MCB Accepted Manuscript Posted Online 25 January 2016 Mol. Cell. Biol. doi:10.1128/MCB.01013-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Evolution and Functional Trajectory of Sir1 in Gene Silencing

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Aisha Ellahi1 and Jasper Rine1#

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California Institute of Quantitative Biology

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UC Berkeley, Berkeley California 94720

Department of Molecular and Cell Biology

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#

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Phone: 510-642-7047

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Email: [email protected]

Corresponding author.

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Key words: Sir proteins, heterochromatin, sirtuins, epigenetics, bistability

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Running head: Sir1 Evolution

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Word Count: 823 (Materials and Methods), 6563 (Introduction, Results, Discussion)

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ABSTRACT (200 words)

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We used the budding yeasts Saccharomyces cerevisiae and Torulaspora delbrueckii to examine the evolution

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of Sir-based silencing, focusing on Sir1, silencers, the molecular topography of silenced chromatin,

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and the roles of SIR and RNAi genes in T. delbrueckii. Chromatin immunoprecipitation followed by

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deep sequencing (ChIP-Seq) analysis of Sir proteins in T. delbrueckii revealed a different topography

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of chromatin at the HML and HMR loci than observed in S. cerevisiae. S. cerevisiae Sir1 enriched at the

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silencers of HMLα and HMRa, was absent from telomeres, and did not repress subtelomeric genes.

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In contrast to S. cerevisiae SIR1’s partially dispensable role in silencing, the T. delbrueckii SIR1 paralog

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KOS3 was essential for silencing. KOS3 was also found at telomeres with Td-Sir2 and Td-Sir4, and

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repressed subtelomeric genes. Silencer mapping in T. delbrueckii revealed single silencers at HML and

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HMR, bound by Td-Kos3, Td-Sir2, and Td-Sir4. The KOS3 gene mapped near HMR, and its

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expression was regulated by Sir-based silencing, providing feedback regulation of a silencing protein

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by silencing. In contrast to the prominent role of Sir proteins in silencing, RNAi did not function in

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heterochromatin formation. These results highlighted the shifting role of silencing genes and the

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diverse chromatin architectures underlying heterochromatin.

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INTRODUCTION

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Heterochromatin-based gene silencing in Saccharomyces cerevisiae and its close relatives among

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the budding yeasts use the four Sir proteins to bind to nucleosomes throughout specific regions on

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chromosomes and block accessibility of other DNA binding proteins in that region (1–3). In these

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species, the Sir1 protein is perhaps most enigmatic. In contrast to Sir2, Sir3, and Sir4, which are the

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structural proteins of heterochromatin necessary for its establishment, maintenance, and inheritance,

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Sir1’s main role in S. cerevisiae seems to be in the establishment of heterochromatin at HMLα and

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HMRa (4), though it contributes somewhat to the maintenance of heterochromatin (5). sir1∆ cells

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exhibit a phenotype whereby 50-80% of individual cells within the mutant population completely

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lack silencing at HMLα and HMRa, whereas the remaining cells are fully silenced at these loci. The

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unsilenced sir1∆ cells express transcripts from the silent mating type loci to the same extent as sir4∆

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mutants, are mating defective, and in the case of MATa haploids, lose sensitivity to α-factor (4, 5).

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Furthermore, individual sir1∆ cells can switch transcriptional states at HML and HMR, switching

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from unsilenced to silenced once every 250 cell divisions, and somewhat slower in the reverse

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direction. Biochemical and structural data revealed that Sir1 directly interacts with Orc1 and Sir4,

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suggesting that its localization is restricted to the silencers, where it facilitates efficient establishment

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of silencing (6, 7).

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In addition to its bistable mutant phenotype, SIR1 has a dynamic evolutionary history. SIR1

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has been duplicated more than once among Saccharomyces yeasts, and some species have lost paralogs,

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while others have retained them (8). As a result, SIR1 paralogs vary widely among these species in

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number and in the level of protein sequence similarity between paralogs, which is typically < 50%.

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At one end of the spectrum, Saccharomyces bayanus v. uvarum has four SIR1 paralogs: SIR1 and three

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Kin-Of-SIR1 (KOS1-3). All four paralogs contribute to silencing in this species (8). At the other end

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of the spectrum, K. lactis lacks an identifiable SIR1 paralog, and silencing is mediated by SIR2, SIR4,

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ORC1, and SUM1 (9, 10). Candida glabrata is another yeast that lacks SIR1, yet like S. cerevisiae, has

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SIR2, SIR3, and SIR4 orthologs that function in silencing (11). Yeast species seem to have innovated

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multiple solutions for how to establish gene silencing, with some having no need for a SIR1 gene,

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whereas others have employed up to four SIR1 genes. Analyses of SIR1 orthologs among the

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species of this clade indicate that KOS3 is the most ancestral form of SIR1(8).

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RNAi is by far the most common mechanism of gene silencing. Key components of the

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RNAi machinery include Argonaute, and Dicer, and in most other organisms an RNA-dependent

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RNA polymerase (12). RNAi mechanisms involve the production of double-stranded RNAs

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generated either by DNA-dependent RNA polymerases or an RNA-dependent RNA polymerase.

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These double-stranded RNAs are cleaved by Dicer and bound by Argonaute proteins, which use

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them to direct the modification of DNA and histones occupying sequences complementary to the

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RNAs bound by the Argonaute protein. RNAi is found widely in plants, animals and many fungi,

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including Schizosaccharomyces pombe, but is completely missing from S. cerevisiae.

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Torulospora delbrueckii is a budding yeast species evolutionarily well positioned to explore

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some of the most enigmatic questions concerning the origins of Sir-based silencing, and especially

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the role of Sir1/Kos3. This species diverged from the Saccharomyces species before the whole-

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genome duplication, and has Td-Kos3, the most ancestral form of Sc-Sir1. T. delbrueckii also has pre-

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whole-genome-duplication orthologs of SIR2 and SIR4, and a single gene orthologous to the

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ORC1/SIR3 gene pair of S. cerevisiae, which we referred to as ORC1/SIR3. In addition, this species

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has orthologs of key RNAi components: a gene encoding Argonaute, AGO1, and a budding-yeast

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Dicer-like gene called DCR1. These RNAi-like genes are orthologous to the AGO1 and DCR1

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present in Naumovozyma castellii, a species in which they repress transcription of repetitive Ty

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elements (13). T. delbrueckii thus offers a chance to explore possible connections between, or

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divergence of, the two major mechanisms of heterochromatic gene silencing.

