Rpd3-dependent boundary formation at by removal of Sir2 substrate

Stefan Ehrentrauta, Jan M. Webera, J. Nikolaj Dybowskib, Daniel Hoffmannb, and Ann E. Ehrenhofer-Murraya,1

aAbteilung für Genetik and bAbteilung für Bioinformatik, Zentrum für Medizinische Biotechnologie (ZMB), Universität Duisburg-Essen, D- 45117 Essen, Germany

Edited by Jasper Rine, University of California at Berkeley, Berkeley, CA, and approved November 27, 2009 (received for review August 13, 2009) Boundaries between euchromatic and heterochromatic regions tion in addition to its function in local gene repression (18). Rpd3 until now have been associated with chromatin-opening activities. is present in two different HDAC complexes: Rpd3(L) is targeted Here, we identified an unexpected role for deacetylation in to gene promoters and establishes gene repression via promoter this process. Significantly, the (HDAC) Rpd3 was deacetylation, whereas Rpd3(S) provides deacetylation in the necessary for boundary formation in Saccharomyces cerevisiae. body of genes and prevents intragenic transcription (19, 20). The rpd3Δ led to silent information regulator (SIR) spreading and repres- deletion of RPD3 leads to higher global acetylation levels (21). sion of subtelomeric genes. In the absence of a known boundary Intriguingly, Rpd3-mediated histone deacetylation also has been factor, the histone acetyltransferase complex SAS-I, rpd3Δ caused invoked in Hog1-dependent activation of osmosensitive genes inappropriate SIR spreading that was lethal to yeast cells. Notably, (22), and rpd3Δ causes increased silencing in yeast as well as in Rpd3 was capable of creating a boundary when targeted to hetero- Drosophila (23–25). chromatin. Our data suggest a mechanism for boundary formation Here, we performed a screen for factors that become essential in whereby histone deacetylation by Rpd3 removes the substrate for the absence of the HAT Sas2. We found that deletion of RPD3 is the HDAC Sir2, so that Sir2 no longer can produce O-acetyl-ADP lethal in sas2Δ cells and that excessive spreading of hetero- ribose (OAADPR) by consumption of NAD+ in the deacetylation reac- chromatic SIR complexes is responsible for the lethality. Rpd3 is tion. In essence, OAADPR therefore is unavailable for binding to Sir3, necessary to restrict the SIR proteins to the telomeres, and tar- preventing SIR propagation. geting Rpd3 to normally silent chromatin creates a barrier to the

