Sds3 (Suppressor of Defective Silencing 3) Is an Integral Component of the Yeast Sin3·Rpd3 Histone Deacetylase Complex and Is
Required for Histone Deacetylase Activity
LECHNER, Thomas, et al .
SDS3 (suppressor of defective silencing 3) was originally identified in a screen for mutations that cause increased silencing of a crippled HMR silencer in arap1 mutant background. In addition, sds3mutants have phenotypes very similar to those seen in sin3and rpd3 mutants, suggesting that it functions in the same genetic pathway. In this manuscript we demonstrate that Sds3p is an integral subunit of a previously identified high molecular weight Rpd3p·Sin3p containing yeast histone deacetylase complex. By analyzing an sds3Δ strain we show that, in the absence of Sds3p, Sin3p can be chromatographically separated from Rpd3p, indicating that Sds3p promotes the integrity of the complex. Moreover, the remaining Rpd3p complex in the sds3Δ strain had little or no histone deacetylase activity. Thus, Sds3p plays important roles in the integrity and catalytic activity of the Rpd3p·Sin3p complex.
LECHNER, Thomas, et al . Sds3 (Suppressor of Defective Silencing 3) Is an Integral
Component of the Yeast Sin3·Rpd3 Histone Deacetylase Complex and Is Required for Histone Deacetylase Activity. Journal of Biological Chemistry , 2000, vol. 275, no. 52, p. 40961-40966
DOI : 10.1074/jbc.M005730200
Disclaimer: layout of this document may differ from the published version.
1 / 1
Sds3 (Suppressor of Defective Silencing 3) Is an Integral Component of the Yeast Sin3 䡠 Rpd3 Histone Deacetylase Complex and Is
Required for Histone Deacetylase Activity*
Received for publication, June 29, 2000, and in revised form, October 2, 2000 Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M005730200
Thomas Lechner,a,bMichael J. Carrozza,a,cYaxin Yu,dPatrick A. Grant,a,eAnton Eberharter,a David Vannier,fGerald Brosch,g,hDavid J. Stillman,dDavid Shore,iand Jerry L. Workmana,j From theaHoward Hughes Medical Institute, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802-4500, thedDivision of Molecular Biology and Genetics, Department of Oncological Sciences, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, thefDepartment of Microbiology, Columbia University, New York, New York 10032, thegDepartment of Microbiology, University of Innsbruck-Medical School, Innsbruck A-6020, Austria, and theiDepartment of Molecular Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland
SDS3(suppressor of defective silencing 3) was origi- nally identified in a screen for mutations that cause increased silencing of a crippledHMRsilencer in arap1 mutant background. In addition,sds3mutants have phe- notypes very similar to those seen insin3andrpd3mu- tants, suggesting that it functions in the same genetic pathway. In this manuscript we demonstrate that Sds3p is an integral subunit of a previously identified high molecular weight Rpd3p䡠Sin3p containing yeast histone deacetylase complex. By analyzing an sds3⌬strain we show that, in the absence of Sds3p, Sin3p can be chro- matographically separated from Rpd3p, indicating that Sds3p promotes the integrity of the complex. Moreover, the remaining Rpd3p complex in thesds3⌬strain had little or no histone deacetylase activity. Thus, Sds3p plays important roles in the integrity and catalytic ac- tivity of the Rpd3p䡠Sin3p complex.
Numerous studies in the past have linked acetylation of core histones to transcriptional regulation (1). The identification of co-activator proteins as histone acetyltransferases (HATs)1has strengthened the connection between histone acetylation and transcription (2–5). In yeast several distinct HAT complexes have been identified that modify nucleosomal histones (5–9).
Acetylation of nucleosomal histones by these HAT complexes stimulates transcription from preassembled chromatin tem- plates (10, 11), and these complexes are targeted by direct
interactions with transcriptional activators (12, 13).
To counteract the effect of HAT complexes, it is necessary to reverse acetylation by efficiently deacetylating nucleosomal histones. Histone deacetylase complexes (HDACs) perform this reaction. HDACs have been isolated and characterized in sev- eral organisms as multiprotein complexes that are associated with DNA binding repressors and co-repressors (14 –22). The majority of these complexes contain members of the Rpd3䡠 HDAC-related protein family as catalytic subunits (23). In addi- tion to these Rpd3p-related HDAC complexes, two non-Rpd3p- related deacetylase complexes have been identified inZea mays (24, 25). Moreover, yeast HDAC complexes containing Hda1p and Hos3p as catalytic subunits have been identified (26, 27).