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To date, no one has uncovered a sexual cycle for T. delbrueckii. However, the genome

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sequence of the T. delbrueckii type strain contains a MAT locus on chromosome III, an HMLα locus

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on the same chromosome, and two HMRa loci (one on chromosome V and the other on

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chromosome VII) (14). To explore the functions of T. delbrueckii silencing genes, we first created

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marked strains, protocols, and vectors to allow molecular genetic investigations (Ellahi and Rine,

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manuscript in preparation). We then compared the functions of presumptive silencing genes of T.

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delbrueckii to the functions of their S. cerevisiae orthologs. These experiments offered an unbiased

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view of the genome-wide function of T. delbrueckii SIR genes, revealing a distinctly different

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molecular topography of silenced chromatin than seen in S. cerevisiae. Additionally, we constructed

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ago1∆ and dcr1∆ single mutants and an ago1∆dcr1∆ double-mutant and performed deep-sequencing

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of mRNAs to uncover all loci that were possibly subject to transcriptional repression by the T.

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delbrueckii RNAi pathway. This study began with a genome-wide analysis of the roles of Sc-Sir1 in

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Saccharomyces to set the stage for studies of Td-Kos3 in T. delbrueckii. Collectively, these experiments

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lead to a new conceptualization for the evolution of Sir1’s role in silencing, and contribute to an

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expanded appreciation of the roles of RNAi components. These data provide the most complete

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picture to date of how the earliest SIR1-containing SIR silencing complex functioned and the

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evolutionary trajectories it may have followed.

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RESULTS

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S. cerevisiae Sir1 Localized to the Autonomous Silencers of HML and HMR-E Previous studies of genome-wide Sir protein localization in S. cerevisiae have focused on Sc-

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Sir2, Sc-Sir3, and Sc-Sir4 (1, 15). To study Sc-Sir1’s evolution, we first established the molecular

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topography of Sc-Sir1 across the S. cerevisiae genome. Chromatin immunoprecipitation of tagged Sc-

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Sir1-3xV5 followed by deep sequencing (ChIP-Seq) revealed several important features of Sc-Sir1’s

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genome-wide binding profile. First, Sc-Sir1 displayed a sharp, narrow, largely silencer-restricted

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binding profile at HML-E, HML-I, and HMR-E (Figure 1A and B; No Tag control shown in Figure

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S1). This distribution was in agreement with previous ChIP-PCR data suggesting that Sc-Sir1 is

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restricted to the HMR-E silencer (16). Sc-Sir1’s binding profile was strikingly different from

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previous data on Sc-Sir2, Sc-Sir3, and Sc-Sir4 (Sir2 shown in green in Figure 1A and B). Those

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proteins exhibit strong co-enrichment in discrete peaks both at the pair of silencers flanking HMLα

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and HMRa as well as within the silent loci (1). Sc-Sir1 enrichment overlapped with Sc-Sir2, Sc-Sir3,

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and Sc-Sir4 enrichment at three of the silencers and at a smaller peak located in the promoter region

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of HMLα but not within HMRa (Figure 1A). Each silencer at HML is sufficient, on its own, for

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silencing HML (17). At HMR, the E silencer is required for HMR silencing. HMR-I contributes to

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silencing when the locus is carried on a plasmid, but on its own is insufficient to silence HMR and

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can be deleted from the chromosome with no obvious impact on silencing (18, 19). No Sc-Sir1

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enrichment was detected at the HMR-I silencer.

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S. cerevisiae Sir1 Was Absent From Telomeres Telomeres in S. cerevisiae recruit the Sc-Sir2, Sc-Sir3, and Sc-Sir4 proteins through interactions

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with Rap1 (20). Mutations in SIR2, SIR3, and SIR4, but not SIR1, disrupt transcriptional repression

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of reporter genes placed adjacent to artificially truncated telomeres (1, 21). These early studies

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suggested SIR1 has no role in gene silencing near artificial telomeres. However, one study of a

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URA3 reporter gene at a native telomere (TEL11L) indicated a role for Sir1 in repressing genes at

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native telomeres (22). Thus, SIR1’s role in telomeric and subtelomeric silencing warranted further

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genome-wide evaluation.

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Strikingly, our results showed that the Sc-Sir1 protein was undetectable at all telomeres and

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subtelomeric regions (TEL15L shown in Figure 1C; see Figure S2 for all 32 telomeres). The sole

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exceptions to this rule are the Sc-Sir1 peaks at the silencers of HMLα, which fall within 20 kbp of

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the left end chromosome III (Figure 1A and Figure S2). In contrast, Sc-Sir2, Sc-Sir3, and Sc-Sir4 are

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all highly enriched at the telomeres, where they repress ~6% of subtelomeric genes (Figure 1C and

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(1, 15)). To test the possibility that Sc-Sir1 might bind telomeres transiently, long enough to repress

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genes but not long enough to be detectably enriched, we performed deep-sequencing of mRNAs

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from wild-type and sir1Δ strains. Genes at HMLα and HMRa were de-repressed in the sir1Δ strain,

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as expected, as were a handful of genes under a/α control (Table S2). However, consistent with a

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lack of Sc-Sir1 binding at and/or near telomeres, no subtelomeric genes were de-repressed in the

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sir1Δ mutant.

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The Torulaspora delbrueckii Genome Contains KOS3, an Ancestral SIR1 Paralog A reconstruction of the evolutionary history of the SIR1 gene (8) yielded two important

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findings: (1) SIR1 has undergone at least two to three gene duplications among post-whole-genome-

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duplication yeast species; and (2) SIR1 may itself may also be the product of an internal duplication

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of a shorter SIR1 paralog called KOS3 (Kin of Sir1), first recognized in S. bayanus v. uvarum. This

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paralog dates back to pre-whole genome duplication yeast species (8). Torulaspora delbrueckii, like

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Zygosaccharomyces rouxii, has a KOS3 paralog as its only Sir1-related gene (Figure 2). KOS3 is

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approximately half the sequence length of SIR1 and best aligns to the C-terminal Orc1-interacting

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region of Sir1. S. bayanus v. uvarum, N. castellii, and N. diarenesis also have KOS3 paralogs of similar

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size (Figure 2). The KOS3 paralog in S. bayanus v uvarum participates in silencing, though its function

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is partially shared with the other three paralogs in that species (8). All identified SIR1 paralogs are

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highly divergent at the protein sequence level (8). Similarly, Sc-Sir1 and Td-Kos3 share only 16%

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protein similarity.

