ORIGINAL PAPER

Multiple histone deacetylases are recruited by corepressor Sin3and contribute to gene repression mediated by Opi1 regulatorof phospholipid biosynthesis in the yeast Saccharomyces cerevisiae

Mathias Grigat • Yvonne Jaschke •

Felix Kliewe • Matthias Pfeifer •

Susanne Walz • Hans-Joachim Schuller

Received: 12 September 2011 / Accepted: 13 April 2012 / Published online: 28 April 2012

� Springer-Verlag 2012

Abstract Yeast genes of phospholipid biosynthesis are

negatively regulated by repressor protein Opi1 when pre-

cursor molecules inositol and choline (IC) are available.

Opi1-triggered gene repression is mediated by recruitment

of the Sin3 corepressor complex. In this study, we sys-

tematically investigated the regulatory contribution of

subunits of Sin3 complexes and identified Pho23 as

important for IC-dependent gene repression. Two non-

overlapping regions within Pho23 mediate its direct inter-

action with Sin3. Previous work has shown that Sin3

recruits the histone deacetylase (HDAC) Rpd3 to execute

gene repression. While deletion of SIN3 strongly alleviates

gene repression by IC, an rpd3 null mutant shows almost

normal regulation. We thus hypothesized that various

HDACs may contribute to Sin3-mediated repression of

IC-regulated genes. Indeed, a triple mutant lacking

HDACs, Rpd3, Hda1 and Hos1, could phenocopy a sin3

single mutant. We show that these proteins are able to

contact Sin3 in vitro and in vivo and mapped three distinct

HDAC interaction domains, designated HID1, HID2 and

HID3. HID3, which is identical to the previously described

structural motif PAH4 (paired amphipathic helix), can bind

all HDACs tested. Chromatin immunoprecipitation studies

finally confirmed that Hda1 and Hos1 are recruited to

promoters of phospholipid biosynthetic genes INO1 and

CHO2.

Keywords Histone deacetylase � Phospholipid

biosynthesis � Saccharomyces cerevisiae � Opi1 � Sin3 �Paired amphipathic helix

Introduction

Transcriptional corepressor complexes in eukaryotes are of

central importance for negative regulation of numerous

structural genes by creating a local structure of chromatin

inhibitory to gene expression. In the yeast Saccharomyces

cerevisiae, two pleiotropic corepressor complexes, Sin3

and Ssn6/Tup1, have been described (reviewed by Grzenda

et al. 2009; Silverstein and Ekwall 2005; Malave and Dent

2006), both of which execute gene repression by recruiting

histone deacetylases (Kadosh and Struhl 1997; Watson

et al. 2000; Davie et al. 2003). Since neither of these

complexes displays DNA binding, promoter targeting is

achieved through interactions with sequence-specific

DNA-binding proteins.

We have previously shown that the specific repressor

Opi1 of yeast phospholipid biosynthesis functions by

recruiting Sin3 and Ssn6 (Wagner et al. 2001; Jaschke et al.

2011). Structural genes of phospholipid biosynthesis such

as INO1, CHO1 and CHO2 are repressed when precursor

molecules inositol and choline (IC) are available in excess.

In contrast, limiting concentrations of IC allow activation

of these genes which is triggered by the heterodimeric

transcription factor Ino2/Ino4 (Schwank et al. 1995;

reviewed by Chen et al. 2007). Both Ino2 and Ino4 are

members of the basic helix-loop-helix (bHLH) family of

Communicated by H. Ronne.

M. Grigat and Y. Jaschke contributed equally to this work.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00438-012-0692-x) contains supplementarymaterial, which is available to authorized users.

M. Grigat � Y. Jaschke � F. Kliewe � M. Pfeifer � S. Walz �H.-J. Schuller (&)

Institut fur Genetik und Funktionelle Genomforschung,

Jahnstrasse 15a, 17487 Greifswald, Germany

e-mail: schuell@uni-greifswald.de

123

Mol Genet Genomics (2012) 287:461–472

DOI 10.1007/s00438-012-0692-x

DNA-binding proteins and specifically interact with the

UAS element ICRE (inositol/choline-responsive ele-

ment = UASINO; Schuller et al. 1992; Ambroziak and

Henry 1994; Hoppen et al. 2005) found upstream of

phospholipid biosynthetic genes. While Ino4 is responsible

for nuclear import of the heterodimer and its DNA binding

(Kumme et al. 2008), Ino2 mediates transcriptional acti-

vation (Schwank et al. 1995; Dietz et al. 2003) and regu-

latory input via its Opi1 interaction domain (Heyken et al.

2005). Thus, Opi1 as the specific repressor of ICRE-

dependent genes is indirectly recruited to target promoters

by activator Ino2 (Wagner et al. 2001).

Genetic analysis initially has shown that negative reg-

ulation of phospholipid biosynthetic genes by IC requires

the pleiotropic repressor Sin3, which can bind to the

N-terminus of Opi1 (containing OSID: Opi1-Sin3 interac-

tion domain; Slekar and Henry 1995; Wagner et al. 2001).

Sin3 contains four paired amphipathic helix motifs (PAH1–

PAH4; Wang et al. 1990) which via protein–protein

interactions support its function as a scaffold for various

unrelated regulatory networks. Interestingly, OSID is

responsible for interaction with PAH1 of Sin3 and for

contacting tetratricopeptide repeat motifs (TPR) of core-

pressor Ssn6 (Jaschke et al. 2011). Being recruited to

defined control regions via interaction with specific regu-

lators, Sin3 executes repression by associated histone

deacetylases (HDACs) such as Rpd3 in yeast (Kadosh and

Struhl 1997) and HDAC1 and HDAC2 in mammalian

systems (Laherty et al. 1997). Methods of proteomic

analysis led to the identification of two multiprotein com-

plexes (designated Sin3/Rpd3L and Sin3/Rpd3S; Loewith

et al. 2001; Carrozza et al. 2005b), varying with respect to

auxiliary proteins. While Rpd3L preferentially localizes to

promoter regions (Rundlett et al. 1998), Rpd3S is associ-

ated with transcribed regions (Joshi and Struhl 2005),

possibly preventing internal initiations. Importantly, the

genome of S. cerevisiae encodes five Zn2?-containing

HDACs which deacetylate histones by hydrolysis (Rpd3,

Hda1, Hos1, Hos2 and Hos3; Rundlett et al. 1996), while

Sir2 is a NAD-dependent enzyme. Based on sequence

similarities, HDACs have been classified into four groups

(class I, Rpd3, Hos1 and Hos2 in S. cerevisiae; class II,

Hda1 and Hos3; class III, Sir2 and related sirtuins; class

IV, no member in yeasts; reviewed by Hildmann et al.

2007; Yang and Seto 2008).