Two yeast multiprotein HDAC complexes have been found to contain Rpd3p (26, 28). The larger of these complexes was also found to contain the Sin3p co-repressor (28). Sin3p is thought to mediate interactions of the Sin3䡠Rpd3 complex with se- quence-specific DNA binding repressors such as Ume6p, which leads to a localized deacetylation of histones H3 and H4 and repression of transcription in vivo(19, 29).RPD3 and SIN3 were both originally identified in genetic screens as regulators of gene expression (30, 31). Mutations inRPD3andSIN3affect transcription of the same set of genes (32). SIN3 andRPD3 were identified among at least 19 other genes in a screen for extragenic suppressors of a silencing defectiveRAP1allele (rap 1-12) (33, 34). Another gene identified in this screen isSDS3 (suppressor of defective silencing 3). Although mutations in SDS3were shown to cause several phenotypes in common with sin3 and rpd3 mutants, they did not appear to derepress a plasmid-borneTRK2 gene, raising the possibility thatSDS3 might function independently of SIN3 and RPD3(33). How- ever, a recent study found that a mutation in SDS3reduced Sin3p-mediated repression and that Sds3p and Sin3p could be co-immunoprecipitated from cell extracts (35). These results illustrated thatSDS3functions in the same genetic pathway as SIN3and that Sds3p and Sin3p can interact in some way.
In this manuscript we demonstrate that Sds3p is an integral subunit of a Rpd3p䡠Sin3p-containing yeast HDAC complex with an apparent molecular mass of 1.2 MDa. By analyzing a sds3⌬strain we show that, in the absence of Sds3p, Sin3p can be chromatographically separated from Rpd3p, indicating that Sds3p promotes the integrity of this complex. In addition, the remaining Rpd3p complex in thesds3⌬strain had little or no histone deacetylase activity. Thus, Sds3p plays important roles in the integrity and catalytic activity of the Rpd3p䡠Sin3p complex.
* This work was supported in part by a grant from the National Institute of General Medical Sciences (to J. L. W.). The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
bSupported by a fellowship from the Austrian Science Foundation (FWF).
cAn Howard Hughes Medical Institute Postdoctoral Associate.
eSupported by Postdoctoral Fellowship PF-98-017-01-GMC from the American Cancer Society and by Burroughs Wellcome.
hSupported by an grant from the Austrian Academy of Science (APART fellowship).
jAn Associate Investigator of Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Dept. of Biochemistry and Molecular Biology, The Pennsyl- vania State University, University Park, PA 16802-4500. Tel.: 814-863- 8256; Fax: 814-863-0099; E-mail: firstname.lastname@example.org.
1The abbreviations used are: HATs, histone acetyltransferases;
HDAC, histone deacetylase complex; HA, hemagglutinin; bp, base pair(s); PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol;
PAGE, polyacrylamide gel electrophoresis.
This paper is available on line at http://www.jbc.org
Yeast Strains—Thesds3⌬strain in the W303 background (36) was generated as described previously (33). The Sds3-HAp construct was made by inserting three copies of the HA epitope in the 3⬘-end of the SDS3open reading frame. A uniqueNotI site was engineered into⫹978 of the 984-bpSDS3open reading frame. ANotI cassette containing three copies of the HA epitope (YPYDPDYA) was inserted and cloned into the 2-m plasmid pRS423. The resulting clone, DV-246, comple- ments thesds3⌬mutation in a manner identical to the untaggedSDS3 gene.
Preparation of Whole Cell Extracts and Purification of the Rpd3p Complex—Strains were grown to an optical density of 1.5 as described previously (37), and cells of a 12-liter culture were harvested by cen- trifugation at 3000 ⫻ g for 10 min. The cell pellets were washed, resuspended, and lysed by using a glass bead-beater (Biospec), and the resulting extracts were loaded onto Ni2⫹-agarose as described (37).
After Ni2⫹-agarose chromatography, the imidazole eluate was loaded directly onto a Mono-Q HR5/5 column (Amersham Pharmacia Biotech) to separate HDAC complexes. Bound proteins were eluted with a linear 25-ml gradient of 100 to 500 mMNaCl in buffer B (50 mMTris-HCl, pH 8.0, 10% glycerol, 0.1% Tween 20, 2g/ml leupeptin, 2g/ml pepstatin A, 1 mMPMSF, 0.5 mMDTT). Fractions of 0.5 ml were collected and assayed for HDAC activity as described previously (38). In general, 10
l of each fraction was incubated with 3g of tritium-labeled chicken reticulocyte core histones (38). Samples were incubated for 30 min at 30 °C, and the released radioactivity was measured as described (38).