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KOS3 Was Indispensible for Silencing in T. delbrueckii In S. cerevisiae, deletion of SIR1 causes a partial loss of silencing at HMLα and HMRa when

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evaluated at the population level. At the single-cell level, 50-80% of sir1∆ cells lack silencing at

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HMLα and HMRa, whereas these loci are fully silenced in the remaining cells (5). Thus, expression

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of HMRa1 in a sir1Δ strain, as measured in bulk RNA from a population of cells, was less than the

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expression level seen in S. cerevisiae sir2Δ, sir3Δ, or sir4Δ mutants (Figure 3A).

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To evaluate whether KOS3 was also only partially required for silencing in T. delbrueckii, or

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played a more prominent role, we measured expression of the HMRa1 locus in a MATα strain

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containing deletion alleles of KOS3, SIR2, or SIR4 (the SIR3 ortholog in T. delbrueckii is ORC1,

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which appears to be essential; unpublished observation). In contrast to the partial de-repression of

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HMRa1 seen in S. cerevisiae sir1Δ, T. delbrueckii kos3Δ cells showed complete de-repression of HMRa1,

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indistinguishable from that in sir2Δ and sir4Δ (Figure 3B). Thus, KOS3 played a more central role in

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silencing in T. delbrueckii as compared to S. cerevisiae SIR1.

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T. delbrueckii Kos3 Co-Enriched with Td-Sir2 and Td-Sir4 at all Heterochromatic

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Locations

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The genome-wide binding profiles of Td-Kos3, Td-Sir2, and Td-Sir4 in T. delbrueckii were

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striking with respect to the differences with Sir protein distributions in S. cerevisiae. At HMR, Td-

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Kos3 was most enriched in a pair of close but discrete peaks beginning approximately 670 base pairs

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3’ of HMRa1, which were also the positions most enriched for Td-Sir2 and Td-Sir4 (Figure 4B). The

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first of these peaks corresponded to a tRNA-Val gene. The distribution of Td-Kos3, Td-Sir2 and

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Td-Sir4 at HMLα showed only a single prominent peak of enrichment 770 base pairs from the 3’

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end of HMLα1 (Figure 4A). At neither HML nor HMR of T. delbrueckii was there evidence of two

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flanking enrichment peaks analogous to the two silencers flanking the silent mating-type loci in S.

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cerevisiae.

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In addition to examining Td-Kos3 binding at HML and HMR, we also interrogated Td-

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Kos3 enrichment at presumptive telomeres in T. delbrueckii to determine whether it was absent from

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telomeres, as Sc-Sir1 was in S. cerevisiae. At least eleven out of sixteen telomeres showed enrichment

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of Td-Kos3, Td-Sir2, and Td-Sir4: TEL01L, TEL02L, TEL04L, TEL07L, TEL08L, TEL01R,

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TEL04R, TEL05R, TEL06R, and TEL08R (Figure 4C shows TEL01R; see Figures S3 for all 16

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telomeres). Td-Kos3’s presence at telomeric sequences in T. delbrueckii was a marked difference to

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Sc-Sir1’s absence from telomeres. Likewise, many genes within 20 kilobases of chromosome ends

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increased in expression in all three T. delbrueckii sir mutants examined (kos3Δ, sir2Δ, and sir4Δ) (Table

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S9). Thus, similar to its more extensive role in silencing at T. delbrueckii HML and HMR, Td-Kos3

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was also required to repress expression of subtelomeric genes.

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T. delbrueckii SIR2 Had Roles Outside of Its Functions with KOS3 and SIR4

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We interrogated genome-wide functions for T. delbrueckii KOS3, SIR2, and SIR4 by

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performing mRNA-Seq in kos3∆, sir2∆, and sir4∆ mutants. Overall, twenty-two genes increased in

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expression across all three mutants (Table S9). These twenty-two genes were either at the silent

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mating type loci, adjacent to the silent mating type loci, or were subtelomeric genes within 20kb of a

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chromosome end. No centromere-adjacent genes changed expression among this set of mutants.

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When comparing the overlap between genes across all three sir mutants, we found that the majority

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of the changes in expression in the kos3∆ and sir4∆ mutants completely overlapped the changes in

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expression in the sir2∆ mutant, suggesting that KOS3 and SIR4 did not have any function outside of

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their role in the Sir complex (Figure S4B). There were 124 genes that increased specifically in the

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sir2∆ mutant, however, indicating that like SIR2 in S. cerevisiae, T. delbrueckii SIR2 had roles beyond

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heterochromatin.

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To examine potential roles that T. delbrueckii SIR2 may have, we performed GO term analysis

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on the 85 sir2∆-specific genes that had orthologs in S. cerevisiae. Using the S. cerevisiae functional

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annotations for these genes, we found 21 genes that were associated with meiosis and sporulation,

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and 9 genes that were associated with carbohydrate metabolism (starred genes, Table S4). Since T.

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delbrueckii SIR2 is the pre-whole genome ortholog of S. cerevisiae SIR2 and HST1, we also

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interrogated whether like HST1, T. delbrueckii SIR2 functioned as a promoter-specific repressor by

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examining whether any genes contained statistically significant Td-Sir2 peaks in their promoters. Of

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the 124 Td-Sir2-regulated genes, 66 had a Td-Sir2 peak in their promoters (genes marked ‡, Table

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S4).

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T. delbrueckii Kos3 Bound to the Silencers of HMLα and HMRa

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The largely silencer-restricted binding profile of Sc-Sir1 correlated with Sc-Sir1’s importance

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in establishing silencing. To determine whether or not the regions bound by TdKos3 corresponded

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to the silencers of T. delbrueckii, we created a reporter-based silencing assay using a plasmid

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containing the entire T. delbrueckii HMLα locus plus 1000 base pairs on either side and transformed

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this plasmid into T. delbrueckii. In this plasmid the α2 coding region was replaced with K. lactis

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URA3. Strains auxotrophic for uracil yet containing this plasmid were unable to grow on medium

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lacking uracil due to silencing of the K. lactis URA3 gene. Deletion of KOS3, SIR2, or SIR4 relieved

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this repression, leading to URA3 expression and growth on medium lacking uracil (Figure 5A).

