Homo-oligomerisation and nuclear localisation of mouse histone deacetylase 1

doi:10.1006/jmbi.2001.4569 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 308, 27±38

Homo-Oligomerisation and Nuclear Localisation ofMouse Histone Deacetylase 1

Jan Taplick1, Vladislav Kurtev1, Karin Kroboth1, Markus Posch1

Thomas Lechner2 and Christian Seiser1*

1Institute of MedicalBiochemistry, Division ofMolecular Biology, ViennaBiocenter, University ofVienna, Austria2Institute of MicrobiologyUniversity of InnsbruckMedical School, Austria

J.T. & V.K. contributed equally toAbbreviations used: HAT, histon

HDAC, histone deacetylase; HAD,domain; GST, glutathione S-transferlocalisation signal; TK, thymidine k¯uorescence protein; DPAI, 40,6-diaphenylindole.

E-mail address of the [email protected]

0022-2836/01/010027±12 $35.00/0

Reversible histone acetylation changes the chromatin structure and canmodulate gene transcription. Mammalian histone deacetylase 1 (HDAC1)is a nuclear protein that belongs to a growing family of evolutionarilyconserved enzymes catalysing the removal of acetyl residues from corehistones and other proteins. Previously, we have identi®ed murineHDAC1 as a growth factor-inducible protein in murine T-cells. Here, wecharacterise the molecular function of mouse HDAC1 in more detail. Co-immunoprecipitation experiments with epitope-tagged HDAC1 proteinreveal the association with endogenous HDAC1 enzyme. We show thatHDAC1 can homo-oligomerise and that this interaction is dependent onthe N-terminal HDAC association domain of the protein. Furthermore,the same HDAC1 domain is also necessary for in vitro binding ofHDAC2 and HDAC3, association with RbAp48 and for catalytic activityof the enzyme.

A lysine-rich sequence within the carboxy terminus of HDAC1 iscrucial for nuclear localisation of the enzyme. We identify a C-terminalnuclear localisation domain, which is suf®cient for the transport ofHDAC1 and of reporter fusion proteins into the nucleus. Alternatively,HDAC1 can be shuttled into the nucleus by association with anotherHDAC1 molecule via its N-terminal HDAC association domain. Ourresults de®ne two domains, which are essential for the oligomerisationand nuclear localisation of mouse HDAC1.

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Keywords: chromatin; histone acetylation; histone deacetylase; nuclearlocalisation; oligomerisation
*Corresponding author
Introduction

Modi®cation of core histones by acetylation oftheir N-terminal tails has been recognised as animportant mechanism in the regulation of geneexpression. In concert with histone acetyltrans-ferases (HATs), histone deacetylases (HDACs) con-trol the dynamic acetylation of core histones and anumber of important nuclear regulatory proteinssuch as p53 and E2F-1 (reviewed by Kouzarides,2000). The identi®cation of the short-chain fatty

this work.e acetyltransferase;HDAC associationase; NLS, nuclearinase; GFP, greenmidino-2-

ing author:

acid butyrate as an inhibitor of deacetylatingactivities (Boffa et al., 1978; Candido et al., 1978;Reeves & Candido, 1978; Sealy & Chalkley, 1978;Vidali et al., 1978) provided an important tool toinvestigate transcriptional regulation by reversiblehistone acetylation. Later, trichostatin A and tra-poxin were found to be more speci®c deacetylaseinhibitors (Yoshida et al., 1990, 1995). Af®nity puri-®cation on a trapoxin matrix led to the identi®-cation of the ®rst histone deacetylase HDAC1, themammalian homologue of yeast Rpd3p (Tauntonet al., 1996).

The superfamily of mammalian deacetylasesconsists currently of three subfamilies (recentlyreviewed by Gray & EkstroÈm, 2001). The RPD3-like class I enzymes are represented by HDAC1,HDAC2 (formerly known as mRPD3; Yang et al.,1996), HDAC3 (Dangond et al., 1998; Emiliani et al.,1998; Yang et al., 1997) and HDAC8 (Hu et al.,2000; Buggy et al., 2000; Van den Wyngaert et al.,

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28 Oligomerisation and Nuclear Localisation of HDAC1

2000). The HDA1-like class II enzymes HDAC4 toHDAC7 share homology with the yeast histonedeacetylase HDA1 (Rundlett et al., 1996, Fischleet al., 1999; Grozinger et al., 1999; Kao et al., 2000;Miska et al., 1999; Verdel & Khochbin, 1999; Wanget al., 1999; Zhou et al., 2000). Class III enzymes areNAD�-dependent deacetylases with homology tothe yeast silencing protein Sir2 (Imai et al., 2000;Landry et al., 2000; Smith et al., 2000). While HDA1and RPD3-related HDACs show signi®cant hom-ology, Sir2-related proteins form a distinct groupof deacetylating enzymes.