Fractions containing HDAC activity were pooled separately, concen- trated to 500l using Centriprep 10 (Millipore) concentrators, and then loaded onto a Superose 6 HR10/30 size exclusion column (Amersham Pharmacia Biotech) to determine the native molecular weight. The column was run in 350 mMNaCl in 40 mMHepes, pH 7.8, 10% glycerol, 0.1% Tween 20, 2g/ml leupeptin, 2g/ml pepstatin A, 1 mMPMSF, 0.5 mMDTT at a flow rate of 0.2 ml/min. Fractions of 0.5-ml volume were collected and assayed for HDAC activity. Aliquots of fractions contain- ing HDAC activity were applied to SDS-PAGE and subject to Western blotting as described (39). In addition, 50l of Mono-Q concentrate was applied to a Superose 6 PC 3.2/30 size exclusion column (Amersham Pharmacia Biotech). The column was run in 500 mMNaCl in 40 mM
Hepes, pH 7.8, 10% glycerol, 0.1% Tween 20, 2g/ml leupeptin, 2g/ml pepstatin A, 1 mMPMSF, 0.5 mMDTT at a flow rate of 0.02 ml/min.
50-l fractions were collected and tested for HDAC activity, and West- ern blot analysis was performed as described. The flow-through fraction of Ni2⫹-agarose chromatography was dialyzed three times against 10 volumes of buffer B (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 10%
glycerol, 0.1% Tween 20, 2g/ml leupeptin, 2g/ml pepstatin A, 1 mM
PMSF, 0.5 mM DTT) and processed over a Mono-Q column and a Superose 6 size exclusion column. Western blot analyses described in this study were performed using antibodies against Rpd3p (Upstate Biotechnology), Hda1p (Santa Cruz Biotechnologies), HA (Covance), and Sin3p (28).
Immunoprecipitation, Modified HDAC Assay for Immunoprecipita- tions—Antibodies for HA (Covance) and Rpd3p (Upstate Biotechnology) were coupled to protein A-Sepharose beads. 40l of 50% bead slurry was washed twice with 200l of binding buffer (150 mMNaCl, 40 mM
Hepes, pH 7.5, 10% glycerol, 0.1% Tween 20, 2g/ml pepstatin A, 2
g/ml leupeptin, 0.2 mMDTT, and 1 mMPMSF). Beads were recovered by centrifugation at 110⫻gfor 1 min in a table-top centrifuge after each wash step. 20l of antibody solution (20g) was added to the beads, and the antibodies were bound to the beads by rotation on a wheel for 1 h at room temperature. Antibodies were cross-linked to beads as described previously (39). Superose 6 chromatography peak fractions were diluted to the appropriate salt concentration of 150 mM
NaCl with binding buffer, reconcentrated with Microcon 10 concentra- tors (Amicon) to the original volume, and 20l of sample was added to 20l of antibody beads. The binding reaction was performed for 4 h or overnight with a rotation wheel at 4 °C. After the binding reaction, beads were recollected by centrifugation, the supernatant was removed and saved, and beads were washed twice with binding buffer. HDAC assays were performed with beads and supernatant. 20l of 1g/l radiolabeled chicken erythrocyte core histones were added to samples and were incubated for 3 h at 30 °C with rotation. HDAC activity was measured as described previously (38).
Whole cell extract for immunoprecipitation was prepared from 100 ml of the Sds3-HAp expression strain by glass bead disruption into extraction buffer (40 mMHEPES, pH 7.5, 350 mMNaCl, 10% glycerol, 0.1% Tween 20, 1 mMPMSF, 1 mMDTT, 2g/ml pepstatin A, 2g/ml leupeptin). For immunoprecipitation, 200g of whole cell extract total
protein, as determined by Bradford assay, was diluted to 150 mMNaCl in extraction buffer lacking NaCl. The diluted extract was mixed for several hours at 4 °C in the presence or absence of 4g of antibody directed against Rpd3 or 1l of antibody directed against Sin3. Im- mune complexes were collected by mixing antibody- or mock-treated extracts with protein A-Sepharose for several hours at 4 °C. After several washes in binding buffer (see above), input extract, unbound supernatants, and bound bead material were subjected to Western analysis using antibody against the HA epitope tag.
For Western blot analysis, beads and supernatant were boiled in Laemmli SDS sample buffer for 10 min and applied to 10% SDS-PAGE with subsequent Western blotting as described (39).
Identification of Yeast HDAC Complexes—Several yeast HAT complexes bind to Ni2⫹-agarose, which concentrates these activities from yeast whole cell extract for further purification (37). We tested if HDAC activities might also be found in the Ni2⫹-agarose eluant to determine if this material might also serve as a starting point for purifying HDAC multiprotein complexes. Whole yeast cell extract of a Sds3-HAp strain was prepared and bound to Ni2⫹-agarose. The flow-through and eluates from Ni2⫹-agarose were then subjected to Mono-Q chro- matography followed by Superose 6 size exclusion chromatog- raphy (Fig. 1). We detected three HDAC activities by this procedure. These included an Rpd3-containing complex of ap- proximately 0.6 MDa (data not shown), which was found in the Ni2⫹-agarose flow-through and is presumably related to that described by Rundlett and co-workers (26). We did not observe co-fractionation of Sds3-HAp with this Rpd3 complex (data not shown). Two additional HDAC complexes eluted together from the Ni2⫹-agarose but were separated on the subsequent Mono-Q column (Fig. 2A). Using Western blot analysis we found that Rpd3p, Sin3p, and Sds3-HAp co-fractionated with the HDAC complex eluting at approximately 0.35MNaCl (Fig.
