of America, 3 Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, United States of America Abstract Deacetylases of the Sir2 family regulate lifespan and response to stress. We have examined the evolutionary history of Sir2 and Hst1, which arose by gene duplication in budding yeast and which participate in distinct mechanisms of gene repression. In Saccharomyces cerevisiae, Sir2 interacts with the SIR complex to generate long-range silenced chromatin at the cryptic mating-type loci, HMLa and HMRa. Hst1 interacts with the SUM1 complex to repress sporulation genes through a promoter-specific mechanism. We examined the functions of the non-duplicated Sir2 and its partners, Sir4 and Sum1, in the yeast Kluyveromyces lactis, a species that diverged from Saccharomyces prior to the duplication of Sir2 and Hst1. KlSir2 interacts with both KlSir4 and KlSum1 and represses the same sets of target genes as ScSir2 and ScHst1, indicating that Sir2 and Hst1 subfunctionalized after duplication. However, the KlSir4-KlSir2 and KlSum1-KlSir2 complexes do not function as the analogous complexes do in S. cerevisiae. KlSir4 contributes to an extended repressive chromatin only at HMLa and not at HMRa. In contrast, the role of KlSum1 is broader. It employs both long-range and promoter-specific mechanisms to repress cryptic mating-type loci, cell-type–specific genes, and sporulation genes and represents an important regulator of cell identity and the sexual cycle. This study reveals that a single repressive complex can act through two distinct mechanisms to regulate gene expression and illustrates how mechanisms by which regulatory proteins act can change over evolutionary time. Citation: Hickman MA, Rusche LN (2009) The Sir2-Sum1 Complex Represses Transcription Using Both Promoter-Specific and Long-Range Mechanisms to Regulate Cell Identity and Sexual Cycle in the Yeast Kluyveromyces lactis. PLoS Genet 5(11): e1000710. doi:10.1371/journal.pgen.1000710 Editor: Hiten D. Madhani, University of California San Francisco, United States of America Received June 1, 2009; Accepted October 5, 2009; Published November 6, 2009 Copyright: ß 2009 Hickman, Rusche. 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 research was supported by a grant by the National Institute of Health (GM073991) to LNR. 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: lrusche@biochem.duke.edu Introduction Deacetylases of the Sir2 family are key regulators of lifespan and stress resistance in many organisms ranging from yeast to humans [1]. These enzymes couple deacetylation with hydrolysis of NAD + and consequently their activity is linked to the metabolic state of the cell [2]. Despite having a well-conserved enzymatic activity, Sir2 family members act on a wide variety of substrates and serve a diverse set of biological functions [3,4]. To explore the process by which Sir2 deacetylases have diversified, we examined the evolutionary history of two family members from budding yeast, Sir2 and Hst1 [5,6], which arose in a whole-genome duplication [7,8,9], yet have distinct functions. Gene duplication is an important force in evolution because it allows variation to occur without compromising the original function of the gene. Preservation of duplicate genes, or paralogs, is proposed to occur through at least two mechanisms, neofunctio- nalization and subfunctionalization. In the neofunctionalization model, one duplicate retains the original function, leaving the other gene free of selective constraint and able to evolve a new function [10]. Alternatively, in the subfunctionalization model, if the ancestral gene had multiple functions, duplicated genes could each lose one of the original functions and together retain the entire set of ancestral functions [11]. Only a few studies have characterized the path by which paralogs have diverged [12,13,14,15]. To investi- gated how Sir2 and Hst1 diverged, we have characterized the function of a representative non-duplicated Sir2 from Kluyveromyces lactis, a budding yeast species that diverged from S. cerevisiae prior to the whole-genome duplication [16]. The functions of Sir2 and Hst1 in S. cerevisiae are well understood. Sir2 interacts with the histone-binding proteins Sir3 and Sir4, and together these proteins generate an extended silenced domain at the telomeres and cryptic mating-type loci, HMLa and HMRa [17]. The HM loci are flanked by silencers that recruit Sir proteins through DNA binding proteins to initiate the formation of silenced chromatin. The telomere repeats also recruit Sir proteins. Sir2, Sir3, and Sir4 spread from sites of recruitment through a sequential deacetylation mechanism that is independent of DNA sequence [18,19,20]. Sir2 deacetylates nearby nucleo- somes, creating high affinity binding sites for Sir3 and Sir4, which bind preferentially to deacetylated tails of histones H3 and H4. Sir3 and Sir4 then recruit additional Sir2 to newly deacetylated PLoS Genetics | www.plosgenetics.org 1 November 2009 | Volume 5 | Issue 11 | e1000710