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To map the silencers at HMLα, we deleted a 284 base-pair fragment (region E)

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corresponding to the major Td-Kos3, Td-Sir2 and Td-Sir4 binding peak adjacent to the coding

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genes and evaluated its impact on URA3 silencing. This deletion completely abolished silencing at

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HMLα (Figure 5C). Formally, silencers are defined as cis-acting regulatory sites. Because of the

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nature of the assay, there was an intact copy of the E-region in the chromosome, which nevertheless

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could not maintain silencing in cells with a deletion of this region on a plasmid-borne HML locus.

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Therefore the deleted region contained a silencer for HML, or at least a critical component of one.

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A similar assay was developed to map silencer elements at HMRa by cloning a ~5 kb

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fragment containing the HMR from T. delbrueckii chromosome V and replacing the a1 coding with

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the K. lactis URA3 gene. Silencing of this reporter was also dependent on KOS3, SIR2, and SIR4

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(Figure 6A). The binding profile of Td-Kos3 at HMRa at the putative silencer region showed two

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peaks, corresponding to regions A and B. Region C included regions A plus B and some

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surrounding sequence (Figure 6B). Region A was centered on the first peak and contained a valine

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tRNA gene. Deletion of region A had a modest effect on silencing, resulting in weak growth on

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medium lacking uracil, but not to the extent as in the kos3∆ mutant (Figure 6C). Deletion of region

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B had a slight to almost no effect on silencing, and deletion of region C led to a complete loss of

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silencing (Figure 6C). For the reasoning described above, the deletion of the C region must have

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removed all or a critical part of a silencer for HMR.

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T. delbrueckii Silencers Contained Rap1 Binding Sites That Were Important for Silencing In S. cerevisiae, the E and I silencers contained combinations of binding sites for Rap1, Abf1,

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and the Origin Recognition Complex (ORC). The silencers of K. lactis contain binding sites for

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Reb1, Ume6, as well as an additional “C-box” sequence (23). Since T. delbrueckii lies in a position

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between S. cerevisiae and K. lactis on the phylogenetic tree, we evaluated whether T. delbrueckii silencers

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contained binding sites that resembled those of K. lactis or S. cerevisiae, potentially illuminating how

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this major evolutionary transition of transcription-factor binding sites occurred. The T. delbrueckii

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silencer region E defined by the deletion at HML contained a high-scoring Rap1 DNA binding

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motif, 797 base pairs away from the 3’ end of the α1 gene: GACCTGTACA. A high-scoring Rap1

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site was also found in the promoter region of HML, between the α2 and α1 genes, reminiscent of

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the Rap1 binding site in the promoter region of HML in S. cerevisiae. To test the importance of the

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Rap1 binding site within region E, a triple mutant that disrupted the three most conserved base pairs

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of this Rap1 motif (GACCTGTACA to GAAATATACA) was evaluated (Figure 5C). This mutant

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diminished silencing to the same extent as deleting the entire E region, suggesting that this Rap1

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binding site was a key component of the silencer. A Rap1 binding site was also found in the T.

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delbrueckii HMR region immediately adjacent to the valine tRNA, residing just outside of region A.

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Disrupting this Rap1 binding site via a complete deletion, or mutating it from CATCCATACA to

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CATAAATACA, also greatly reduced silencing at HMRa (Figure 6D).

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The DNA-binding domain of the S. cerevisiae Rap1 protein has been mapped to amino acid

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residues 358-602 (24, 25). Alignment of the Sc-Rap1 and Td-Rap1 protein sequences revealed that

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this region is highly conserved between both species, displaying 81% sequence identity, suggesting

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that Td-Rap1 may bind to the conserved Rap1 binding motifs at the T. delbrueckii silencers. To test

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directly if Td-Rap1 bound to the silencers, chromatin immunoprecipitation followed by qPCR was

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performed on tagged Td-Rap1-3xV5. Td-Rap1 was enriched at the silencers of both HML and

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HMR, most highly at regions that included the conserved Rap1 binding site (Figure 7).

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In addition to Rap1 binding sites, a motif search also revealed the presence of three putative

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Abf1 binding sites clustered within region B of HMR (green lines under black arrow, Figure 6B), as

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well as one site within the promoter region of HML (overlapping the putative Rap1 site). Mutations

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of the highest-scoring of these putative binding sites in the B region, or deletion of all three, had no

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effect on silencing (data not shown). A search for ARS consensus sequences revealed a potential

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candidate AT-rich sequence of 13 base pairs in length in the C region of HMR (Figure 6B, black

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arrow marked “ARS”). This C region was also found to have a functional ARS; however, deleting

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the sequence that may represent this functional ARS had no effect on silencing (data not shown).

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KOS3 Expression Was Autoregulated The KOS3 gene itself is located ~1kb away from the copy of HMR carried on chromosome

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V (Figure 8A). Interestingly, in sir2∆ and sir4∆ mutants, the expression of KOS3 itself doubled

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(Figure 8B). Neither Td-Sir2 nor Td-Sir4 was enriched at the promoter of the KOS3 gene, indicating

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that these proteins do not directly repress it. Genes adjacent to silent mating-type cassettes are often

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de-repressed when losses in silencing occur, presumably because repressive chromatin at the silent

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locus exerts transcriptional repression on nearby genes; for example, the CHA1 gene adjacent to

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HML in S. cerevisiae increases in expression in sir mutants (15). When the KOS3 gene was moved

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from its native location to a plasmid, there was no increase in its expression in a sir2∆ mutant

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(Figure 8C). The location of the KOS3 gene and its increased expression when HMR is de-repressed

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suggests that in a wild-type strain, occasional lapses in silencing at HMR would increase the

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expression of its repressor, KOS3, providing an autoregulatory method of maintaining silencing.

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KOS3 Was Necessary For Efficient Recruitment of Sir2 and Sir4 To Silenced Loci

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In S. cerevisiae, Sc-Sir2, Sc-Sir3, and Sc-Sir4 can be recruited to the silencers of HMR in the

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absence of Sc-Sir1 (16), presumably due to the interactions between Sc-Rap1 at the silencer and a

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Sc-Sir2-Sir4 dimer, which, in turn, recruits Sc-Sir3. These interactions do not require Sc-Sir1 and

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may allow silencing to be re-established, albeit inefficiently, in a sir1∆ strain. ChIP-Seq of V5-tagged

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alleles of SIR2 and SIR4 in kos3Δ strains showed that KOS3 was required for efficient enrichment of

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Td-Sir2 and Td-Sir4 at HML and HMR and at telomeres (HMRa shown in Figure 9; see Figure S5

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for HMLα and TEL01R).