Previous work focusing on the negative regulatory ele-

ment URS1 and its specific binding factor Ume6 proposed

the simple regulatory hierarchy URS1-Ume6-Sin3-Rpd3

(Kadosh and Struhl 1997). However, a more complex sit-

uation may be effective with other genes repressed by Sin3.

Our previous analysis of ICRE-dependent gene regulation

showed that repression by IC is severely alleviated in a sin3

null mutant, while loss of Rpd3 causes almost no change

(Wagner et al. 2001). We thus hypothesized that Sin3

should be able to recruit not only Rpd3, but also additional

HDACs which together may be required to transform

chromatin into a repressed state. In this study, we indeed

could show that loss of HDACs Rpd3, Hda1 and Hos1

phenocopies deletion of SIN3. This genetic result is further

confirmed by demonstration of physical interaction of Sin3

with Rpd3, Hda1 and Hos1 in vitro and in vivo. Finally, we

identify two additional HDAC interaction domains within

Sin3.

Materials and methods

Yeast strains, media and growth conditions

All strains of the yeast S. cerevisiae used in this study are

isogenic to the regulatory wild-type SIRP3 (Schwank et al.

1995), containing a chromosomally integrated ICRE-CYC1-

lacZ reporter gene. Strains differ with respect to the status of

subunits of Sin3 complexes and HDACs. Strains SIRP3.-

Dopi1, SIRP3.Dsin3, SIRP3.Dume6 and SIRP3.Drpd3 have

been described (Wagner et al. 2001). Null mutations were

introduced into SIRP3 by transformation with deletion

mutant alleles described below. Transformants obtained

were verified for the presence of the desired mutant allele

and the absence of the wild-type allele. Yeast extracts used

for protein–protein interaction assays were prepared from

strain C13-ABY.S86, lacking four vacuolar proteinases

(pra1 prb1 prc1 cps1; De Antoni and Gallwitz 2000). A

compilation of strains and genotypes is available as sup-

porting online Table 1.

Synthetic complete (SC) media used for selective

growth of transformants and conditions of IC repression or

derepression have been described (Schuller et al. 1992).

Although doubling times of mutants lacking multiple

HDACs were severely delayed with respect to the wild

type (275 min for the rpd3 hda1 hos1 triple mutant com-

pared with 100 min for the wild type), strains were uni-

formly harvested at a cell density of 2 9 107 cells/ml.

Plasmid constructions

The following plasmids were constructed by established

procedures to disrupt genes encoding subunits of Sin3

complexes and HDACs: pRH2 (Dsap30::HIS3), pRH4

(Dsds3::HIS3), pRH6 (Dpho23::kanMX), pSW2 (Ddep1::

LEU2), pSW21 (Dume1::LEU2), pTN2 (Drxt2::LEU2),

pTN4 (Deaf3::LEU2), pYJ11 (Dhos1::TRP1), pYJ16

(Dhos3::LEU2), pYJ18 (Dhda1::HIS3), pYJ20 (Dhos2::

hph) and pSS20 (Dino2::LEU2). To construct these plas-

mids, flanking sequences upstream and downstream of the

respective coding regions were amplified by PCR and

462 Mol Genet Genomics (2012) 287:461–472

123

inserted on both sides of the selection marker, allowing

total deletion of reading frames.

To perform interaction assays, Escherichia coli expres-

sion plasmids (derived from pGEX-2TK; GE Healthcare)

encoding various glutathione S-transferase (GST) fusions

were constructed. Length variants of coding regions of

PHO23, SDS3, SAP30, RPD3, HDA1 and HOS1 were

amplified by PCR and fused behind GST. Similarly,

HA-tagged length variants of Sin3 representing PAH and

HID domains were expressed in yeast using plasmid

p426-MET25HA (Mumberg et al. 1994). For bacterial

expression of selected Sin3 variants, plasmid pASK-IBA5

(tetR-regulated; IBA, Gottingen, Germany) was used.

Plasmid constructs encoding minimal length variants used

for interaction studies were verified by DNA sequencing to

confirm authenticity of gene fragments obtained by PCR.

Plasmid names and fused sequences are mentioned in the

legends of figures.

In vitro interaction assays (GST pull-down)

GST- and HA-tagged proteins used for interaction assays

by affinity chromatography were synthesized by E. coli

strain BL21 (Stratagene/Agilent). The tac promoter con-

trolling GST fusion genes was induced with 1 mM IPTG.

Similarly, tetR-dependent gene expression was activated

by 0.2 mg/l anhydrotetracycline. Derepression of MET25-

dependent gene fusions was achieved by cultivating yeast

transformants in the absence of methionine.

GST fusion proteins synthesized in E. coli were released

by sonication, immobilized on glutathione (GSH) Sephar-

ose and subsequently incubated with yeast or bacterial total

protein extracts containing HA fusions. To avoid unspecific

interactions, protein extracts were pre-cleared by treatment

with GSH Sepharose beads prior to incubation with GST

fusions. Details on washing steps at intermediary strin-

gency have been described (Wagner et al. 2001). After

release of GST fusions with free GSH (10 mM), eluates

were separated by SDS/PAGE and proteins transferred to a

filter. Following incubation with anti-HA-peroxidase con-

jugate, HA fusion proteins were detected with POD

chemiluminescent substrate (antibody conjugate and sub-

strate from Roche Biochemicals).

Two-hybrid assays

To perform two-hybrid assays, strain PJ69-4A was used

(MATa trp1 leu2 ura3 his3D gal4D gal80D GAL2-ADE2

LYS2::GAL1-HIS3 met2::GAL7-lacZ; James et al. 1996).

DNA fragments encoding interaction domains of Sin3,

Hda1, Hos1 and Rpd3 were inserted into plasmids pGBD-

C1 (2 lm GAL4DBD TRP1) and pGAD-C1 (2 lm GAL4TAD

LEU2), respectively. Double-transformed strains containing

both types of fusion plasmids were first selected on med-

ium lacking leucine and tryptophan (-L-T) and subse-

quently transferred to medium devoid of adenine (-L-T-A).

Chromatin immunoprecipitation

Essentially, chromatin immunoprecipitation (ChIP) analy-

sis followed the procedure described by Cobb and van

Attikum (2010). Chromosomal loci HDA1 and HOS1 were

modified such that they expressed His-tagged Hda1 and

Hos1 without alteration of gene copy number or control

region. Tagging was performed by transformation of strain

C13-ABY.S86 with a gene-specific modification fragment

and selection for resistance against geneticin. The modifi-

cation fragment was amplified by PCR, using gene-specific

primers and plasmid pU6H3HA as a template (contains a

His6-HA3-kanMX cassette; De Antoni and Gallwitz 2000).