2A). A second HDAC activity eluted at approximately 0.25 M NaCl, and subsequent Western blot analysis indicated that it co-elutes with Hda1p (Fig. 2A).
Co-fractionation of Sds3p, Rpd3p, and Sin3p—To further test if Sds3p is an actual component of the Rpd3p䡠HDAC com- plex, we tested for co-elution of Sds3-HAp with Rpd3p, Sin3p, and HDAC activity by gel filtration chromatography. Mono-Q fractions containing the Rpd3p complex from the Sds3-HA strain (fractions 28 –34) were pooled, concentrated, and applied to a Superose 6 size exclusion column. The HDAC activity eluted at an apparent molecular mass of 1.2 MDa (Fig. 2B).
This was consistent with our earlier findings showing the same FIG. 1.Fractionation scheme for HDAC complexes used in this study.Shown is the fractionation of 0.6- and 1.2-MDa Rpd3p com- plexes, a 0.6-MDa Hda1p complex, and a 0.9-MDa breakdown Rpd3p complex found in ansds3deletion strain.
Sds3p Is an Essential Component of the Sin3 䡠 Rpd3 HDAC Complex 40962
molecular weight for this particular HDAC complex from a wild type strain (data not shown). Aliquots of fractions 16 –24 of Superose 6 size exclusion chromatography were applied to SDS-PAGE. Subsequent Western blotting with antibodies for Rpd3p, Sin3p, and HA (Sds3p) demonstrated co-elution of all three of these proteins with the deacetylase activity peak (Fig.
2B). The high molecular weight of this complex and the fact that it contains Sin3p and Rpd3p suggest that it is related to or identical to the complex described by Kasten and colleagues (28).
Co-immunoprecipitation of Sds3p, Rpd3p, Sin3p, and HDAC Activity—To confirm that Sds3p is a bona fide subunit of the 1.2-MDa Rpd3p complex, we tested for co-immunoprecipitation of these two proteins and HDAC activity from the Superose
fractions. We used fraction 19 of the Superose 6 size exclusion chromatography of the Sds3-HA strain, which corresponds to the HDAC activity peak (Fig. 2B) and performed co-immuno- precipitation experiments. Samples were incubated with HA antibodies coupled to beads, the beads were pelleted, and the HDAC activity of supernatant and beads was tested. Fig. 3A shows that the HDAC activity was clearly immunoprecipitated with the HA antibodies, illustrating that Sds3p is part of the HDAC complex. As a control we incubated samples of a HDAC peak fraction of a Superose 6 size exclusion chromatography of aSDS3⫹(WT) strain with HA antibodies coupled to beads. Fig.
3B (group 1) shows that HDAC activity was not immunopre- cipitated with the HA antibodies from this strain, ruling out any nonspecific interaction of HA antibodies with the Rpd3p complex. Moreover, the HDAC activity of the Rpd3p complex from both the Sds3-HA and wild type strains immunoprecipi- tated with Rpd3p antibodies coupled to beads (Fig. 3B,groups 2 and 3). To further confirm the co-immunoprecipitation of Sds3p and Rpd3p, Western blots of the immunoprecipitations with anti-HA were performed. In Fig. 3C(upper panel), Rpd3p and HA-Sds3p can be detected in the bead fraction using frac- tion 19 of the Superose 6 column of the Sds3p-expressing strain, whereas no signal is detectable in the supernatant.
Lanes 2and3of Fig. 3C(lower panel) show co-immunoprecipi- tation of Rpd3p with antibody against Rpd3 using Superose 6 peak fraction of the WT, untagged strain. By contrast, no Rpd3p protein was immunoprecipitated with antibodies for HA from this strain (Fig. 3C,lower panel,lanes 4and5).
Further confirmation that Sds3 associates with the Rpd3䡠Sin3 complex came from immunoprecipitation experiments per- formed in crude extracts. Antibodies directed against either Rpd3 or Sin3 immunoprecipitated Sds3 from whole cell extract (Fig. 3D) (35). As expected, antibody directed against Rpd3 was also able to immunoprecipitate Sin3 from the same extract (data not shown). These results indicate that, in addition to highly fractionated preparations, Rpd3, Sin3, and Sds3 associ- ate within cell extracts.
Deletion of SDS3 Alters the Chromatographic Behavior of the Rpd3䡠Sin3 Complex—To investigate if the integrity of the 1.2- MDa HDAC complex was dependent on SDS3, we prepared whole cell extracts from asds3⌬strain and applied it to Ni2⫹- agarose followed by Mono-Q chromatography. The HDAC ac- tivity in the fractions normally containing the WT 1.2-MDa Rpd3p complex (28 –32) was decreased (Fig. 4A, HDAC activity ofsds3⌬) compared with that of wild type (compare with Fig.