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Sc-Sir1 and T. delbrueckii Kos3, Sir2, and Sir4 May Be Enriched at Centromeres

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Sc-Sir1 had previously been found at six centromeres by locus-specific ChIP (CEN1, CEN2,

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CEN3, CEN4, CEN11, and CEN16), and sir1Δcac1Δ mutants show elevated rates of nondisjunction

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(26). When examining the Sir1 IP track separately from the input track, we saw a consistent under-

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representation of centromere sequences, hinting that centromere DNA was systematically under-

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recovered in our samples (representative example shown in Figure S6). To account for this under-

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recovery, we plotted Sir1 enrichment in terms of IP/ input and compared those values to the IP/

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input of the no-tag control. This analysis revealed Sc-Sir1 enrichment at all sixteen S. cerevisiae

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centromeres (Figure S7). ChIP-Seq datasets have been shown to contain certain reproducible but

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artifactual signals, implying the association of proteins to sequences that they do not actually bind in

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vivo (27, 28). To rigorously test whether these Sc-Sir1 peaks at centromeres represented ChIP-Seq

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artifacts, we compared Sc-Sir1 enrichment to enrichment of GFP tagged with a nuclear localization

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sequence (GFP-NLS) at centromeres (data from (27)). GFP is not expected to bind in a meaningful

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way to any portion of the yeast genome, yet control experiments show that it co-localizes with

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multiple common ChIP-Seq artifacts. Only one centromere, CEN13, showed GFP-NLS IP over

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input enrichment. Thus, although the Sc-Sir1 signal present at that centromere may be spurious

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(Figure S7, panel marked with *), there was no indication of artifactual enrichment at the others.

315

Additionally, despite the presence of Sc-Sir1 at centromere sequences, there was no indication of any

316

Sir-dependent gene silencing adjacent to any centromere (see also (15)).

317

Because we saw Sc-Sir1 enrichment at S. cerevisiae centromeres, we evaluated whether Td-

318

Kos3, Td-Sir2, and Td-Sir4 were present at centromeres in T. delbrueckii. T. delbrueckii, like S. cerevisiae,

319

has point centromeres that have been annotated based on conservation of the centromere DNA

320

elements (CDEI, CDEII, and CDEIII) and by synteny (29). We confirmed function for two of these

321

centromeres (T. delbrueckii CEN1 and CEN3) by observing their ability to functionally replace S.

322

cerevisiae CEN6 in the pRS316 vector, allowing strains to maintain the plasmid in the absence of

323

selection in an S. cerevisiae host (centromeres appear to be compatible between the two species). We

324

then examined Td-Kos3, Td-Sir2, and Td-Sir4 enrichment at presumptive T. delbrueckii centromeres

325

in terms of IP/ input and detected enrichment of all three proteins at centromeres (Figure S8). As in

326

S. cerevisiae, we observed no evidence of silencing of genes adjacent to the centromeres.

327 328 329

T. delbrueckii AGO1 and DCR1 Had No Function in Silencing Most Saccharomyces yeasts lack the machinery for RNAi, a mechanism of gene silencing found

330

in Schizosaccharomyces pombe and many other organisms, including plants and animals. The Argonaute

331

and Dicer proteins are required for heterochromatin formation in S. pombe, and presumably in all

332

organisms using the RNAi mechanism. Ago1 is a necessary component of the RNA-induced

333

initiation of transcriptional gene silencing (RITS) complex, and Dcr1 cleaves double-stranded RNA

334

into small interfering RNAs (siRNAs) that serve as guide RNAs, directing the heterochromatin

335

machinery to the locus targeted for silencing (30). The Naumovozyma castellii genome contains an

336

AGO1 ortholog and a DCR1-like gene (DCR1-like because it is not directly orthologous to the S.

337

pombe DCR1, but rather a duplicate of RNT1, a ribonuclease specific for double-stranded RNA). N.

338

castellii AGO1 and DCR1 together degrade Ty transcripts (13).

339

The T. delbrueckii genome also contains an AGO1 and a DCR1-like gene, orthologous to

340

those of N. castellii. Given that AGO1 and DCR1 repress Ty elements in N. castellii, we tested

341

whether the AGO1 and DCR1 genes functioned in silencing in T. delbrueckii by deep sequencing of

342

mRNAs in T. delbrueckii ago1∆, dcr1∆, and ago1∆dcr1∆ double-mutants. These mutants displayed no

343

defect in transcriptional repression of HML, HMR, or of any genes near telomeres (Figure 10A-B),

344

and thus, these genes displayed no overlap in function with the SIR genes. Additionally, no genes

345

showed a clear signal of de-repression in the RNAi mutants—i.e., no genes went from 0 FPKM in

346

wild type to an FPKM > 0 in the mutant. Overall, fifteen genes significantly changed in expression

347

in the ago1∆ mutant, nine in the dcr1∆ mutant, and 53 in the ago1∆dcr1∆ double mutant (Figure 10B

348

and Tables S6-S8). Among the genes changing in expression in RNAi mutants, little to no overlap

349

was seen among these gene sets (Figure 10C and D). The most striking observation was the bigger

350

impact that the double mutant had on expression of genes than either of the single mutants,

351

discussed below. For the genes that had S. cerevisiae orthologs, we performed GO term analysis for

352

the ago1∆dcr1∆ double-mutant and found that several genes were associated with oxidation-

353

reduction processes and/or small molecule metabolism, indicating a possible coordinating role in

354

metabolic function (genes marked with black and orange dots, Figure 10B).

355 356

DISCUSSION

357

In this study we exploited four opportunities provided by Torulaspora delbrueckii to explore

358

theme and variation in the evolution of gene silencing. Specifically, T. delbrueckii, as a pre-whole-

359

genome duplication ascomycete, has one of the oldest versions of the SIR1 gene, perhaps the most

360

enigmatic of all budding yeast silencing genes. We explored the functional trajectory of this gene

361

from its earliest recognized appearance in Torulaspora delbrueckii to its reduced role in Saccharomyces

362

cerevisiae. Interestingly, we found that although the overall function of SIR1 in the formation of

363

heterochromatin has remained constant, its precise role in that process has evolved considerably.

364

The effect of deleting SIR1 on silencing in S. cerevisiae is relatively minor on a cell population basis.