The resulting strains FKY40 (HDA1-His6-HA3) and

FKY42 (HOS1-His6-HA3) and their isogenic ino2 deriva-

tives FKY44 and FKY46 grew until mid-log phase and

were treated with formaldehyde for 15 min. The cross-

linking reaction was subsequently quenched for 5 min by

addition of glycine to a final concentration of 125 mM.

After lysis, cells were sonicated five times for 30 s to shear

chromatin, using a Bandelin Sonoplus UW 70 microtip

(35 % power). After sonication, lysates were centrifuged

for 10 min at 16,0009g to remove insoluble material and

incubated for at least 4 h with His-Tag Dynabeads�

(Invitrogen/Dynal�). After elution of affinity-purified pro-

teins and bound DNA with a buffer containing 300 mM

imidazole, cross-linking was reversed by heating to 65 �C

overnight. DNA was recovered and analyzed by PCR (27

amplification cycles), using specific primers against INO1

(-315/?5) and CHO2 (-360/-40) promoters or ACT1

gene (?841/?1165) as a control.

Miscellaneous procedures

Transformation of S. cerevisiae strains, PCR amplification

and b-galactosidase assays have been described (Schwank

et al. 1995; Wagner et al. 2001). Oligonucleotides used in

this study are presented in supporting online Table 2.

Results

Importance of Sin3 binding proteins and multiple

HDACs for ICRE-dependent gene expression

We have previously shown that repression of ICRE-

dependent genes requires recruitment of pleiotropic

corepressor complexes Sin3 and Ssn6/Tup1 by the path-

way-specific repressor Opi1 (Wagner et al. 2001; Jaschke

Mol Genet Genomics (2012) 287:461–472 463

123

et al. 2011). We thus asked whether additional subunits of

Sin3 complexes, Rpd3L and Rpd3S (Carrozza et al. 2005a,

b), also contribute to repression of ICRE-dependent genes.

The possible importance of subunits Ash1, Raf60, Dot6

and Tod6 (Colina and Young 2005; Shevchenko et al.

2008) also identified in the Rpd3L complex was not

characterized in this study. For this investigation, we

constructed a set of isogenic single mutants, each con-

taining a synthetic minimal promoter, solely driven by

ICRE activating motifs (integrated ICRE-CYC1-lacZ

reporter gene; Schwank et al. 1995). To avoid confusion

among two regulatory pathways comprising Sin3, we did

not use an INO1-lacZ reporter gene, which in addition to

ICRE motifs is also affected by the URS1-Ume6-Sin3

pathway (Lopes et al. 1993; Kadosh and Struhl 1997). As a

means of deregulation in mutants, we used the ratio of

b-galactosidase activities assayed in cells grown under

derepressing and repressing conditions, respectively (D/R).

As is apparent from data shown in Table 1, subunits of

Sin3 complexes unequally affect ICRE-dependent gene

expression. In agreement with previous results (Wagner

et al. 2001), Sin3 is clearly of outstanding importance

(D/R = 2.0), but loss of subunits Pho23 (4.4) and Sap30

(6.1) also weakens the degree of regulation and increases

gene expression under repressing conditions at least by

1.5-fold.

It has been suggested that Sin3 executes its regulatory

influence by recruiting the HDAC Rpd3 (Kadosh and

Struhl 1997). In contrast to what was shown for a sin3

mutant, regulated expression of the reporter gene was not

significantly altered in an rpd3 null mutant (Wagner et al.

2001; Table 2). Apparently, Opi1-dependent repression of

Ino2-activated ICRE motifs is still effective even in the

Table 1 Subunits of Sin3/Rpd3 complexes differently influence

ICRE-dependent gene expression

Genotype (complex) Specific b-gal. activity (U/mg) D/R

D (SD) R (SD)

Wild type 220 (35) 20 (4) 11.0

sin3 (3L/3S) 86 (28) 44 (7) 2.0

rpd3 (3L/3S) 125 (20) 15 (2) 8.3

pho23 (3L) 215 (40) 49 (15) 4.4

sap30 (3L) 197 (35) 32 (8) 6.1

sds3 (3L) 191 (74) 28 (6) 6.8

dep1 (3L) 157 (40) 20 (5) 7.8

rxt2 (3L) 205 (25) 25 (6) 8.2

rxt3 (3L) 188 (36) 25 (3) 7.5

cti6 (3L) 293 (55) 26 (4) 11.3

ume6 (3L) 170 (35) 29 (3) 5.9

ume1 (3L/3S) 309 (32) 31 (3) 10.0

rco1 (3S) 345 (45) 35 (5) 9.9

eaf3 (3S) 245 (45) 20 (4) 12.2

Mutants used are deleted for a single gene and contain a chromo-

somally integrated ICRE-CYC1-lacZ reporter gene. Strains were

grown under repressing (R, 200 lM inositol ? 2 mM choline) and

derepressing conditions (D, 5 lM inositol ? 5 lM choline). Specific

b-galactosidase activities are given in nmol oNPG hydrolyzed per

min per mg of protein (U/mg). Each experiment represents the mean

value of at least 12 independent strain cultivations and enzyme

assays. 3L and 3S refer to complexes Rpd3L and Rpd3S, respectively

SD standard deviation

Table 2 Loss of HDACs, Rpd3, Hda1 and Hos1, imitates the

regulatory defect of a sin3 null mutation

Genotype Specific b-gal. activity (U/mg) D/R

D (SD) R (SD)

Wild type 220 (35) 20 (4) 11.0

opi1 240 (30) 250 (35) 1.0

sin3 86 (28) 44 (7) 2.0

rpd3 125 (20) 15 (2) 8.3

hda1 172 (30) 20 (3) 8.6

hos1 260 (30) 20 (2) 13.0

hos2 165 (25) 15 (2) 11.0

hos3 245 (35) 23 (3) 10.6

rpd3 hda1 113 (28) 33 (8) 3.4

rpd3 hos1 146 (30) 22 (5) 6.6

rpd3 hos2 67 (15) 8 (2) 8.4

rpd3 hos3 174 (25) 26 (5) 6.7

hda1 hos1 226 (28) 29 (4) 7.8

hda1 hos2 223 (30) 20 (4) 11.2

hda1 hos3 307 (38) 38 (6) 8.1

hos1 hos2 259 (24) 20 (4) 13.0

hos1 hos3 219 (32) 15 (3) 14.6

hos2 hos3 123 (20) 11 (2) 11.2

rpd3 hda1 hos1 80 (25) 42 (8) 1.9

rpd3 hda1 hos3 91 (27) 24 (5) 3.8

rpd3 hos1 hos2 90 (25) 11 (3) 8.2

rpd3 hos1 hos3 59 (17) 6 (2) 9.8

rpd3 hos2 hos3 59 (14) 8 (2) 7.4

hda1 hos1 hos2 215 (30) 21 (4) 10.2

hda1 hos1 hos3 260 (42) 22 (3) 11.8

hda1 hos2 hos3 94 (18) 6 (3) 15.7

hos1 hos2 hos3 56 (10) 5 (2) 11.2

rpd3 hda1 hos1 hos3 73 (20) 33 (8) 2.2

hda1 hos1 hos2 hos3 36 (15) 18 (6) 2.0

Strains with various combinations of HDAC gene deletions contain a

chromosomally integrated ICRE-CYC1-lacZ reporter gene. Strains

were grown under repressing (R, 200 lM inositol ? 2 mM choline)

and derepressing conditions (D, 5 lM inositol ? 5 lM choline).