spreading of SIR-dependent repression. Our data suggest a mech- GENETICS gene silencing | histone deacetylase | O-acetyl-ADP-ribose | Sas2 | Sir3 anism for boundary formation, in that Rpd3 effectively removes the substrate for Sir2 at the –euchromatin boundary. he functional distinction between euchromatic and hetero- Therefore, heterochromatin spreading is stopped by the inability Tchromatic domains within eukaryotic genomes is essential to of Sir2 to perform histone deacetylation, to produce OAADPR, maintain gene expression programs that drive development and and thus to support heterochromatin spreading. differentiation in higher organisms (1). Each expression domain Results must maintain its identity, and thus junctions exist that separate SAS2 RPD3 active from inactive regions and maintain opposing transcriptional Deletion of and Is Synthetically Lethal. The SAS-I complex states. Barriers between chromatin states have been described in a globally acetylates H4 K16 (26, 27). To search for factors that variety of organisms (1). In Saccharomyces cerevisiae, histone become essential in the absence of Sas2, we performed a syn- thetic lethal screen with sas2Δ cells. Surprisingly, we found that acetylation by several histone acetyltransferase (HAT) complexes RPD3 A (2–5), H3 K79 methylation by the histone methyltransferase Dot1 the deletion of is lethal in the absence of Sas2 (Fig. 1 and B), a finding that is in agreement with an earlier study (28). At (6), and H3 K4 methylation by Set1 (7) have been associated with rpd3Δ sas2Δ boundary formation. Some modifications also may cooperate with higher temperatures, double mutants are nonviable. chromatin remodeling activities to implement barrier function (4). Furthermore, the lethality requires the acetyl-CoA binding site as well as the atypical zinc finger of Sas2 (Fig. S1). We found that Other chromatin alterations such as the complete loss of nucleo- sas4Δ sas5Δ rpd3Δ somes (4, 5) or incorporation of the histone variant H2A.Z (8) also and also are synthetically lethal with (Fig. 1A), showing that the whole SAS-I complex is involved in the are associated with a block to silencing. rpd3Δ Although some boundaries are fixed to a certain genomic position, lethality with . Furthermore, the lethality depends upon the Rpd3(L), not the Rpd3(S), complex, because sas2Δ shows syn- others are characterized by a balance of opposing enzymatic activ- thetic growth defects in the absence of the Rpd3(L) components ities and the competition between chromatin-opening and -con- Dep1 and Sds3 but not the Rpd3(S) components Rco1 and Eaf3 densing complexes. One such example is telomeric heterochromatin (Fig. 1B and Table S1), and the additional deletion of RCO1 does in S. cerevisiae, where histone deacetylation by the NAD+-depend- not exacerbate the growth defect of sas2Δ sds3Δ cells (Fig. 1B). ent histone deacetylase (HDAC) Sir2 (9) is required for the However, set2Δ does not cause a growth defect in sas2Δ cells (Fig. repressive silent information regulator (SIR) complexes to bind to 1C), indicating that neither Set2-dependent recruitment of Rpd3 the chromatin (10). Deacetylation by Sir2 at telomeres is counter- (S) (20) nor an Rpd3(S)-independent function of Set2 at telo- acted by the HAT complex SAS-I, which contains the MYST family mere boundaries (29) is involved in the synthetic lethality HAT Sas2 and acetylates H4 K16. Thus, the competing activities of sas2Δ rpd3Δ. fl fl between and The lethality is re ected further in an SAS-I and Sir2 create exible boundaries between eu- and hetero- increased sensitivity of sas2Δ cells to treatment with the HDAC chromatin at telomeres via de-/acetylation of H4 K16 (11, 12). Interestingly, the deacetylation reaction of Sir2 is distinct from that of non–NAD+-dependent HDACs in that it produces an Author contributions: S.E. and A.E.E.-M. designed research; S.E., J.M.W., and A.E.E.-M. per- unusual compound, O-acetyl-ADP ribose (OAADPR) (13), formed research; J.N.D. and D.H. contributed new reagents/analytic tools; S.E. and D.H. an- which has been proposed to influence SIR complex stability (14). alyzed data; and S.E. and A.E.E.-M. wrote the paper. Intriguingly, the Sir3 protein carries a domain that resembles the The authors declare no conflict of interest. + ATP binding pocket of AAA ATPases but lacks certain cata- This article is a PNAS Direct Submission. lytic residues (15). It therefore has been hypothesized that this 1To whom correspondence should be addressed. E-mail: ann.ehrenhofer-murray@uni- domain constitutes an OAADPR binding site (16). due.de. SAS-I globally acetylates H4 K16 in subtelomeric regions (17). This article contains supporting information online at www.pnas.org/cgi/content/full/ Similarly, the HDAC Rpd3 provides global chromatin deacetyla- 0909169107/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0909169107 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 A sas2Δ rpd3Δ sas4Δ rpd3Δ sas5Δ rpd3Δ A sas2Δ rpd3Δ + pURA3-SAS2 - sir1Δ sir4Δ

sir2Δ sir3Δ B sas2Δ + pURA3-SAS2 rpd3Δ +pLEU2- YM 5-FOA RPD3 rco1Δ rpd3Δ Rpd3 (S) sas2Δ rpd3Δ + H3/H4 allele sds3Δ rco1Δ B Rpd3 (L) sds3Δ control H4 rco1Δ K->Q sds3Δ wt H3 K->Q