2A, HDAC activity of the Sds3-HAp-expressing strain). Subse- quent Western blotting of fractions 19 –32 of this Mono-Q col- umn showed a different elution profile of Sin3p and Rpd3p.
Both proteins eluted at a lower salt concentration from the column compared with wild type (0.2– 0.3M NaCl instead of 0.35MNaCl). Rpd3p and Sin3p eluted in the range of Hda1p.
Hda1p peaked with the main HDAC activity at fractions 22–24, which was comparable to the wild type elution pattern for Hda1p.
The HDAC Activity of the Rpd3 Complex Depends on SDS3—To determine if the deletion of SDS3affects not only the elution profile of Sin3p and Rpd3p, but also the integrity and activity of the complex, we pooled fractions 20 –32 of the Sds3⌬ Mono-Q chromatography, concentrated the material, and applied it to Superose 6 size exclusion chromatography.
The HDAC activity was examined as described, and corre- sponding aliquots were analyzed by SDS-PAGE and subse- quent Western blotting. Fig. 4Bshows that the main HDAC activity eluted at approximately 0.6 MDa and corresponds to Hda1p, indicating that the deletion ofSDS3does not affect a 0.6-MDa Hda1p complex. However, the remaining Rpd3 com- FIG. 2.HDAC activity profile of a Sds3-HAp expressing strain.
A, the HDAC elution profile from a Mono-Q column shows two major HDAC activities. Subsequent Western blotting revealed that the com- plex eluting at 0.25MNaCl co-eluted with Hda1p, whereas the complex eluting at 0.35MNaCl co-eluted with Rpd3p, Sin3p, and Sds3-HAp.B, the HDAC activity profile of the 0.35MMono-Q complex when subse- quently run on a Superose 6 gel-filtration column is shown. The HDAC activity, Sin3p, Rpd3p, and Sds3-HAp all co-eluted at a molecular weight of approximately 1.2.
plex had a smaller molecular weight compared with wild type complex (Fig. 2B). Western blot analysis revealed a signal for Rpd3 at fractions 20 and 21, which corresponds to an approx- imate molecular mass of 0.9 MDa. Importantly, this 0.9-MDa Rpd3 breakdown did not have HDAC activity.
Deletion of SDS3 Decreases the Interaction between Sin3p and Rpd3p—The elution of Sin3p from the Superose 6 column (Fig. 4B) overlapped with that of Rpd3p. However, these pro- FIG. 4.HDAC activity profile of thesds3⌬strain.A, whole cell extract of asds3⌬strain was prepared and applied to Ni2⫹-agarose and subsequently to a Mono-Q column. The HDAC elution profile shows one broad HDAC peak, which corresponds to the Hda1p complex found from wt cells (Fig. 2). Western blotting revealed the elution of Hda1p, Sin3p, and Rpd3p from thesds3⌬strain (compare with Fig. 2A).B, the Super- ose 6 elution profiles from thesds3⌬strain. Fractions 20 –32 of Mono-Q of thesds3⌬strain (A) were pooled and applied to Superose 6 size exclusion chromatography at 0.35 M NaCl (B). The HDAC activity eluted at approximately 0.6 MDa with Hda1p. No HDAC activity was eluted with Sin3p or Rpd3p from this strain.
in the bead-bound fraction immunoprecipitated with antibody for HA (lane 5). D, co-immunoprecipitation of Sds3p with Rpd3p and Sin3p from whole cell extracts. Whole cell extract from a Sds3-HAp expressing strain was subjected to immunoprecipitation conditions in the presence or absence of antibody directed against Rpd3 or Sin3. The bead-bound material (B) and one-fifth of the input extract and unbound superna- tants (S) were subjected to Western analysis for Sds3-HAp using anti- body recognizing the HA-epitope tag.
FIG. 3.Co-immunoprecipitation of Sds3p, Rpd3p, Sin3p, and HDAC activity. A, the peak fraction, fraction 19 of the Superose 6 column from the Sds3-HAp-expressing strain (Fig. 2A) was used to perform immunoprecipitation experiments. The HDAC activity of this fraction was found to co-immunoprecipitates with Sds3-HAp.B, immu- noprecipitation of the HDAC activity with antibody for HA is specific for the Sds3-HAp-expressing strain. Superose 6 HDAC peak fraction of the 1.2-MDa complex prepared from anSDS3⫹strain (WT) was incubated with antibody for Hap (group 1). Superose 6 HDAC peak fractions from the Sds3-HA-expressing strain and anSDS3⫹strain (Sds3-HAandWT) were precipitated with antibody for Rpd3p (groups 2and3, respective- ly). Input (I), supernatant (S), and bead-bound fraction (B) were mon- itored for HDAC activity in all three cases. HA antibody does not precipitate HDAC activity of WT strain (group 1), whereas Rpd3p antibody precipitates HDAC activity of Sds3-HA and WT strain (groups 2and3).C, the Western blot analysis of these immunoprecipitation experiments is shown.Top panel, the Superose 6 peak fraction from the Sds3-HA strain (Input, see Fig. 2B) was incubated with HA beads.