Specific b-galactosidase activities are given in nmol oNPG hydro-

lyzed per min per mg of protein (U/mg). Each experiment represents

the mean value of at least 12 independent strain cultivations and

enzyme assays

SD standard deviation

464 Mol Genet Genomics (2012) 287:461–472

123

absence of Rpd3. We thus reasoned that a functional

redundancy among yeast HDACs, Rpd3, Hda1, Hos1, Hos2

and Hos3, may exist (Rundlett et al. 1996; no consideration

of Sir2 and related NAD-dependent HDACs). Conse-

quently, we constructed deletion cassettes for HDAC genes

to obtain all viable combinations of mutations rpd3, hda1,

hos1, hos2 and hos3. However, we failed to obtain the

following combinations of mutant alleles: triple mutant

rpd3 hda1 hos2; quadruple mutants rpd3 hda1 hos1 hos2,

rpd3 hda1 hos2 hos3 and rpd3 hos1 hos2 hos3; pentuple

mutant rpd3 hda1 hos1 hos2 hos3.

In contrast to HDAC single mutants which showed

normal repression, gene expression of the rpd3 hda1 dou-

ble mutant under repressing conditions was increased

(D/R = 3,4; cf. Table 2). Importantly, the triple mutant

rpd3 hda1 hos1 (1.9) copied the regulatory phenotype of a

sin3 null mutant (2.0), indicating that the corresponding

HDACs may be recruited by Sin3 to trigger repression of

ICRE-dependent genes.

Two non-overlapping sequences of Pho23 directly

interact with Sin3

Pho23 is required for negative regulation of ICRE-depen-

dent gene expression (Table 1). Interestingly, the C-ter-

minal region of Pho23 (amino acids 276-330) comprising

the zinc finger-related C4-HC3 plant homeodomain (PHD)

shows strong similarity to the mammalian family of ING

tumor suppressor proteins (inhibitor of growth) involved in

control of cellular proliferation. We thus wished to inves-

tigate whether Pho23 binds to Sin3 and to establish a

physical map of interacting domains in both proteins.

A GST-Pho23 fusion protein (full-length) immobilized at

glutathione (GSH) Sepharose was incubated with

HA-tagged Sin3, synthesized either in E. coli or in S. cerevi-

siae. As shown in Fig. 1, Sin3 from either source could

bind to Pho23, arguing for a direct interaction of both

proteins. For a more precise mapping, GST fusions with

Pho23 length variants were incubated with Sin3. Interest-

ingly, two non-overlapping domains of Pho23 outside of

ING similarity could bind to Sin3, each independent of the

other. These domains were designated PSID1 and PSID2

(Pho23-Sin3 interaction domain; amino acids 80-151 and

208-250, respectively).

Vice versa, we also mapped Sin3 domains required for

Pho23 interaction. GST fusions of Pho23 comprising either

PSID1 or PSID2 were incubated with HA-tagged length

variants of Sin3. Sin3 fragments used for the interaction

assay represent individual structural and functional domains

described previously (PAH1-PAH4, HID). As shown in

Fig. 2, both PSID sequences could bind aa 801-1100 of

Sin3 but not aa 601-950, indicating aa 951-1100 as the Sin3

core domain responsible for Pho23 recruitment. Thus, nei-

ther PAH domain is required for binding of Pho23, but

instead sequences following the HDAC interaction domain

are involved.

Although deregulation of ICRE-dependent genes was

less evident for sap30 and sds3 mutants than with a pho23

mutant, we also investigated whether the corresponding

proteins could directly interact with Sin3. Similar to what

was found for Pho23, GST fusions of full-length Sds3 and

Sap30 were able to bind HA-tagged Sin3 from yeast and

bacterial protein extracts, arguing for direct interaction

(Fig. 3a, b). The C-terminus of Sds3 (aa 201-327) interacts

with the same internal domain of Sin3 which is also bound

by Pho23 (aa 801-1100). The importance of the C-terminus

of Sds3 for binding Sin3 agrees with the existence of a

shortened protein in the yeast Saccharomyces kluyveri (173

amino acids; Cliften et al. 2003) lacking aa 1-144 of Sds3

from S. cerevisiae.

PS1 PS2

1

1

1

1 330

151

100

PHD

Pho23interactionwith Sin3

1 200

1 200

+Ec

+Sc

+Sc

Sc

Ec

++

Sc

80

330

151 +Sc

+Sc330154

330208 +Sc

330251 Sc

200154 Sc

+Sc250154

+Sc250

330208 +Ec

208

ScGST vector

Input HA3-Sin3

Input HA3-Sin3

Sc

Ec

Fig. 1 Mapping of Pho23 domains responsible for interaction with

Sin3. Length variants of Pho23 were fused with GST, immobilized on

GSH Sepharose and incubated with full-length HA3-Sin3 in total

protein extracts, synthesized by S. cerevisiae (Sc, strain C13-

ABY.S86, plasmid pCW117) or E. coli (Ec, strain BL21, plasmid

pSW11). GST-Pho23 fusions are encoded by plasmids pSW5 (aa

1-330), pSW23 (aa 1-100), pSW24 (aa 1-151), pSW25 (aa 1-200),

pSW26 (aa 251-330), pSW27 (aa 208-330), pSW28 (aa 154-330),

pSW32 (aa 208-250), pSW33 (aa 154-200), pSW35 (aa 154-250) and

pSW40 (aa 80-151). Input controls are shown at the bottom of the

figure (20 % of protein used for the interaction assay). PS1, PS2

(=PSID1, 2): Pho23-Sin3 interaction domains 1 and 2; PHD plant

homeodomain

Mol Genet Genomics (2012) 287:461–472 465

123

HDAC Hda1 binds to Sin3 via new interaction domains

As shown above, the absence of Rpd3, Hda1 and Hos1

imitates the regulatory defect observed in a strain lacking

Sin3. Thus, in addition to Rpd3, HDACs Hda1 and Hos1

may also directly contact the Sin3 corepressor to induce a

more compact chromatin. Class II HDAC Hda1 contains a

long C-terminus of unknown function. To investigate

a possible interaction of Hda1 with Sin3, we used GST-Hda1

fusions comprising amino acids 1-353 and 354-706,

respectively. Indeed, the N-terminus of Hda1 with its

deacetylase domain could bind HA-Sin3 full-length protein

(cf. Fig. 4a). In subsequent studies, we therefore used the

GST-Hda1 (1-353) fusion plasmid to map the Sin3 domain

required for interaction. To allow a high resolution of

mapping, HA-tagged length variants of Sin3 described

above together with smaller fragments each synthesized in

yeast were used for the interaction assay. As is shown in

Fig. 4a, two non-overlapping Sin3 fragments representing

amino acids 301-600 and 1101-1536 (comprising PAH2

and PAH4, respectively) were able to bind to Hda1.