YM 5-FOA H4 H3 K9R K5,8R H4 sas2Δ + pURA3-SAS2 H3 K14R C H4 K5,12R K16R rpd3Δ

rpd3Δ Fig. 2. The synthetic lethality between the Rpd3(L) and the SAS-I complex is +pLEU2- caused by inappropriate SIR spreading. (A) Deletions of subunits of the RPD3 sas2Δ rpd3Δ set2Δ telomeric SIR complex suppress the synthetic lethality. Deriva- tives of an sas2Δ rpd3Δ pURA3-SAS2 strain (AEY 3923) with deletions for SIR1, SIR2, SIR3,orSIR4 were grown on minimal plates (YM, growth assay) YM 5-FOA and on 5-FOA plates to select against pURA3-SAS2 for 2 days at 30°C. (B) Mutations within the histone H3 and H4 N-termini suppress the sas2Δ rpd3Δ wt sas2Δ synthetic lethality. Alleles of H3 and H4 were introduced into an rpd3Δ D sas2Δ pLYS2-SAS2 strain by plasmid shuffle (AEY3945; SI Materials and Methods for details), and the ability of the derivatives to survive in the absence of the SAS2 plasmid was tested. “wt” refers to WT copies of H3 and H4 in AEY3945. “Control” refers to a WT strain. H4 K -> Q designates H4 K5, 8, 12, and 16 Q. H3 K -> Q designates H3 K4, 9, 14, 18, 23, and 27 Q. TSA SIR1 HM A ,whichaffects but not telomeric silencing, does not sup- Fig. 1. Synthetic lethality between the Rpd3(L) and the SAS-I complex. ( ) sas2Δ rpd3Δ A Cells disrupted for subunits of the SAS-I complex are synthetically lethal with press the lethality between and (Fig. 2 ). Also, dele- rpd3Δ. Tetrad dissection of crosses of sas2Δ, sas4Δ or sas5Δ with rpd3Δ isogenic tion of HMR in a MATα sas2Δ rpd3Δ sir2Δ strain, which reverses the W303 strains. The four spores from individual asci are aligned in vertical lines. pseudodiploid cell type, does not abrogate the viability of the strain. Double mutants are marked with circles. (B) The lethality between rpd3Δ and The binding of SIR complexes to the telomeres depends on the sas2Δ is specific for the Rpd3(L) complex. sas2Δ cells with pURA3-SAS2 and acetylation state of the amino-terminal of H3 and H4 RCO1, SDS3 additional deletions of , or both were incubated on supplemented and, in particular, on H4 K16 (10). Therefore, mutation of critical yeast minimal medium (YM) or 5-fluoroorotic acid (5-FOA) medium. (C) set2Δ sas2Δ sas2Δ D sas2Δ histone residues that abrogate silencing should suppress the does not cause synthetic lethality in .( ) cells are sensitive to tri- rpd3Δ chostatin A (TSA). Filter discs with DMSO control (Upper row) or increasing synthetic lethality. In fact, we observed that mutation of the amounts (16, 12 and 8 μg) of TSA (Lower row) were placed on lawns of 2 × 105 acetylatable lysine residues in the tails of H3 or H4 allows growth cells of a WT or sas2Δ strain. Plates were incubated for 1 day at 30°C. of sas2Δ rpd3Δ strains and that a sole mutation of H4 K16R is sufficient for the suppression (Fig. 2B). Such mutations have been shown previously to abrogate silencing (Fig. S2) (30). The H4 inhibitor trichostatin A (Fig. 1D). No other HAT is lethal in K16R suppression may seem counterintuitive, given that muta- combination with rpd3Δ, and no other HDAC is lethal with sas2Δ tions of lysine to arginine generally are thought to mimic the (Table S1), indicating that the lethality between sas2Δ and rpd3Δ deacetylated state of the lysine residue and therefore would be is specific to these two enzymes. expected to improve SIR binding and silencing. Contrary to this assumption, H4 K16R mutations cause a strong loss of telo- The Lethality Between sas2Δ and rpd3Δ Is Caused by Increased SIR meric silencing and defects in HM silencing (Fig. S2)(26,30), Spreading. SAS-I exerts a boundary function at telomeres in that it showing that the K16R mutation is not equivalent to a deace- acetylates H4 K16 in subtelomeric regions, preventing the heter- tylated lysine. We propose that H4 K16R suppresses the sas2Δ ochromatin-like SIR complex from spreading toward more cen- rpd3Δ lethality through its derepressing effect on telomeric tromere-proximal regions (11, 12). We hypothesized that Rpd3 silencing. also might exert a boundary function similar to that of the SAS-I To assess boundary function of Rpd3, we measured SIR levels at complex. The combined effect of SIR spreading by rpd3Δ and the telomeres by performing ChIP. The absence of Rpd3 leads to sas2Δ thus might cause increased SIR spreading to a degree that is more Sir2 and Sir3 bound to telomeres and the presence of more lethal to the cells because of the repression of one or several Sir2 and Sir3 in -proximal regions of the right arm of essential subtelomeric genes. If this supposition is true, then re- chromosome VI (Fig. 3A). The effect of rpd3Δ is distinct from that lieving telomeric silencing by deleting one of the SIR components of sas2Δ, which leads to a shift of Sir2 toward centromere-proximal should abrogate the sas2Δ rpd3Δ lethality. Remarkably, we found sequences with less Sir2 at sequences close to the (11, that the deletion of SIR2, SIR3,orSIR4 completely suppresses the 12), (Fig. 3A). lethality of sas2Δ rpd3Δ, showing that the lethality between sas2Δ In agreement with the observed Sir spreading in rpd3Δ cells, we and rpd3Δ depends on the SIR proteins (Fig. 2A). This suppression observed that rpd3Δ causes a loss of colony sectoring in strains with is the result of the absence of the SIR complex at the telomeres ADE2 inserted at the telomere (Fig. 3B), reflecting increased rather than of changes in cell type or HM derepression that result telomeric silencing (23). Furthermore, subtelomeric genes are from deletion of SIR2, SIR3,orSIR4.Specifically, the deletion of more repressed in the absence of Rpd3, and the repression