Input (I), supernatant (S), and bead-bound fraction (B) were separated on SDS-PAGE followed by Western blotting. Blots were probed with antibodies to HAp and Rpd3p. Bottom panel, the Superose 6 peak fraction from theSDS3⫹strain (Input) was incubated with␣Rpd3 beads and␣HA beads and further processed as described above. Rpd3 in the Superose 6 peak fraction from the WT strain immunoprecipitates with antibody to Rpd3p (lane 3). By contrast, no Rpd3p signal can be observed
Sds3p Is an Essential Component of the Sin3 䡠 Rpd3 HDAC Complex 40964
teins did not co-elute to the extent that they did from the wild type strain (Fig. 2B). This suggests that the association of Sin3p and Rpd3p was weakened in the absence of Sds3p. To confirm that Sin3p and Rpd3p could be chromatographically separated in the sds3⌬ strain, we applied the concentrated Mono-Q pool (fractions 20 –32, see Fig. 4A) to an additional Superose 6 size exclusion column, using the Amersham Phar- macia Biotech SMART (Sensitive Methods And Recovery Tech- nology) system, at 0.5MNaCl. HDAC activity was measured, and Western blot analysis was performed (Fig. 5A). The re- maining HDAC activity eluted at 0.6 MDa and corresponded to Hda1p. Rpd3p was detected in fractions 24 and 25, which correspond to a molecular mass of approximately 0.9 MDa.
Sin3p was now detected in the molecular range of 0.7– 0.5 MDa.
Thus, under these chromatographic conditions, Rpd3p and Sin3p were separated into distinct subcomplexes in the absence of Sds3p (Fig. 5A). As a control, the concentrated Mono-Q pool
of the Sds3-HA strain (fractions 20 –32, see Fig. 2A) was ap- plied to Superose 6 on the SMART system under the same conditions. Fig. 5Bshows that two HDAC activities were sep- arated. One activity corresponded to a 1.2-MDa complex, con- taining Sin3p, Rpd3p, and Sds3p, whereas the second activity, eluting at 0.6 MDa, corresponded to the Hda1p complex. This confirmed that the higher salt concentration used on this col- umn did not cause a disruption of the WT Rpd3p䡠Sin3p䡠Sds3p complex.
To understanding the detailed roles of Rpd3p䡠HDAC com- plexes, it will be necessary to identify the functions of other subunits of these multiprotein complexes. It was shown previ- ously that Sin3p targets Rpd3p-dependent HDAC activity through interaction with the repressor protein Ume6 to certain promoters (19, 29). Therefore, it is very likely that other sub- units are required for the regulation of the enzymatic activity of HDAC complexes. Sap 30, for example, was identified as part of Sin3p䡠HDAC and Rpd3p䡠HDAC complexes in mammals and yeast (20, 40). It appears to be required for the normal function of these complexes. Furthermore, Sap30 is capable of repress- ing transcription when tethered to DNA. This might indicate that Sap30 facilitates interactions of Sin3p䡠HDAC and Rpd3p䡠HDAC and might even recruit HDAC activity in the absence of Sin3p (20). Disruption of SAP30 in yeast shows phenotypes comparable to disruption ofSIN3andRPD3, sug- gesting that it works in the same genetic pathway. Moreover, Sap30p and Rpd3p have been shown to co-immunoprecipitate with antibodies against Rpd3p (40).
Deletion of another yeast gene,SDS3, also showed similar phenotypes to deletions ofSIN3 andRPD3. The yeastSDS3 gene was originally identified in a screen for mutations that cause increased silencing of a crippledHMRsilencer in arap1 mutant background (33, 41). This screen identified more than 20 other genes, includingSIN3andRPD3. Subsequent analy- sis showed thatSDS3shares several transcription regulation properties with SIN3/RPD3, although epistasis tests of the silencing effect suggested thatSDS3might differ in function, at least subtly, from RPD3/SIN3 (33). However, more recent work has shown thatsds3mutants have phenotypes very sim- ilar to those seen in sin3and rpd3mutants, with transcrip- tional regulation of the same set of genes affected in all three mutants (35). The changes in transcriptional regulation seen in rpd3/sds3and sin3/rpd3 double mutants are no more severe than the single mutants. This genetic analysis indicates that SDS3is in the same functional pathway asRPD3andSIN3, and this idea is supported by co-immunoprecipitation experi- ments showing that Sds3p can associate with Sin3p (35).