Importantly, these fragments do not contain the previously

characterized HDAC interaction domain (HID, aa

729-1048). By using N- and C-terminal truncations of the

fragment 301-600, we were able to show that it was not

PAH2 (406-472), but instead a region in its C-terminus (aa

473-600) that was sufficient to bind Hda1. The same result

was obtained with the Sin3 fragment comprising aa

1101-1210. We thus conclude that PAH4 (core sequence:

aa 1131-1208) can also bind to Hda1. Obviously, Sin3

contains at least three HID regions which we now desig-

nate HID1 (former HID), HID2 and HID3 (cf. Fig. 4).

We next wanted to map the region within Hda1 which is

required for interaction with Sin3. Length variants of Hda1

were fused with GST and used for interaction assays with

Sin3 sequences representing HID2 and HID3 (aa 301-600

and 1101-1300, respectively). Hda1 fragments aa 201-300

and aa 251-353 could interact with HID2 and HID3 of Sin3

(Fig. 5a), indicating that the region of aa 251-300 may

function as the core binding domain. Since identical results

were obtained with proteins entirely produced in E. coli,

we concluded that aa 251-300 of Hda1 mediated its inter-

action with HID2 and HID3 of Sin3.

HDAC Hos1 also binds to HID2 and HID3 within Sin3

In further studies, we investigated whether class I HDAC

Hos1 could also bind to Sin3. Immobilization of GST-Hos1

(full-length) on GSH Sepharose indeed allowed retention of

full-length Sin3. Subsequently, HA-tagged length variants

1

1 1536

Sin3 bait proteins

301 600

601 950

300

15361101

InputSin3

Pho23PSID1

Pho23PSID2

+ +

P1 P2 P3 P4

801 1100 + +

Fig. 2 Mapping of Sin3 domains interacting with PSID1 and PSID2

of Pho23. GST-Pho23 fusion plasmids pSW25 and pSW32 were used

to synthesize residues 1-200 (comprising PSID1) and 208-250

(comprising PSID2). The following expression plasmids representing

individual PAH domains were used for synthesis of HA-tagged Sin3

length variants in S. cerevisiae: pCW117 (aa 1-1536), pCW83 (aa

1-300), pYJ91 (aa 301-600), pYJ90 (aa 601-950), pYJ89 (aa

801-1100) and pMP20 (aa 1101-1536). For input controls (shown in

the right panel of the figure), 20 % of protein used for the interaction

assay was analyzed. P1, P2, P3 and P4 (=PAH1-4): paired amphi-

pathic helices 1–4

1

1 327

Sds3 Interaction with Sin3

1 200

201 327

+Ec

+

+

Sc

Sc +

327

Sc 1 - 1536

1 - 1536

1 - 1536

1 - 1536

801 - 1100201 327

1

1 201

Sap30 Interaction with Sin3

++

201

Sc 1 - 1536

1 - 1536

1 - 1536GST vector

(a)

(b)

Ec

Sc

Sc

GST vector 801 - 1100Sc

Fig. 3 Direct interaction of Sds3 (a) and Sap30 (b) with Sin3.

Fusion proteins GST-Sds3 (aa 1-327, encoded by pSW4), GST-Sds3

(aa 1-200, pMG4), GST-Sds3 (aa 201-327, pMG6) and GST-Sap30

(aa 1-201, pSW3), respectively, were immobilized on GSH Sepharose

and incubated with protein extracts containing epitope-tagged length

variants of Sin3. Protein extracts were prepared from transformants of

S. cerevisiae (Sc, plasmids pCW117 or pYJ89, 801-1100 of Sin3) or

E. coli (Ec, pSW11). Sin3 input controls are shown in Figs. 1 and 2

466 Mol Genet Genomics (2012) 287:461–472

123

of Sin3 described above were also used to map minimal

interaction domains. As is shown in Fig. 4b, minimal

fragments of Sin3, comprising HID2 and HID3 (=PAH4),

were also able to bind to Hos1, while HID1 again failed to

interact with Hos1. Thus, an identical interaction pattern

was observed for Hda1 and Hos1. Similar to what was

found for Hda1, the enzymatic deacetylase core domain of

Hos1 (amino acids 236-400) is sufficient for this interaction

(Fig. 5b). Again, binding of the Hos1 core region also

occurs with HID2 and HID3 of Sin3 synthesized in E. coli,

arguing for a direct interaction.

HDACs, Rpd3, Hda1 and Hos1, all bind to HID3/PAH4

of Sin3

Initially, HID(1) was identified as a conserved region of

mSin3A (amino acids 524-851, similar to aa 729-1048 of

1

1 1536

Interactionwith Hda1

301 600

601 950

300

InputSin3

P1 P2 P3 P4

801 1100

472301

600473

575473

15361101

13001101

12101101

13001140

+

+

+

+

+

+

1

1 1536

Interactionwith Hos1

301 600

601 950

300

801 1100

600473

15361101

12101101

+

+

+

H2

++

Sin3

(a) Pull-down assays with GST-Hda1

1

Interactionwith Rpd3

301 600

601 950

300

+++

++

15361101

12101101

H1(= H3)

13001101

801 1100

(b) Pull-down assays with GST-Hos1

(c) Pull-down assays with GST-Rpd3

Fig. 4 Mapping of Sin3 domains interacting with histone deacety-

lases Hda1, Hos1 and Rpd3. a GST-Hda1 fusion protein (residues

1-353 of Hda1, plasmid pMP1) was immobilized on GSH Sepharose

and incubated with yeast protein extracts containing epitope-tagged

length variants of Sin3. The following plasmids were used for

synthesis of Sin3 length variants: pCW117 (aa 1-1536), pCW83 (aa

1-300), pYJ91 (aa 301-600), pYJ90 (aa 601-950), pYJ89 (aa

801-1100), pMP20 (aa 1101-1536), pYJ105 (aa 301-472), pMG7

(aa 473-600), pMG8 (aa 473-575), pMP22 (aa 1101-1300), pMG13

(aa 1101-1210) and pMG15 (aa 1140-1300). b Incubation of

immobilized GST-Hos1 full-length fusion protein (470 amino acids,

plasmid pYJ26) with yeast protein extracts containing epitope-tagged

length variants of Sin3. c Incubation of immobilized GST-Rpd3

fusion protein (residues 141-300 of Rpd3, plasmid pMG17) with yeast

protein extracts containing epitope-tagged length variants of Sin3.