365

In contrast, in T. delbrueckii, deletion of KOS3 completely abolished silencing. Second, in addition to

366

having the oldest SIR-silencing components, T. delbrueckii also has genes orthologous to budding

367

yeast AGO1 and DCR1, whose function(s) in T. delbrueckii were not known. Third, the silencer

368

composition of the only other pre-duplication species examined, K lactis, differs from S. cerevisiae.

369

Hence, T. delbrueckii offered the chance to explore whether the S. cerevisiae composition originated

370

before or after the whole-genome duplication event. Finally, T. delbrueckii offered the opportunity to

371

explore to what extent unusual features of the molecular topography of silenced chromatin were

372

intrinsic to the mechanism of silencing.

373 374 375

Sc-Sir1 Associated with Silencers Except For The HMR-I Silencer Sc-Sir1 clearly bound to three of the four silencers in S. cerevisiae: it was strikingly enriched at

376

HML-E, HML-I, and HMR-E, but not at HMR-I. It bound to those silencers that are sufficient on

377

their own to maintain silencing (17). Sc-Sir1 directly interacts with Orc1, a component of the Origin

378

of Recognition Complex, and this interaction likely brings Sc-Sir1 to the silencer (7, 31). However,

379

the ORC complex presumably associates with all four silencers, as an ARS consensus sequence is

380

present at each one, and all four are capable of functioning as an origin of replication when on

381

plasmids. Moreover, both HMR-E and HMR-I are origins of replication in their chromosomal

382

context (32, 33). Therefore, it is perplexing why Sir1 enrichment was absent from HMR-I.

383

Interestingly, HMR-I lacks a Rap1 binding site. It is possible that Sc-Rap1 stabilizes the interactions

384

between Sc-Sir1, ORC, and Sc-Sir4, and that Sc-Sir1’s absence is due to Sc-Rap1’s absence at this

385

silencer.

386 387 388

Td-Kos3 was Essential For Silencing, Whereas Sc-Sir1 is Not Two observations emphasize the importance of Kos3 in silencing: (1) T. delbrueckii kos3∆

389

strains exhibited a complete loss of silencing at HML, HMR, and telomeres; and (2) in the absence

390

of Td-Kos3, enrichment of Td-Sir2 and Td-Sir4 at these positions was greatly reduced. In S.

391

cerevisiae, Sc-Sir1 and Sc-Sir4 interact (6). Sc-Rap1 is also present at the silencer, and the interactions

392

between Sc-Rap1 and Sc-Sir4 and Sc-Rap1 and Sc-Sir3 are well documented (34). Therefore, in

393

addition to the interaction between Sc-Sir1 and Sc-Sir4, interactions between Sc-Rap1 and Sc-Sir4

394

and Sc-Rap1 and Sc-Sir3 boost the efficiency with which silencing proteins associate with the

395

silencer in S. cerevisiae. Td-Rap1 bound silencers in T. delbrueckii and contributed to silencing the

396

adjacent loci. The absence of a Sir3 paralog and/or the lack of a Td-Sir4-Td-Rap1 interaction in T.

397

delbrueckii may explain why Td-Kos3 is essential for silencing in that species: Td-Kos3 may be the

398

primary protein mediating an interaction between Td-Sir4/Td-Sir2 and the silencer.

399 400

Td-Kos3 Functioned At Telomeres, Whereas Sc-Sir1 Did Not

401

Early studies of telomeric silencing in S. cerevisiae found no role for Sir1 in “telomere position

402

effect,” as measured by reporter genes adjacent to synthetic telomeres. Our ChIP-Seq data of Sc-Sir1

403

and RNA-Seq data of the sir1∆ mutant corroborated these early observations and extended them to

404

all telomeres. We saw no Sc-Sir1 protein enrichment at or near telomeres (except for at HMLα) and

405

no subtelomeric genes were de-repressed in the sir1∆ mutant. In contrast, Td-Kos3 bound to at least

406

eleven out of sixteen telomeric and subtelomeric sequences in T. delbrueckii, where it’s enrichment

407

pattern closely matched that of Td-Sir2 and Td-Sir4. These data suggest that the ancestral SIR1 was

408

once a part of a core silencing complex composed of TdOrc1/Kos3/Sir4/Sir2, functionally

409

equivalent to the ScSir2/Sir3/Sir4 complex. For the five telomeres where Td-Kos3 was absent, Td-

410

Sir2 and Td-Sir4 were also absent. It may be that the genome assembly for these five telomeres is

411

less complete; sequencing using longer genomic inserts (> 1kb) would be required to fully assemble

412

the remaining five telomeres and assess whether Td-Kos3, Td-Sir2, and Td-Sir4 are present at those

413

ends as well.

414 415

T. delbruekii SIR2 Had Roles In Addition to Silencing

416

SIR2 in S. cerevisiae has other roles in the cell in addition to its role in heterochromatin at

417

telomeres and the silent mating-type loci, such as suppression of recombination at rDNA repeats

418

and lifespan regulation (35, 36). Our RNA-Seq data suggested that even in T. delbrueckii, SIR2

419

regulates many genes and likely performs functions outside of silencing, as there were 146

420

expression changes that were specific to the sir2∆ mutant (124 genes increased and 22 decreased in

421

expression). T. delbrueckii SIR2 is the pre-whole-genome-duplication ancestor of the S. cerevisiae SIR2

422

and HST1 duplicates; thus, T. delbrueckii SIR2 may also repress genes that in S. cerevisiae are repressed

423

by HST1. S. cerevisiae Hst1, in complex with Sum1 and Rfm1, functions in promoter-specific

424

repression of middle-sporulation genes (37). K. lactis SIR2, another pre-whole-genome duplication

425

ortholog of S. cerevisiae SIR2 and HST1, possesses functions of both S. cerevisiae SIR2 and HST1 (9,

426

38). Interestingly, T. delbrueckii othologs of two middle-sporulation genes repressed by Hst1 in S.

427

cerevisiae were de-repressed in the T. delbrueckii sir2∆ mutant: SPS4 and DIT1. Many other orthologs

428

of meiotic genes were also de-repressed (21 marked genes in Table S4), and six of these had Sir2

429

peaks in their promoters: DIT2, SPO19, SPS101, SPS2, SPS4, and IME2. The presence of promoter-

430

specific Sir2 peaks suggests that, like K. lactis SIR2, T. delbrueckii SIR2 is capable of acting as both a

431

promoter-specific repressor as well as a long-range, promoter-independent repressor of gene

432

expression.

