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Rpd3-Dependent Boundary Formation at Telomeres by Removal of Sir2 Substrate

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Rpd3-Dependent Boundary Formation at Telomeres by Removal of Sir2 Substrate Rpd3-dependent boundary formation at telomeres 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 histone deacetylation in to gene promoters and establishes gene repression via promoter this process. Significantly, the histone deacetylase (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 heterochromatin–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 histones 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.
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