H1, H2, H3, histone deacetylase interaction domains HID1-3; P1, P2,

P3 and P4: paired amphipathic helices PAH1-4

1

Hda1

Interaction with

354 706

201 353

353

DAC

201 300

+(301-600)

Sin3(301-600)

Sin3

+

+

+(1101-1300)

Sin3(1101-1300)

Sin3

+

+

EcInput HA3-Sin3

201 300 + +Ec

Sc

Sc

Sc

Sc

251 353 + +Sc

Hos1

Interaction with

236 470

DAC

236 400

+(301-600)

Sin3(301-600)

Sin3

++

(1101-1300)Sin3

(1101-1300)Sin3

+

1 470

1 350

n. t.

n. t.

n. t.

Sc

Sc

Sc

Sc

Ec + +236 400

Rpd3

Interaction with

141 300

DAC

+(801-950)

Sin3(801-950)

Sin3

++

(1101-1300)Sin3

(1101-1300)Sin3

+ScSc

Ec141 300

+

EcInput HA3-Sin3

(a) Pull-down assays with GST-Hda1 variants

(b) Pull-down assays with GST-Hos1 variants

(c) Pull-down assays with GST-Rpd3

Fig. 5 Mapping of HDAC domains interacting with Sin3 domains

HID1, HID2 and HID3. a Length variants of Hda1 were fused with

GST, immobilized on GSH Sepharose and incubated with protein

extracts from yeast or E. coli. The following GST expression

plasmids were used: pMP1 (aa 1-353), pMP2 (aa 354-706), pMP12

(aa 201-353), pMP14 (aa 201-300) and pMP15 (aa 251-353). Epitope-

tagged Sin3 domains representing HID2 and HID3 were expressed in

yeast (Sc; plasmids pYJ91, aa 301-600 and pMP22, aa 1101-1300,

respectively) or in E. coli (Ec; pMG22 and pMG23, respectively).

b Length variants of Hos1 fused with GST were incubated with

protein extracts from yeast or E. coli. The following GST expression

plasmids were used: pYJ26 (aa 1-470), pMP9 (aa 1-350), pMP4 (aa

236-470) and pYJ85 (aa 236-400). c GST-Rpd3 fusion representing

residues 141-300 of Rpd3 (plasmid pMG17) was incubated with

protein extracts from yeast or E. coli. The epitope-tagged Sin3

domain representing HID1 was expressed in yeast (Sc; plasmid

pYJ92, aa 801-950) or in E. coli (Ec; pMG26). Yeast input controls

are shown in Fig. 4. DAC deacetylase core domain, n. t. not tested

Mol Genet Genomics (2012) 287:461–472 467

123

yeast Sin3), which turned out as sufficient to bind the

Rpd3-related mammalian HDAC2 (Laherty et al. 1997).

We thus wished to test whether Rpd3 can also interact with

a second domain of Sin3 and to perform a more precise

mapping of the yeast HID1. Assuming that a conserved

segment within HDACs may be responsible for Sin3

binding, we constructed a GST-Rpd3 fusion containing

amino acids 141-300. Indeed, this fusion protein could

interact with two Sin3 fragments covering HID1 (aa

601-950 and aa 801-1100; Fig. 4c). Using proteins entirely

produced in E. coli, we could finally confirm that amino

acids 801-950 indeed defined the functional core of HID1,

which was directly bound by Rpd3 without yeast-specific

auxiliary factors (Fig. 5c). In addition, GST-Rpd3 was also

able to bind Sin3 fragments containing HID3/PAH4, syn-

thesized either in yeast or in E. coli. Consequently, HID3/

PAH4 functions as an interaction domain for at least three

HDACs from yeast.

In vivo interaction of Sin3 with various HDACs

In addition to in vitro interaction assays, we also performed

two-hybrid analyses to verify binding of Sin3 to HDACs,

Hda1, Hos1 and Rpd3. Length variants of Sin3 comprising

HID2 (aa 301-600) and HID1, HID2 and HID3 (aa

301-1536), respectively, were fused with the DNA-binding

domain (DBD) of Gal4. Core deacetylase domains of

Hda1, Hos1 and Rpd3 which have been shown to bind Sin3

in vitro were fused with Gal4 transcriptional activation

domain (TAD). Sin3-HDAC interactions in vivo should

reconstitute a functional Gal4 activator being able to

induce the GAL2-ADE2 reporter gene of the recipient

strain, thereby allowing growth of transformants in the

absence of adenine. As is shown in Fig. 6, both DBD

fusions of Sin3 in combination with empty TAD vector

were unable to mediate growth on a medium lacking ade-

nine. In contrast, co-transformation of DBD-Sin3 (aa

301-1536) with TAD fused to HDACs, Hda1, Hos1 or

Rpd3, restored growth on adenine-free medium. We con-

clude that Sin3 comprising HID1-3 is able to interact with

these HDACs in vivo. On the other hand, a minimal Sin3

(aa 301-600) with HID2 but devoid of HID1 and HID3

could interact with Hda1 and Hos1, but not with Rpd3.

To finally investigate whether Hda1 and Hos1 are

indeed recruited to ICRE-containing promoters regulated

by Ino2 and Opi1, we performed ChIP analyses. We thus

constructed strains containing variants of HDA1 and HOS1

which encode epitope-tagged proteins (His6). To analyze

protein occupation of ICRE-containing promoters, we

selected INO1 (biosynthesis of inositol; three ICRE motifs)

and CHO2 (biosynthesis of choline, three ICRE motifs).

The ACT1 gene encoding actin served as a negative con-

trol. As shown by pull-down experiments and two-hybrid

studies, binding of HDACs follows a defined order of

interaction events (ICRE-Ino2-Opi1-Sin3-HDACs; Wagner

et al. 2001; this work). We thus also constructed ino2

deletion strains which should no longer allow recruitment

of HDACs to ICRE-containing promoters. As shown in

Fig. 7, Hda1 and Hos1 indeed occupy ICRE-dependent

promoters INO1 and CHO2 under conditions of gene

repression by inositol and choline (lines 1 and 3; INO2

intact), while binding to ACT1 is substantially less effec-

tive. The figure also shows that binding of HDACs, Hda1

and Hos1, requires a functional INO2 gene (lines 2 and 4,

ino2 deletion mutants). These findings agree with results

derived from in vitro interaction studies and provide in

vivo evidence that ICRE-dependent promoters are affected

by more than a single histone deacetylase.