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.0909169107 Ehrentraut et al. Downloaded by guest on October 1, 2021 A Sir2 Sir3 B Tel VII-L::ADE2 40 wt wt 4 sas2Δ rpd3Δ 30 rpd3Δ 3 20 2 10

Sir2 enrichment Sir3 enrichment 1

0 0 wt rpd3Δ 0.5 1 2.5 5 7.5 15 0.5 1 2.5 5 7.5 15

COS8 YCR106W YDR541C IRC7 SOR1 HXK1 C 300 75 75 600 15 80

200 50 50 400 10 60 gene expression 40 100 25 25 200 5 20 wt sas2Δ expression relative to SPS2 0 0 0 0 0 0 rpd3Δ rpd3Δ sir2Δ 5 2.5 4 2 2 sas2Δ rpd3Δ sir2Δ 3 4 2 3 1.5 1.5 3 1.5 2 2 1 1 2 1 Sir2 Sir2 enrichment 1 1 0.5 association 1 0.5 0.5

0 0 0 0 0 0 1 kb to 3 kb to 5 kb to 5 kb to 8 kb to 15 kb to Tel VIII-L Tel III-R Te l I V- R Te l V I - R Te l X - R Te l V I - R D H4K5Ac/H4 H4K16Ac/H4 H4K12Ac/H4

20 10 1 wt rpd3Δ 10 5 0.5 GENETICS ratio Ac / H4 ratio 0 0 0 0.5 1 2.5 5 7.5 15 0.5 1 2.5 5 7.5 15 0.5 1 2.5 5 7.5 15 E H4K5Ac/H4 H4K16Ac/H4 H4K12Ac/H4

20 10 1 wt sir2Δ

10 5 0.5 rpd3Δ sir2Δ ratio Ac / H4 ratio 0 0 0 0.5 1 2.5 5 7.5 15 0.5 1 2.5 5 7.5 15 0.5 1 2.5 5 7.5 15

YFR056C YFR054C HXK1 RET2 F Chromosome VI YFR057W IRC7 RPN12 right telomere 0.5 1 2.5 5 7.5 15

0 4 8 12 16 20 kb

Fig. 3. Deletion of RPD3 causes mislocalization of Sir2 and Sir3, gene silencing, and changes in histone acetylation in subtelomeric regions. (A) Sir2 and Sir3 binding at the right telomere of chromosome VI is shown as enrichment in ChIP experiments relative to their enrichment at the control gene SPS2. The amount of enrichment is given as a function of the distance to the telomere end in kb in strains with the indicated genotype. ChIP was performed with antibodies against myc-Sir2 and HA-Sir3. Error bars give SD (SI Materials and Methods). (B) Telomeric ADE2 is repressed in rpd3Δ strains. WT and rpd3Δ strains carrying telomeric ADE2 were grown for 2 days at 30°C. (C) Subtelomeric genes are repressed in rpd3Δ cells in a SIR-dependent fashion that correlates with Sir2 association. The upper row of diagrams shows the amount of cDNA of selected subtelomeric genes in the indicated strains as X-fold expression level relative to SPS2. Error bars give standard deviations of at least three PCR analyses from at least two independent reverse transcriptase reactions. The lower row shows ChIP analysis of Sir2 at the respective genes and is represented as in (A). (D) rpd3Δ causes changes in subtelomeric histone acetylation levels. Acetylation was measured by ChIP using antibodies against the indicated residues. Values indicate enrichment of a modification relative to enrichment of H4 (SI Materials and Methods). (E) Sir2-independent changes in subtelomeric histone acetylation by rpd3Δ. Experiments were performed as described in (D). (F) Schematic representation of the telomere VI-R with ORFs and fragments amplified in the ChIP experiments. Genes drawn above the upper line are transcribed from left to right; those below that line are transcribed in the opposite direction.