During the last few years, class I histone de-acetylases have been studied intensively. HDAC1and its homologues have been identi®ed as com-ponents of numerous protein complexes regulatinggene expression during cell-cycle progression,differentiation and development (reviewed by Ng& Bird, 2000; Ahringer, 2000) and several studiessuggest a potential role of class I enzymes inhuman cancer (reviewed by Cress & Seto, 2000).While the interaction of HDAC1 with differenttranscriptional regulators has been well studied,the protein itself and its domains are less wellunderstood.

Here, we characterise two important functionalregions of the HDAC1 protein: The N terminuscontains a motif required for HDAC1 homo-oligo-merisation and for hetero-oligomerisation ofHDAC1 with HDAC2 and HDAC3. Deletion of theHDAC association domain (HAD) or mutation of

conserved residues lead to loss of enzymaticactivity.

The C terminus of the protein (residues 438-482),on the other hand, is crucial for the nuclear localis-ation of HDAC1 and contains a lysine-richsequence that can function as an ef®cient nuclearlocalisation signal when fused to a marker protein.

Results

HDAC1 self-association in vitro and in vivo

To test the importance of histone deacetylatingactivity for the transcriptional repression byHDAC1-containing protein complexes, we createda number of HDAC1 mutants with single ordouble amino acid exchanges of highly conservedresidues. Previously, HDAC1 was shown to be aZn-binding protein with crucial aspartate and histi-dine residues within the active-site pocket,suggesting the importance of charged amino acidsfor the activity of the enzyme (Finnin et al., 1999).Therefore, we individually mutated two highlyconserved histidine residues, H28 and H68, andthe previously described residues H140/141 andH178/179 (Hassig et al., 1998; Kadosh & Struhl,1997) of the murine HDAC1 protein to alanines(Figure 1). Histidine 28 is part of a domain import-ant for the formation of the active-site pocket,while histidine 68 is located near, but outside theactive centre (Finnin et al., 1999). In addition, wemutated aspartate 174, which is part of the charge-

Figure 1. Sequence comparisonof mouse HDAC1, HDAC2 andHDAC3. Multiple sequence align-ment of the cDNA sequencesfor HDAC1 (Bartl et al., 1997),HDAC2/mRPD3 (Yang et al., 1996)and HDAC3 (Yang et al., 1997) wasperformed with the LasergeneMegalign program. Mutated resi-dues and the core nuclear localis-ation signal of HDAC1 (NLS) areshown in bold. The N-terminalHDAC association domain (HAD)and the C-terminal nuclear localis-ation domain (NLD) are under-lined.

Oligomerisation and Nuclear Localisation of HDAC1 29

relay system of HDAC1 (Finnin et al., 1999) to histi-dine (Figure 1).

Wild-type and mutant HDAC1 proteins carryinga C-terminal Myc-epitope were transientlyexpressed from retroviral vectors in Swiss 3T3®broblasts. To test the enzymatic activity of theHDAC1 variants, the proteins were immunopreci-pitated via their Myc-tag and assayed for histonedeacetylase activity. As shown in Figure 2, enzy-matic activity was reduced dramatically when anyof the conserved histidine residues (H28, H68,H140/141 and H178/179) was changed to alanine.We concluded that, in addition to the previouslydescribed histidine residues H141 and H178(Hassig et al., 1998; Kadosh & Struhl, 1997), histi-dine 28 and histidine 68 are also important for thecatalytic activity of HDAC1. Furthermore,mutation of aspartic acid 174 to histidine (D174H)also resulted in strong reduction of histone deace-tylase activity (Figure 2(a)). However, immunopre-cipitates of HDAC1 mutants H28A, H68A andH140/141A displayed enzymatic activity that issigni®cantly higher than the control with extractsfrom untransfected cells.

To characterise the mutated HDAC1 proteins inmore detail, we established stable cell lines expres-sing Myc-tagged versions of wild-type HDAC1and of three HDAC1 mutants (H28A, H68A andD147H). Expression levels of all HDAC1-Mycmutants and wild-type HDAC1-Myc in Swiss 3T3®broblasts were low (5-10 %) when compared to

Figure 2. Mutation of highly conserved residuesstrongly reduces the speci®c activity of mouse HDAC1.Swiss 3T3 cells were infected with recombinant pBABEretroviruses expressing Myc-tagged HDAC1 mutantproteins or Myc-tagged HDAC1 wild-type protein (wt).Comparable amounts of epitope-tagged HDAC1 proteinwere immunoprecipitated. Immunoprecipitates weredivided and (a) two-thirds were assayed for HDACactivity and (b) one-third was analysed on Westernblots. The Myc-speci®c antibody 9E10 was used forboth, immunoprecipitation and Western blot detection.Extracts from non-infected Swiss 3T3 ®broblasts (con-trol) were included as a negative control. The resultsshown are representative of three independent transfec-tion experiments.