Discussion

The importance of Sin3 as a pleiotropic corepressor for

several regulatory systems of gene expression in eukary-

otes is well known (Grzenda et al. 2009). The existence of

Growth on-L -T -L -T -A

Opi1 (1-106)

Sin3 (301-1536)

Sin3 (301-1536)

Sin3 (301-1536)

Sin3 (301-1536)

Sin3 (301-600)

Sin3 (301-600)

Sin3 (301-600)

Sin3 (301-600)

DBD fusion

Sin3 (1-300)

-

Hda1 (201-353)

Hos1 (236-400)

Rpd3 (141-300)

-

Hda1 (201-353)

Hos1 (236-400)

Rpd3 (141-300)

TAD fusion

Fig. 6 Interaction of Sin3 with various HDACs shown by two-

hybrid assays. The Gal4 DNA-binding domain (DBD) was fused with

Sin3 fragments comprising HDAC interaction domains to give

plasmids pMG42 (aa 301-600) and pJW31 (aa 301-1536). Corre-

spondingly, Gal4 transcriptional activation domain (TAD) was fused

with HDAC fragments to give pMG41 (Hda1, aa 201-353), pMG40

(Hos1, aa 236-400) and pMG39 (Rpd3, aa 141-300). Plasmids

encoding fusions DBD-Opi1 and TAD-Sin3 (pJW8 and pJW1,

respectively; Wagner et al. 2001) were used as a positive control.

As a negative control, empty TAD vector pGAD-C1 was used. DBD

and TAD pairs of fusion plasmids (selection markers: TRP1 and

LEU2, respectively) were co-transformed into strain PJ69-4A,

containing a GAL2-ADE2 fusion which allows growth in the absence

of adenine when a functional Gal4 activator is reconstituted. Selection

plates (-L -T and -L -T -A; absence of leucine, tryptophan and

adenine) were incubated for 48 h

468 Mol Genet Genomics (2012) 287:461–472

123

structural motifs suitable for protein–protein interactions

suggested that Sin3 functioned as a recruiting platform for

effector enzymes (e.g., HDAC Rpd3), allowing their access

to specific promoter regions. In this study, we have

investigated the influence of subunits of the Sin3 complex

for regulation of phospholipid biosynthetic genes. Char-

acterization of various combinations of HDAC mutations

led us to conclude that Rpd3 was not the sole enzyme

responsible for gene repression mediated by Sin3. This

hypothesis is further confirmed by our demonstration of

physical interaction of Sin3 with HDACs, Rpd3, Hda1 and

Hos1. The existence of three non-overlapping HDAC

interaction domains (HID1, HID2 and HID3) indicates that

a greater combinatorial variety of Sin3-containing com-

plexes than previously assumed is able to contribute to

target gene repression.

The Sin3/Rpd3L complex comprises at least 12 subunits

which may stabilize interactions of Sin3 with specific

repressors and thus unequally influences different regula-

tory pathways. Surprisingly, phenotypes of the corre-

sponding null mutants differ with respect to the regulatory

system, leading to enhanced repression of yeast silent

mating type loci and at telomeric positions, but increased

initiation at the PHO5 promoter and URS1-containing

promoters (Vannier et al. 1996; Zhang et al. 1998; Sun and

Hampsey 1999; Loewith et al. 2001). To investigate the

importance of 13 subunits identified in complexes Rpd3L

and Rpd3S for ICRE-dependent gene expression, we used

single null mutants for analyzing the regulation of a syn-

thetic minimal promoter which did not contain a URS1

motif such as the widely used INO1 control region. Besides

sin3, deregulation was most apparent with a pho23 mutant,

which was initially isolated because of increased expres-

sion of the PHO5-encoded acid phosphatase (Lau et al.

1998). Pho23 is especially interesting due to the similarity

of its C-terminal PHD finger with mammalian tumor sup-

pressors of the ING family. This structural motif strongly

binds to trimethylated lysine of histone H3 (H3K4me3) and

mediates recruitment of Sin3/Rpd3L to the repressed

PHO5 promoter (Shi et al. 2006; Wang et al. 2011). Our

results show that Pho23 contains two non-overlapping

domains which both can directly bind to a domain of Sin3

close to HID1. In contrast to Rpd3, Pho23 interacts with aa

801-1100 but not with aa 601-950, suggesting that aa

951-1100 are at least necessary for binding of Pho23.

Similarly, Sds3 also binds to aa 801-1100 of Sin3 (sum-

marized in Fig. 8a). We could also demonstrate that Sds3

and Sap30 directly interact with Sin3. For the mammalian

ING1b protein, association with Sin3 through direct inter-

action with Sap30 has been shown (Kuzmichev et al.

2002).

Importantly, comparison of ICRE-dependent gene reg-

ulation in sin3 and rpd3 null mutants revealed a striking

difference, indicating that Rpd3 could not be responsible

for the entire repressing influence of the Sin3 complex.

Inspection of the data published by Kadosh and Struhl

(1997) on URS1-Ume6 dependent repression showed that

similarly loss of gene repression in a sin3 mutant was

clearly stronger than in an rpd3 mutant. Both findings can

be explained by assuming a separate repressing activity

within the Sin3 complex which is not mediated by histone

deacetylation or, alternatively, triggered by recruitment of

HDAC isoenzymes. Our first evidence supporting the latter

view resulted from construction of multiple HDAC null

mutants and subsequent analysis of ICRE-dependent gene

expression. Although we failed to obtain certain combi-

nations of mutant alleles (indicating that total loss of class I

and class II HDACs leads to synthetic lethality), a triple

mutant rpd3 hda1 hos1 showed deregulation similar to that

observed with a sin3 single mutant.

Our studies with the ICRE-dependent reporter gene

revealed that repression and derepression were affected in

the sin3 strain and in several mutants lacking multiple

HDACs (cf. Table 2). These data could mean that activa-

tors as well as repressors may utilize Sin3, as it has been

shown for the Ssn6/Tup1 corepressor which also acts as a

positive cofactor of Gcn4 activator (Kim et al. 2005).

Similarly, a positive role of Sin3-Rpd3 for activation of

osmosensitive genes such as HSP12 has been shown (de

Nadal et al. 2004). Alternatively, pleiotropic deregulation

in a sin3 strain affecting carbon and nitrogen metabolism

may cause a general shortage of important metabolites,

indirectly leading to less efficient derepression.