To determine whether Sds3p is part of the Rpd3p䡠Sin3p complex, we reintroduced an HA-tagged SDS3 gene into the sds3⌬strain. The reintroduced gene rescued the complex. We used this strain to partially purify the complex and used West- ern blot analysis to illustrate co-elution of Sin3p, Rpd3p, and Sds3p. Co-immunoprecipitation experiments using material obtained from the Sds3-HAp strain demonstrated that Sds3p is an integral part of the 1.2-MDa Rpd3p䡠Sin3p complex. We were able to immunoprecipitate Rpd3p and HDAC activity with antibodies against HA-tagged Sds3p. Deletion ofSDS3had a significant effect on the Rpd3p䡠Sin3p complex. First, the size of the Rpd3p portion of the complex was reduced from approxi- mately 1.2 to 0.9 MDa. Second, the association of Sin3p with the Rpd3p complex was weakened. Sin3p was chromatograph- ically separable from Rpd3p in the absence of Sds3p. This result suggests that Sds3p promotes the interactions of Sin3p with Rpd3p or other components of the complex. Sds3p may not, however, be absolutely required for the interaction of FIG. 5.Separation of Rpd3p and Sin3p breakdown complexes
from thesds3⌬ Strain.A, Mono-Q fractions 20 –32 (Fig. 4A) were applied to Superose 6 on the SMART system at 0.5MNaCl. Rpd3 eluted at fractions 23–24, corresponding to an approximate molecular mass of 0.9 MDa. No HDAC activity was detected in this molecular range. Sin3p eluted in the range of 0.7– 0.5 MDa. Hda1p eluted at approximately 0.6 MDa, which corresponds to the measured HDAC activity (A).B, as a control forA, the corresponding fractions from the Sds3-HAp-express- ing strain were analyzed on the Superose 6 column at 0.5MNaCl using the SMART system. Mono-Q fractions 20 –36 (Fig. 2B) were applied to Superose 6 on the SMART system. Two HDAC peaks were separated corresponding to the 1.2-MDa Sin3p, Rpd3p, and Sds3-HAp complex and the 0.6-MDa Hda1p complex, respectively.
Sin3p with the Rpd3p complex. At low salt concentration some Sin3p and Rpd3p co-elute in the absence of Sds3p and they can be co-immunoprecipitated from extract made from an SDS3 deletion strain.2 Finally, in the absence of Sds3p the Rpd3p complex lacked HDAC activity. Thus, the association of Sds3p, Sin3p, or another subunit that might dissociate in the absence of Sds3p is required for the ability of Rpd3p to act as a histone deacetylase. It is important to note, however, that the second Rpd3p-containing complex (0.6 MDa) identified in this study, which does not contain Sds3p, is fully active on histone sub- strates. Thus, in a different context, the HDAC activity of Rpd3 apparently functions in the absence of Sds3p. Alternatively, a different protein in the smaller complex may replace the func- tion of Sds3p.
In conclusion, our observations indicate that Sds3p is neces- sary to maintain the function of a yeast Sin3p䡠Rpd3p complex.
Sds3p facilitates protein-protein interactions within the com- plex to maintain its structure and enzymatic activity. Sds3p might provide part of the linkage between the co-repressor region of the complex (Sin3p) and the deacetylase function (Rpd3p). Furthermore, disruption ofSDS3abrogates the abil- ity of the 1.2-MDa Rpd3䡠Sin3 complex to function as a histone deacetylase.
Acknowledgment—We are grateful to all members of the Workman laboratory for stimulating discussions.
REFERENCES 1. Loidl, P. (1994)Chromosoma103,441– 449
2. Wang, L., Liu, L., and Berger, S. L. (1998)Genes Dev.12,640 – 653 3. Brownell, J. E., Zhou, J., Ranalli, T., Kobayashi, R., Edmondson, D. G., Roth,
S. Y., and Allis, C. D. (1996)Cell84,843– 851
4. Brownell, J. E., and Allis, C. D. (1996)Curr. Opin. Genet. Dev.6,176 –184 5. Grant, P. A., Duggan, L., Coˆte´, J., Roberts, S. M., Brownell, J. E., Candau, R.,
Ohba, R., Owen-Hughes, T., Allis, C. D., Winston, F., Berger, S. L., and Workman, J. L. (1997)Genes Dev.11,1640 –1650
6. Sendra, R., Tse, C., and Hansen, J. C. (2000)J. Biol. Chem.275,24928 –24934 7. Pollard, K. J., and Peterson, C. L. (1997)Mol. Cell. Biol.17,6212– 6222 8. Eberharter, A., Sterner, D. E., Schieltz, D., Hassan, A., Yates, J. R., 3rd,
Berger, S. L., and Workman, J. L. (1999)Mol. Cell. Biol.19,6621– 6631 9. John, S., Howe, L., Tafrov, S. T., Grant, P. A., Sternglanz, R., and Workman,
J. L. (2000)Genes Dev.14,1196 –1208
10. Steger, D. J., Eberharter, A., John, S., Grant, P. A., and Workman, J. L. (1998) Proc. Natl. Acad. Sci. U. S. A.95,12924 –12929