correlates with increased levels of Sir2 at these genes (Fig. 3C). We furthermore determined how the loss of Rpd3 affected his- The repression is abrogated by the additional deletion of SIR2. tone acetylation at the telomeres. Acetylation of lysine 16 of H4 is Expression analysis of the ORFs on TEL VI-R shows that IRC7 is higher in the absence of Rpd3, and H4 K5 acetylation is increased strongly repressed in rpd3Δ and sas2Δ cells and that the repression at some, but not all, sites tested (Fig. 3D). This finding is consistent is relieved by additional deletion of SIR2 (Fig. 3C). HXK1 and with the notion that the loss of a deacetylase causes an increase in RPN12 show no differences in expression of rpd3Δ or sas2Δ, acetylation and shows that Rpd3 directly affects histone acetylation probably because they are more than 15 kb from the telomere and levels in the vicinity of the telomeres. In contrast, acetylation of H4 therefore outside the repressed domain (Fig. 3C). Importantly, K12 is decreased at some, but not all, sites in rpd3Δ cells. This although more Sir2 and Sir3 is bound at the telomeres, this finding is counterintuitive, because the absence of a deacetylase is increase is not the result of increased Sir2 or Sir3 expression in expected to increase acetylation. One explanation is that the Sir2 rpd3Δ cells, and Sir4 expression is also unaltered (Fig. S3). In deacetylase spreads into these regions in rpd3Δ cells and causes summary, our data show that Rpd3 is required to restrict Sir2 and histone deacetylation. In agreement with this notion, the deletion of Sir3 levels and for localization to sequences closest to the telomere RPD3 causes increased histone acetylation levels at the telomeres and that Rpd3 prevents deleterious gene repression by mis- in sir2Δ cells (Fig. 3E). In summary, these results show that Rpd3 localized SIR complexes in subtelomeric regions. performs global histone deacetylation in subtelomeric regions.

Ehrentraut et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 Targeted Rpd3 Establishes a Boundary at Telomeres and the HM Loci. fi A URA3 We next determined whether Rpd3 is suf cient to create a UASGAL boundary when targeted to a normally silenced gene. When a YM +Ura YM -Ura 5-FOA GBD - UAS Gal4 binding site is present between the telomere and the GBD-Rpd3 reporter (3), the expression of a GBD-Rpd3 fusion disrupts URA3 GBD UAS silencing, whereas the expression of GBD alone causes URA3 to GBD-Rpd3 + be silenced by telomeric heterochromatin (Fig. 4A). The boun- GBD-Hos2 dary function of GBD-Rpd3 depends on the catalytic activity of rpd3Δ GBD-Rpd3 Rpd3. Notably, GBD-Rpd3–dependent boundary formation B YM +Ura YM -Ura 5-FOA requires native Rpd3, because rpd3Δ abrogates the boundary vector function of GBD-Rpd3, and GBD-Rpd3 is unable to complement rpd3Δ in other silencing assays. Significantly, WT, but not cata- RPD3 + UAS RPD3 lytically inactive, (22) can restore boundary function of rpd3 GBD-Rpd3 (Fig. 4B). Furthermore, boundary activity also is (HDAC - ) observed for tethering of another Rpd3(L) component, Dep1, to the telomere (Fig. S4), suggesting that recruitment of the cata- ADE2 URA3 C E UASGAL UASGAL I lytically active Rpd3(L) complex creates a telomere boundary. GBD We next asked whether the ability of tethered Rpd3 to disrupt GBD-Rpd3 HML fi GBD-Rpd3 heterochromatin is speci c to the telomeres or whether Rpd3 GBD also is capable of stopping SIR spreading at the HM loci. To GBD HMR GBD-Rpd3 investigate this question, we used established boundary assays YM +Ura, +Ade YM -Ade 5-FOA 5-FOA, -Ade with the reporter genes ADE2 and URA3 inserted at HML (31) HMR C D HML D URA3 and (32) (Fig. 4 and ). At , GBD-Rpd3 causes UASGAL YM +Ura YM -Ura 5-FOA derepression of both reporter genes but no detectable insulation GBD of ADE2 (Fig. 4C). Furthermore, tethered Rpd3 is capable of GBD-Rpd3 insulating ADE2 from SIR-mediated silencing at the HMR locus GBD-Hst2 (Fig. 4C), because it causes ADE2 derepression while main- GBD-Hos1 URA3 GBD-Hos2 + UAS taining repression. This effect of tethered Rpd3 is the GBD-Hos3 same as that of Sas2 at the two loci (32) and shows that Rpd3 is GBD-Hda1 not only a “desilencer” but can be classified as a “true barrier” GBD-Hst1 GBD-Hst3 factor. These results show that targeting of the HDAC Rpd3 GBD-Hst4 disrupts heterochromatin spreading. This finding is surprising, because thus far only HATs and chromatin remodeling