433 434

Silencer Conservation and Diversity Among Budding Yeasts

435

Pairs of silencers flank both HML and HMR in S. cerevisiae, which are all bound by ScSir2,

436

ScSir3 and ScSir4 and, as shown here with the exception of HMR-I, by ScSir1. A single prominent

437

site bound by Td-Kos3, Td-Sir2 and Td-Sir4 adjacent to HML and a close pair of sites adjacent to

438

one side of HMR mediated silencing of these loci in T. delbrueckii. Although the analysis of these

439

binding sites has only just begun, these sites were, in fact, silencers. A Rap1 binding motif was

440

clearly critical for silencing at both loci, and the Rap1 protein itself associated with regions that

441

included this binding motif. The HMR silencer supported autonomous replication of a plasmid,

442

implying the existence of an origin of replication and thus an ORC binding site. Abf1 binding site

443

motifs were also evident. Individual mutations to the putative Abf1 binding sites and the putative

444

ARS had no effect on silencing. While this result might suggest that these binding sites do not

445

contribute to silencing, it is possible that, like in S. cerevisiae, they have partially redundant roles in

446

facilitating transcriptional repression. As in S. cerevisiae, mutating the two sites simultaneously may be

447

required to disrupt repression (39). Further analysis will be required to map more precisely the

448

functional elements of the silencer, but already there are notable differences between the structure of

449

silenced chromatin in T. delbrueckii from that of S. cerevisiae, pointing to alternative means by which

450

silencing can occur.

451

In K lactis, Reb1 substitutes for the Rap1 protein in silencer function (40), even though Rap1

452

is critical for telomeric gene silencing (41). In T. delbrueckii, Rap1 sites were clearly important for

453

silencer function, and Td-Rap1 bound to the silencer regions of both HML and the chromosome V

454

HMR. Thus, the substitution of Reb1 for Rap1 was not associated with the whole-genome

455

duplication. It is possible that the elevated substitution rate at silencers may drive the diversification

456

of transcription factor binding sites at silencers and silencer binding proteins (42). It is curious that

457

the Sir proteins themselves (with the exception of Sir2) are also rapidly evolving. Whatever the

458

driver of this rapid evolution may be, the result is that hemiascomycete species have a variable

459

repertoire of Sir proteins with differing numbers of Sir1 paralogs. Selection may be imposed on

460

whichever set of protein-protein interactions results in the successful recruitment of the Sir2-Sir4

461

dimer (Sir2, being the catalytic component, is the member that can deacetylate H4K16Ac and

462

ultimately repress the locus). Rapid protein evolution may have strengthened some protein-protein

463

interactions and weakened others. Thus, species that require multiple Sir1 paralogs, like S. bayanus v.

464

uvarum, may be those in which Rap1 or Sir3 are insufficient to stably recruit Sir2/Sir4. Species that

465

lack a SIR1 paralog entirely may be those in which silencer-bound proteins have evolved a higher

466

affinity for the Sir2-Sir4 dimer, obviating the need for Sir1.

467 468

The Presence of Sc-Sir1 and Td-Kos3 at Centromeres

469

Heterochromatin is characteristically assembled at centromeres of eukaryotes including

470

Schiszosaccharomyces pombe, yet in Saccharomyces and other organisms with point centromeres,

471

heterochromatin is not found at centromeres and no genes near centromeres were de-repressed in sir

472

mutants in S. cerevisiae or T. delbruekii. Earlier work established that the Sc-Sir1 protein of S. cerevisiae

473

is present at some centromeres, where it contributes to proper chromosome segregation along with

474

the chromatin assembly factor (CAF) complex (26). We found some enrichment of Sc-Sir1 at all but

475

one centromere. All three Sir proteins in T. delbrueckii (Td-Kos3, Td-Sir2, and Td-Sir4) were found at

476

all eight centromeres in this organism. In both species, the enrichment of IP reads over input for

477

the centromere regions did not reach statistical significance as assessed by MACS. However, MACS

478

is designed to detect peaks created by an enrichment of IP reads relative to input at a particular

479

genomic region, not peaks created by greater under-enrichment in the input sample. Viewing the

480

data in terms of IP over input clearly showed peaks at the centromeres. Unfortunately, we have been

481

unable to express the GFP protein in T. delbrueckii and hence were unable to use this established

482

metric to evaluate whether these peaks represented biological or artifactual associations. One

483

interpretation of this data is that Td-Kos3 in T. delbruekii, like Sc-Sir1, plays some conserved

484

function in centromere function. Whether the other Sir proteins with a ChIP-Seq enrichment signal

485

at a subset of centromeres represent some latent centromere function of these proteins, the vestigial

486

presence of silencing proteins at centromeres, or a new class of ChIP-Seq artifacts, awaits further

487

study.

488 489 490

The Role of RNAi in T. delbrueckii Our RNA-Seq data of ago1∆ and dcr1∆ mutants of T. delbruekii revealed that AGO1 and

491

DCR1 did not function in silencing at HML, HMR, or telomeres. Thus, if these proteins contribute

492

to RNAi function in T. delbrueckii, RNAi must have a role other than in heterochromatin function.

493

Of the 77 genes found to significantly change in expression across all candidate RNAi mutants,

494

~32% are genes of unknown function that have no ortholog in S. cerevisiae. Moreover, budding yeast

495

DCR1 is not directly orthologous to S. pombe DCR1, but rather is a duplicate of RNT1 which

496

encodes a ribonuclease involved in the processing of rRNA transcripts (43). Therefore, DCR1 may

497

have inherited a separate set of interaction partners and functional constraints from its RNT1

498

ancestor and may be on a different evolutionary trajectory from AGO1. Additionally, the AGO1 and

499

DCR1 genes of N. castellii that repress Ty elements are thought to mediate repression at the post-

500

transcriptional level, not at the epigenetic level via interactions with chromatin modifying enzymes

501

(such as histone deacetylases and demethylases). Furthermore, Candida albicans DCR1, an ortholog of

502

both the T. delbrueckii and N. castellii DCR1, functions in rRNA and spliceosomal RNA processing,

503

strengthening the possibility for an RNA-processing function for T. delbrueckii DCR1 (44). As of yet,

504

there exists no evidence tying budding yeast RNAi genes with any chromatin factors involved in the

505

establishment or maintenance of heterochromatin, although there are many direct interactions

506

between chromatin modifiers and DCR1 and AGO1 in S. pombe (12).