Similar regulatory phenotypes of mutants sin3 and rpd3

hda1 hos1 do not necessarily mean that the corresponding

HDA1 INO2

HDA1 Δino2

IN IP IN IP IN IP

INO1 CHO2 ACT1

HOS1 INO2

HOS1 Δino2

Fig. 7 In vivo binding of Hda1 and Hos1 to ICRE-containing

promoters INO1 and CHO2 shown by chromatin immunoprecipitation

(ChIP). INO2 wild-type strains FKY40 and FKY42 contain HDA1and HOS1 variants with C-terminal His6 and HA3 epitopes at their

natural chromosomal position. FKY44 and FKY46 were constructed

by subsequent introduction of an ino2 deletion allele. Strains were

grown under conditions of inositol/choline repression. Details of the

procedure are given in ‘‘Materials and methods’’. After reversal of

cross-linking, DNA samples were analyzed by PCR using gene-

specific primers which allow amplification of *320 bp fragments. INinput control (analysis of total lysate samples), IP immunoprecipitate

(analysis of samples enriched for His-tagged Hda1 and Hos1,

respectively)

Mol Genet Genomics (2012) 287:461–472 469

123

HDACs are indeed recruited by Sin3. We have previously

shown that repression by Opi1 is also mediated to a certain

degree by the Ssn6/Tup1 corepressor (Jaschke et al. 2011),

which can recruit HDACs, Rpd3, Hda1, Hos1 and Hos2

(Watson et al. 2000; Wu et al. 2001b; Davie et al. 2003).

Thus, glucose repression of the invertase gene SUC2 was

abolished in an rpd3 hos1 hos2 triple mutant. These results

clearly show that corepressor function may be indeed

mediated by more than a single HDAC, supporting the idea

of some redundancy among them. Consequently, we per-

formed interaction studies to decide whether Sin3 is also

able to recruit additional HDACs. According to the results

of our mutational analysis, we concentrated on HDACs,

Hda1 and Hos1. Indeed, GST fusions of both enzymes

were able to interact with full-length Sin3. In contrast, we

could not reproducibly show interaction of Hos2 and Hos3

with Sin3. These in vitro studies were confirmed in vivo by

two-hybrid experiments, using fusion plasmids which

encode domains of Sin3 and HDACs, Hda1, Hos1 and

Rpd3. Using ChIP analysis, we also demonstrate recruit-

ment of Hda1 and Hos1 to promoter regions of INO1 and

CHO2. We selected these genes because they show strong

regulation by inositol/choline which is mediated by three

ICRE upstream motifs (Hoppen et al. 2005; Kodaki et al.

1991). Promoter recruitment of HDACs required a func-

tional Ino2 activator which directly binds ICRE motifs and

establishes the core for subsequent interactions with Opi1,

Sin3 and finally Hda1/Hos1.

Detailed mapping experiments revealed that Sin3 not

only contains the single HDAC interaction domain initially

mapped in mammalian proteins (HID, aa 801-950 for the

yeast protein), but also two additional segments which we

designate HID2 (aa 473-600) and HID3 (aa 1101-1210; cf.

Fig. 8a). A hydrophobic-amphipathic sequence pattern

which is completely conserved between yeast and human

Sin3 proteins could be identified within the renamed HID1

(Fig. 8b). HID2 immediately follows the PAH2 domain

and may be specific for yeast Sin3 proteins. This conclu-

sion can be derived from the finding that mSin3 proteins

lack sequence motifs convincingly similar to HID2. In

contrast, HID3 which is identical to PAH4 is a conserved

structural feature of Sin3 proteins. The structural pattern of

PAH4 deviates from PAH1, PAH2 and PAH3 and its

function remained unclear. To our knowledge, the work

reported here is the first demonstration of a physical

function for PAH4 within yeast Sin3. For mammalian

mSin3A, interaction of PAH4 with TPR motifs of an

O-glycosyltransferase (OGT) specific for N-acetylgluco-

samine (NAG) has been demonstrated (Yang et al. 2002),

indicating that NAG transfer on transcription factors may

inhibit their activity. In conclusion, the existence of at least

three HDAC interaction domains indicates that a single

Sin3 protein could be able to simultaneously bind Rpd3,

Hda1 and Hos1. Alternatively, Sin3 may only interact with

a single HDAC isoenzyme, leading to the coexistence of

individual HDAC/Sin3 complexes. This latter view would

Sin3P1 P2 P3 P4HID2(HID3)

Opi1 Ume6

Hda1

Hos1

Sds3Sap30

Rpd3

Pho23

HID1 HCR

(a)

Rpd3Hda1Hos1

(b)

(c)

ScSin3HsSin3A

Fig. 8 a Summary of mapped interaction domains within Sin3. Data

were those presented in this work or taken from Wagner et al. (2001)

(for Opi1), Washburn and Esposito (2001) (for Ume6) and Xie et al.

(2011) (for Sap30). HCR highly conserved region of unknown

function, HID histone deacetylase interaction domains; P1, P2, P3 and

P4 (=PAH1-4): paired amphipathic helices 1–4. b Hydrophobic-

amphipathic pattern in an alignment of sequences from Sin3 HID1 of

yeast and H. sapiens. Hydrophobic residues at heptad positions 7/1

and 4/5 are shown in bold. c Hydrophobic-amphipathic pattern in an

alignment of sequences from HDACs, Rpd3, Hda1 and Hos1, shown

to bind Sin3 HID motifs. #, hydrophobic amino acids

470 Mol Genet Genomics (2012) 287:461–472

123

explain why previous purification procedures using tagged

Rpd3 failed to detect Hda1 and Hos1 within the complex

(Carrozza et al. 2005b). It should be mentioned that Hda1

also exists in a distinct complex together with non-catalytic

subunits Hda2 and Hda3 (Wu et al. 2001a).

Interaction experiments with HDAC length variants

showed that Sin3 binding maps within their catalytic

deacetylase domains. However, catalytic and binding

functions are not identical. Two histidine residues (H247,

H248 for Hda1) which are essential for binding the

substrates water and acetyllysine via hydrogen bonds lie

outside the core interaction domain of Hda1 (aa 251-300).

We also searched for hydrophobic-amphipathic sequence

motifs within HDACs which possibly could bind Sin3. As

shown in Fig. 8c, hydrophobic residues appear at positions

7 or 1 and 4 or 5 of heptad repeats placed above sequence

segments of Rpd3, Hda1 and Hos1. By using bacterial

protein extracts, we could show that Rpd3, Hda1 and Hos1

are able to directly interact with Sin3 domains. This does

not rule out that auxiliary proteins of the Sin3/Rpd3L

complex such as Pho23, Sds3 and Sap30 increase Sin3-

HDAC interaction in yeast. The interactions shown for

yeast Sin3 in this work and previous publications are

summarized in Fig. 8a.

Acknowledgments This work has been supported by the Deutsche

Forschungsgemeinschaft (DFG). We thank Marius Wanjek for valu-

able support.

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