com- Fig. 4. Tethered Rpd3 and Hos2 create a boundary against heterochromatin plexes, but not HDACs, are known to create boundaries (3–5). spreading. (A) Cells with URA3 inserted at TEL VII-L and with (+UAS) or without a Gal4 binding site at the telomere-proximal side (−UAS) were transformed Removal of Sir2 Substrate as a Mechanism for Boundary Formation. A with plasmids carrying the RPD3 or HOS2 genes fused to the GAL4 DNA priori, our observation of a boundary function for Rpd3 is binding domain (GBD-HDAC) or with the vector control (GBD). Repression of URA3 counterintuitive, because chromatin deacetylation generally is was tested by growth on plates lacking uracil and on 5-FOA plates. Serial dilutions of cells were grown for 2 days at 30°C. (B) The targeted boundary viewed as necessary for, rather than prohibitive to, SIR spreading function of Rpd3 depends on its catalytic activity. Cells with a Gal4 UAS in telomeric regions. One possibility is that deacetylation by between telomeric heterochromatin and a subtelomeric URA3 reporter (as in Rpd3 is a prerequisite for the onset of another modification of A) were disrupted for endogenous RPD3 and transformed with RPD3 or a − residues deacetylated by Rpd3; alternatively, the boundary catalytically dead rpd3 allele (rpd3-H150:151A, referred to as “rpd3-HDAC ”). function of Rpd3 may influence chromatin remodeling, exchange (C) Tethered Rpd3 disrupts silencing at HML and has insulating activity at of histone variants, or the presence of linker histones. However, HMR. Cells with ADE2 and URA3 inserted at HML or HMR were provided with the analysis of sas2Δ genetic interactions (Table S1) did not GBD or with GBD-Rpd3. (D) Other HDACs did not display boundary function. support these scenarios. Furthermore, the prevention of SIR GBD-HDAC fusions were assayed for boundary function as in A. spreading is not mediated by Bdf1 or Bdf2 (Table S1). We next hypothesized that chromatin deacetylation per se by produces OAADPR in the deacetylation reaction (13), which binds Rpd3 is the cause for the establishment of a chromatin boundary to the SIR complex and has been proposed to be one of the driving by removing the acetyl-lysine substrate for the HDAC Sir2. In this model, the process of deacetylation by Sir2 helps in the prop- forces in the polymerization of SIR complexes on chromatin (14). agation of SIR complexes along the chromatin fiber. Con- In this scenario, removal of Sir2 substrates renders Sir2 unable to produce OAADPR, thus reducing SIR propagation along the sequently, this process is hindered by prior removal of acetyl-lysine fi residues by Rpd3. chromatin ber and stopping heterochromatin spreading. This This model predicts that targeting other HDACs to the telo- model predicts that a mutation in the OAADPR binding site within meres also should create a barrier to heterochromatin, depending the SIR complex should abrogate SIR spreading and silencing. fi Sir3 contains a domain similar to the nucleotide (ATP) binding on their substrate speci city. We found that tethering the HDAC + Hos2 (33) forms a boundary to telomeric silencing (Fig. 4A), domain of AAA ATPases (16), making it a likely candidate region showing that, in principle, histone deacetylation by other HDACs for an OAADPR binding pocket. Our modeling of Sir3 on the + also can cause boundary formation. Other HDACs, however, do structure of AAA ATPases suggests that Sir3 contains an addi- not display this activity (Fig. 4D), whereas targeting of Hst2 or Sir2 tional cavity as compared with other ATPases that may accom- aids in heterochromatin formation (Fig. 4D and Fig. S4). We modate the O-acetyl-ribose moiety of OAADPR (Fig. 5A). propose that these HDACs are unable to create a boundary We tested whether a mutation in this region of Sir3 could because their different substrate specificities are incompatible abrogate its ability to support silencing. Importantly, alleles of with boundary function, although it also is possible that some of SIR3 that deleted amino acids 575–577 or 578–585 in the Sir3 the HDACs lose activity by fusion to the Gal4 GBD. protein or that mutated residues 575–577 to alanine and therefore How does the process of deacetylation by Sir2 contribute to affected the putative OAADPR binding site are unable to restore SIR propagation? One possibility is suggested by the fact that Sir2 the lethality in sas2Δ rpd3Δ sir3Δ cells (Fig. 5B), and they are