507

Argonaute itself has had a complex evolutionary journey. Eukaryotic Argonaute proteins

508

bind short RNA guide molecules to target transcripts. Prokaryotic Argonaute proteins, however,

509

can bind DNA and may participate in genome defense against mobile elements (45). Budding yeast

510

Argonaute co-purifies with small-interfering RNAs generated by Dicer, which suggests that it

511

functions like other eukaryotic Argonaute proteins (13). However, other binding properties for

512

budding yeast Argonaute have yet to be explored. Little overlap was observed in gene sets between

513

ago1∆ and dcr1∆; however, the 48 genes whose expression is altered only in the ago1∆dcr1∆ double

514

mutant imply that these two proteins may share an overlapping function. That overlapping function

515

must not be one that the proteins carry out together; rather, based upon the unique phenotype of

516

the double mutant, either must be able to contribute to that function in the absence of the other.

517 518

MATERIALS & METHODS

519

Identification of SIR1 Paralogs: To identify SIR1 paralogs, the SIR1 protein sequence was used as

520

a BLAST query against sequenced yeast genomes available on the Yeast Gene Order Browser

521

(YGOB). Significant hits included the KOS3 gene in T. delbrueckii (TDEL0E00350), as well as all

522

other previously found SIR1 paralogs (8). T. delbrueckii KOS3 itself, when used as a BLAST query

523

against yeast genomes on YGOB, identified the Zygosaccharomyces rouxii KOS3 gene and the S. bayanus

524

v. uvarum KOS3 gene as the two top matches. Other SIR1 paralogs, including S. cerevisiae SIR1, were

525

among the top 15 matches.

526

Yeast Strains and plasmids

527

Strains are listed in Supporting Information, Table S1. Saccharomyces cerevisiae strains were generated in

528

the W303 background. Deletion mutants and epitope-tagged alleles of SIR genes were made as

529

previously described, using one-step integration of knockout cassettes (46). Torulaspora delbrueckii

530

strains were grown in rich medium (YPD) at 30°C. Gene disruption in T. delbrueckii required ~500

531

base pairs of sequence identity to the target region. Therefore, knockout cassettes and other tagging

532

constructs were first cloned into plasmids containing 500 base pairs of sequence identical to the

533

sequences flanking the genomic target, then amplified via PCR and transformed into strains.

534

Transformations for T. delbrueckii were performed using the same lithium acetate-PEG method used

535

for S. cerevisiae (47).

536

RNA Isolation and Quantitative reverse-transcriptase PCR (qRT-PCR) analysis

537

Strains of both S. cerevisiae and T. delbrueckii were grown to an A600 of 0.8-1.0 at 30°C in YPD. RNA

538

was extracted as described previously using the hot acid-phenol method (15, 48). cDNA and qRT-

539

PCR analysis was performed as described previously (15). Oligos used for ACT1 amplification:

540

GCCGGTGACGACGCTCC and CCTCTCTTGGATTGAGCTTCATCACC; oligos used for

541

KOS3 amplification: TTGGAGAACTATCGCAGAGAGAGC and

542

TCTCTTTGGCTATTGCGGTTGG.

543

Chromatin Isolation and Immunoprecipitation

544

All strains were grown in 100ml YPD and harvested in log phase at an A600 of ~0.7. Cross-linking

545

was performed at 25°C in 1% formaldehyde for 45 minutes. Chromatin was prepared as previously

546

described (49). Sonication was performed to an average genomic fragment size of 300-400 base

547

pairs. Immunoprecipitation of V5 epitope-tagged Sir1, Td-Kos3, Td-Sir2, and Td-Sir4 was

548

performed overnight at 4°C using 800μl of chromatin and 75μl of anti-V5 resin from Sigma

549

(A7345). After several washes, protein and DNA was eluted from beads in TE buffer + 1% SDS at

550

65°C, followed by reversal of crosslinking, then protease treatment. DNA was purified using Qiagen

551

DNA spin columns prior to library preparation. Functions of epitope-tagged SIR alleles in T.

552

delbrueckii were assayed by measuring repression at the silent HMRa1 gene; function of V5-tagged

553

Sir1 was measured by its ability to complement a sir1∆ mutation.

554

Library Preparation and Sequencing

555

ChIP libraries were prepared using the Illumina TruSeq DNA Sample Prep kit. RNA-Seq libraries

556

were prepared using the Illumina TruSeq mRNA Sample Prep kit. 100-bp paired-end libraries were

557

used to accurately assign reads. A Bioanalyzer instrument (Agilent) was used to quantify all libraries.

558

Libraries were sequenced on an Illumina HiSeq 2000 machine. Reads were deposited in the NCBI

559

Sequence Read Archive (SRA) at http://www.ncbi.nlm.nih.gov/sra under accession numbers

560

SRP055208, SRP065348, SRP065349, SRP065572, and SRP065573. See Table S10 and S11 for

561

sequence read information for all libraries.

562

URA3 Reporter-Gene Assay for Silencing

563

Cells were grown to saturation overnight in 2ml of YPD containing hygromycin B drug (to select for

564

plasmids). Cells were then pinned onto plates with three different media: CSM containing

565

hygromycin B (to assay overall growth), CSM medium containing hygromycin B and lacking uracil

566

(to select for cells expressing URA3), and CSM containing uracil and 5-fluoroorotic acid (5FOA) to

567

select for cells not expressing URA3 (50). Cells were pinned in a 5-fold dilution series, and plates

568

were imaged on day three of growth.

569

Data Analysis

570

ChIP-Seq. Reads were mapped using Bowtie2 to either the Saccharomyces cerevisiae S288C reference

571

genome or the T. delbrueckii reference genome sequence (14). Duplicate reads were discarded using

572

Picard, and pileup files were generated using Samtools (51). Data was plotted and visualized using

573

custom Python scripts. Statistically significant peaks of enrichment in IP samples were found by

574

using the MACS peak-calling software (52).

575

RNA-Seq. Data were analyzed as previously described (15). Briefly, Tophat2 was used to map

576

transcripts to their gene of origin. Transcript quantification was performed using Cufflinks (53).

577

DESeq was used to perform tests for differential gene expression (54). Results were filtered for

578

genes that showed differences in expression greater than two-fold relative to wild type, with p-value

579

of