11. Ikeda, K., Steger, D., Eberharter, A., and Workman, J. L. (1999)Mol. Cell.
12. Utley, R. T., Ikeda, K., Grant, P. A., Coˆte´, J., Steger, D. J., Eberharter, A., John, S., and Workman, J. L. (1998)Nature394,498 –502
13. Vignali, M., Steger, D. J., Neely, K. E., and Workman, J. L. (2000)EMBO J.19, 2629 –2640
14. Taunton, J., Hassig, C. A., and Schreiber, S. L. (1996)Science272,408 – 411 15. Alland, L., Muhle, R., Hou, H. J., Potes, J., Chin, L., Schreiber-Agus, N., and
DePinho, R. A. (1997)Nature387,49 –55
16. Hassig, C. A., Fleischer, T. C., Billin, A. N., Schreiber, S. L., and Ayer, D. E.
17. Laherty, C. D., Yang, W.-M., Sun, J.-M., Davie, J. R., Seto, E., and Eisenman, R. N. (1997)Cell89,349 –356
18. Nagy, L., Kao, H.-Y., Chakravarti, D., Lin, R. J., Hassig, C. A., Ayer, D. E., Schreiber, S. L., and Evans, R. M. (1997)Cell89,373–380
19. Kadosh, D., and Struhl, K. (1997)Cell89,365–371
20. Laherty, C. D., Billin, A. N., Lavinsky, R. M., Yochum, G. S., Bush, A. C., Sun, J.-M., Mullen, T. M., Davie, J. R., Rose, D. W., Glass, C. K., Rosenfeld, M. G., Ayer, D. E., and Eisenman, R. N. (1998)Molecular Cell2,33– 42 21. Rossi, V., Hartings, H., and Motto, M. (1998)Mol. Gen. Genet.258,288 –296 22. Lechner, T., Lusser, A., Pipal, A., Brosch, G., Loidl, A., Goralik-Schramel, M., Sendra, R., Wegener, S., Walton, J. D., and Loidl, P. (2000)Biochemistry39, 1683–1692
23. Johnson, C. A., and Turner, B. M. (1999)Semin. Cell Dev. Biol.10,179 –188 24. Brosch, G., Goralik-Schramel, M., and Loidl, P. (1996)FEBS Lett.16,287–291 25. Lusser, A., Brosch, G., Loidl, A., Haas, H., and Loidl, P. (1997)Science277,
26. Rundlett, S. E., Carmen, A. A., Kobayashi, R., Bavykin, S., Turner, B. M., and Grunstein, M. (1996)Proc. Natl. Acad. Sci. U. S. A.93,14503–14508 27. Carmen, A. A., Griffin, P. R., Calaycay, J. R., Rundlett, S. E., Suka, Y., and
Grunstein, M. (1999)Proc. Natl. Acad. Sci. U. S. A.96,12356 –12361 28. Kasten, M. M., Dorland, S., and Stillman, D. J. (1997)Mol. Cell. Biol.17,
29. Rundlett, S. E., Carmen, A. A., Suka, N., Turner, B. M., and Grunstein, M.
30. Vidal, M., and Gaber, R. F. (1991)Mol. Cell. Biol.11,6317– 6327 31. Nasmyth, K., Stillman, D., and Kipling, D. (1987)Cell48,579 –587 32. Stillman, D. J., Dorland, S., and Yu, Y. (1994)Genetics136,781–788 33. Vannier, D., Balderes, D., and Shore, D. (1996)Genetics144,1343–1353 34. Sussel, L., Vannier, D., and Shore, D. (1995)Genetics141,873– 888 35. Dorland, S., Deegenaars, M. L., and Stillman, D. J. (2000)Genetics154,
36. Thomas, B. J., and Rothstein, R. (1989)Cell56,619 – 630
37. Eberharter, A., John, S., Grant, P. A., Utley, R. T., and Workman, J. L. (1998) Methods14,315–312
38. Ko¨lle, D., Brosch, G., Lechner, T., Lusser, A., and Loidl, P. (1998)Methods15, 323–331
39. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, pp.
522–523, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 40. Zhang, Y., Sun, Z.-W., Iratni, R., Erdjument-Bromage, H., Tempst, P., Michael,
H., and Reinberg, D. (1998)Mol. Cell1,1021–1031 41. Thomas, B. J., and Rothstein, R. (1989)Cell65,619 – 630
2D. Stillman, personal communication.
Sds3p Is an Essential Component of the Sin3 䡠 Rpd3 HDAC Complex 40966