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.0909169107 Ehrentraut et al. Downloaded by guest on October 1, 2021 sas2Δ rpd3Δ sir3Δ + pURA3-SAS2 A AAA+ ATPase- B like domain + vector

Fig. 5. The putative OAADPR binding domain of Sir3 + SIR3 + sir3- Δ is necessary for its function in silencing. (A) Model of the 578- 585 AAA+ ATPase-like domain of Sir3. For a computational modeling of Sir3, the primary sequence of Sir3 AAA+ + SIR3 + Δ578-585 + vector domain and structures of other proteins of the AAA Δ575-577 + sir3 + sir3 family were subjected to a sequence-structure align- Mg2+ OAADPR - - SI Materials and Methods AAA Δ575 ment, using Modeler 9v2 (see 19Sir3 532 834 78 - 577 for details). The model was visualized using PyMOL. The YM 5-FOA position of the putative OAADPR binding domain is sir3Δ HML silencing marked by OAADPR. The mutations of Sir3 used in this C D URA3 study are indicated in red and blue. (B) Mutation of the + vector + SIR3 YM +Ura YM -Ura 5-FOA sir3Δ AAA+ domain of Sir3 abrogates its ability to spread at + sir3 + sir3- rpd3Δ sas2Δ sir3Δ URA3-SAS2 + vector - telomeres. p strains car- AAA Δ575 rying the indicated sir3 alleles were tested for their + SIR3 - 577 SAS2 sir3 growth mating ability to lose the plasmidon5-FOA. -AAA + sir3- designates mutation of residues 575–577 to alanine. (C Δ575- 577 Sir3-ChIP and D) sir3-Δ575–577 and sir3-AAA were deficient in + sir3- AAA F 0.08 Sir3 Sir3- Sir3- + 0.07 telomeric and HML silencing. (E) Mutation of the AAA Δ578-585 Δ575-577 Δ – “ ” 0.06 domain of Sir3 (Sir3- 578 585, designated Sir3* ) AD-Sir3 BD E 0.05 reduces its ability to interact with Sir4 and Sir3. Fusions AD-Sir3* BD

of the indicated proteins to the Gal4 DNA-binding (BD) AD-Sir3 BD-Sir3 / Input IP 0.04 and activation domain (AD) were tested for activation AD-Sir3* BD-Sir3 0.03 of the HIS3 reporter gene on medium lacking histidine. AD-Sir3 BD-Sir4 0.02 (F)Sir3Δ578–585 and Sir3Δ575–577 show reduced AD-Sir3* BD-Sir4 0.01 YM +His YM -His 0 binding to the telomere. ChIP analysis was performed 0.5 1 2.5 5 7.5 15 with Sir3-HA. Tel VIR (kb) GENETICS

unable to support telomeric and HM silencing (Fig. 5 C and D), broad histone substrate range: Rpd3 deacetylates all acetylation indicating that they have lost functionality although they are sites in the H3 and H4 N-termini as well as H2B K11, K16, and expressed at levels comparable to endogenous Sir3 (Fig. S3C). H2A K7, and Hos2 is required for the preferential deacetylation of Furthermore, sir3-Δ578–585 causes a reduction, but not complete all sites in H3 and H4 (33, 35). Although currently we cannot dis- loss, of interaction with Sir3 itself and a loss of Sir3 interaction with tinguish which residues are important for boundary function, the Sir4 (Fig. 5E), suggesting that the binding of OAADPR to Sir3 is broad substrate specificity suggests that the more histone residues important for SIR complex integrity. ChIP analysis of WT and the are deacetylated by an HDAC, the more likely the HDAC is to two mutant Sir3 proteins shows that the mutants display reduced possess boundary activity. This notion is in line with the observation binding to telomeric sequences (Fig. 5F). This finding suggests that that mutation of H4 K5 partially abrogates increased silencing by the deletion or mutation of the OAADPR binding site causes rpd3Δ (34). However, boundary activity must be independent of reduced Sir3 function in heterochromatin spreading. In summary, H4 K16, because the acetylation, rather than the deacetylation, of these results show that the AAA+ domain of Sir3 is important for H4 K16 by SAS-I creates a boundary (11, 12). This finding sug- its silencing function, suggesting that OAADPR binding to Sir3 is gests that a functional difference exists between acetylation of critical for heterochromatin formation. H4 K16 and other lysine residues. We propose that K16 is acety- lated by SAS-I before or concomitant with chromatin deposition Discussion and that it becomes inaccessible to Sir2 deacetylation once Barriers between active and inactive chromatin in a variety of deposited, so that it cannot be used by Sir2 for SIR propagation organisms have been associated with chromatin-activating along the chromatin fiber. Perhaps acetylated H4 K16 is inacces- mechanisms as well as with the attachment at nuclear pore sible because it alters the chromatin configuration (36), which is not structures (1, 31). Here, we describe the unexpected finding that changed by other modifications. Alternatively, recruitment of an enzymatic activity generally associated with repression, the H2A.Z by H4 K16 acetylation may alter its accessibility (17). HDAC Rpd3, is necessary to prevent spreading of hetero- By which mechanism does histone deacetylation halt hetero- chromatic SIR proteins into euchromatin at yeast telomeres. In chromatin spreading? SIR binding to chromatin requires principle, one would expect an HDAC to aid in, rather than to deacetylated histone tails (10). Thus, the process of deacetyla- prevent, the formation of heterochromatin, although Rpd3 has tion by Sir2 itself may help in the process of productive SIR been implicated in gene activation previously (22, 33). We pro- propagation along the chromatin fiber. The deacetylation of pose that the boundary function for Rpd3 described here reflects lysine residues on a subsequently may stabilize SIR a global, untargeted (versus a targeted) role for the Rpd3(L) binding to that nucleosome. Consequently, this process is hin- complex in establishing histone acetylation patterns at telomeres dered if acetyl groups have been removed previously by Rpd3 through a transient interaction with chromatin. Our data are thus and thus are no longer available for Sir2 deacetylation. Notably, in synchrony with previous work showing that Rpd3 antagonizes there may be a difference between mere SIR spreading and SIR propagation at telomeres (34). However, the mechanism of concomitant gene silencing, because two studies showed SIR this boundary function thus far has remained unclear. spreading on mutant histones, but this SIR spreading did not How can a histone deacetylase function as a boundary element? provide stable silencing (37, 38). Our data suggest that histone deacetylation as such halts hetero- As an extension of our model, one attractive possibility is sug- chromatin spreading, because two known HDACs display boun- gested by the fact that, in the deacetylation reaction, Sir2 produces dary activity. However, one still can speculate that other OAADPR (13), which binds to the SIR complex and has been nonhistone targets also contribute to the boundary activity (34) or proposed to be one of the driving forces in the polymerization of that Rpd3 deacetylation supports the binding of another factor that SIR complexes on chromatin (14). In this scenario, removal of the hinders SIR spreading. Rpd3 and Hos2 both have a relatively Sir2 substrate renders Sir2 unable to produce OAADPR, thus

Ehrentraut et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 reducing the efficiency of SIR propagation and functional chromatin to telomeric regions. This boundary activity functions silencing along the chromatin fiber and stopping heterochromatin by a mechanism in which the spreading of SIR complexes along spreading and silencing (Fig. S5). This model also suggests that the chromatin fiber is halted by prior removal of acetyl-lysine + OAADPR is not supplied in trans by the other cellular NAD - groups on histones by Rpd3. We propose that the deacetylation dependent HDACs, Hst1–Hst4, in the absence of OAADPR reaction of Sir2 per se, the generation of OAADPR by Sir2, and production by Sir2. This possibility is quite conceivable, because the binding of the metabolite to Sir3 are essential for SIR OAADPR most likely is unstable in the cellular environment and spreading and are abrogated by the competing histone deacety- because several nucleotide-cleaving enzymes exist in the cell. lation activity of Rpd3 in subtelomeric regions (Fig. S5). We observed that mutations in the putative OAADPR binding site of Sir3 abrogate its silencing function and reduce its ability to Materials and Methods interact with itself and Sir4 and spread on chromatin, thus pro- Yeast Strains and Plasmids. Yeast strains and plasmids used in this study are viding experimental support for the notion that binding of listed in Tables S2 and S3, respectively. Yeast was grown and manipulated OAADPR to Sir3 carries out an important function in hetero- according to standard protocols (SI Materials and Methods). chromatin formation. This notion is in agreement with a recent study that showed increased in vitro binding of the SIR complex Chromatin Immunoprecipitation. ChIP was performed essentially as described, to chromatin in the presence of OAADPR (39). Perhaps the with modifications and analysis performed as described in SI Materials metabolite is necessary for SIR–SIR complex interactions, which and Methods. are important for propagation of heterochromatin along the chromatin fiber. However, it also is possible that these Sir3 Expression Analysis. The expression of subtelomeric genes was determined by mutations disrupt other aspects of Sir3 function. reverse transcription followed by quantitative real-time PCR. Total RNA from Interestingly, a recent study reported apparently OAADPR- 0.5 OD units of yeast cells was reverse transcribed using SuperScript III reverse independent silencing by a fusion protein between the NAD+- transcriptase (Invitrogen) according to the manufacturer’s protocol. independent HDAC Hos3 and Sir3 (40). One explanation to rec- ACKNOWLEDGMENTS. We thank D. Gottschling, L. Guarente, R. Kamakaka, oncile this observation with our data is that OAADPR is required M. Keogh, R. Morse, L. Pillus, F. Posas, J. Rine, and D. Rivier for reagents; for the interaction and recruitment of Sir2/Sir4 to Sir3. Thus, the J. Franke for help with strain constructions; U. Marchfelder, S. Gerber, direct Sir3-Hos3 fusion in the previous study might obviate the M. Rübeling, O. Valdau, and M. Müller for technical assistance; the biology necessity for OAADPR in the recruitment of the HDAC Sir2. students of the Justus-Liebig-University Giessen for help with the synthetic ′ lethal screen; J. Franke and F. Seifert for comments on the manuscript; and In summary, in this work we have expanded the view of Rpd3 s all members of the laboratory for discussions. This work was supported by the function in the establishment of global histone acetylation pat- Max-Planck-Society, the Justus-Liebig-University Giessen, the University of terns, in that we found the Rpd3(L) complex to restrict hetero- Duisburg-Essen, and the Deutsche Forschungsgemeinschaft.

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