the level of endogenous HDAC1 (see Figure 7(b);and data not shown). When mutant HDAC1 pro-tein was precipitated with the Myc-tag speci®cantibody from relatively large amounts of totalprotein extracts (1.5 mg), we observed againresidual HDAC activity associated with the mutantproteins. As shown in Figure 3(a), the enzymaticactivity associated with the HDAC1 mutants(H28A, H68A and D147H) was lower than theactivity of precipitated Myc-tagged wild-type pro-tein (wt) but signi®cantly higher than the activityin immunoprecipitates from extracts of non-infected cells (Figure 3(a), control). When theimmunoprecipitated fractions were analysed onWestern blots with serum against HDAC1, weobserved endogenous HDAC1 associated with theHDAC1-Myc mutants (Figure 3(b), upper panel)and the Myc-tagged wild-type protein (Figure 3(b),lower panel). The co-immunoprecipitated protein(indicated by an asterisk) corresponded in sizeto the endogenous HDAC1 obtained in immuno-precipitates from uninfected cells with the HDAC1antibody (Figure 3(b), lower panel, control).Endogenous HDAC1 was absent from Myc-

Figure 3. Exogenous HDAC1-Myc protein is associ-ated with endogenous HDAC1 protein in retrovirallyinfected Swiss 3T3 cells after stable integration. Mutantand wild-type HDAC1-Myc protein was immunopreci-pitated with the Myc-speci®c 9E10 antibody. Immuno-precipitates of non-infected cells (control) with Myc-speci®c and HDAC1-speci®c antibodies served as con-trols. Immunoprecipitates (a) were assayed for HDACactivity and (b) analysed by Western blotting. Anti-bodies used for immunoprecipitations (IP) and Westernblot detection (W) are indicated. Co-immunoprecipitatedendogenous HDAC1 is indicated by an asterisk (*). Datashown are representative of three independent exper-iments.


immunoprecipitates with extracts from uninfectedcontrol cells (Figure 3(b), lower panel), which is inagreement with the data from the enzyme assayshown in Figure 3(a). Taken together, these datademonstrate that endogenous HDAC1 is associatedwith epitope-tagged HDAC1 in stably expressingSwiss 3T3 ®broblasts.

To con®rm the observed self-association ofHDAC1 in vitro and to determine the domainwithin the HDAC1 protein required for self-interaction, we performed glutathione S-transferase(GST) pull-down experiments. Full-length HDAC1fused to GST was bound to glutathione beads andincubated with similar amounts of in vitro trans-lated, radiolabeled HDAC1 full-length protein anddifferent HDAC1 deletion mutants, respectively(representations and inputs are shown in

Figure 4. In vitro interaction of HDAC1 with HDAC1, HDHDAC1 constructs used for pull-down experiments. The puInputs of the different radiolabelled HDAC1 mutants. (c) HHDAC1 mutants containing the N terminus. GST-HDAC1,protein was incubated with in vitro translated HDAC1 deletwas performed as described in Materials and Methods. (dresidues 1-53 of the HDAC1 protein. GST pull-down of radioGST-HDAC1 fusion proteins containing HDAC1 mutantswith radiolabelled HDAC1 full-length protein and GST-LGST-HDAC3 in the presence of 100 mg/ml ethidium bromide

Figure 4(a) and (b). Strongest binding wasobserved for deletion mutants B and D, indicatingthat the N-terminal 130 amino acid residues ofHDAC1 are important for HDAC oligomerisation(Figure 4(c)). GST and an unrelated GST fusionprotein (GST-LR8) were used as negative controlsand showed only background signal (Figure 4(c)and (e). In addition, the two closely related histonedeacetylases HDAC2 and HDAC3 were also testedas GST fusion proteins in the GST pull-down assayfor their ability to directly interact with HDAC1.As can be seen in Figure 4(c), all three histone dea-cetylases bound in vitro translated HDAC1 withcomparable af®nity and speci®city. To exclude anunspeci®c association via DNA or DNA-bindingproteins, the binding assay was performed in thepresence of ethidium bromide (100 mg/ml). As

AC2 and HDAC3. (a) A representation of the differenttative HDAC association domain (HAD) is indicated. (b)

DAC1, HDAC2 and HDAC3 interact speci®cally withGST-HDAC2 and GST-HDAC3 fusion proteins or GSTion mutants A-E shown in (a). Pull-down and detection) The HDAC association domain (HAD) encompasseslabelled HDAC1 full-length protein was performed with

F-I depicted in (a). (e) GST pull-down was performedR8 (negative control), GST-F, GST-J, GST-HDAC2 and.


shown in Figure 4(e), HDAC1 bound to GST-HDAC2 and HDAC3 in the presence of ethidiumbromide with similar ef®ciency. Taken together,these data prove that HDAC1 can form homo-oligomers and hetero-oligomers with HDAC2 andHDAC3.

To further de®ne the HDAC association domainwithin the N terminus of HDAC1, different por-tions of the protein (constructs F-I, Figure 4(a))were expressed as GST-fusion proteins and testedwith radiolabeled full-length HDAC1 in GST pull-down assays. As depicted in Figure 4(d), GST-F(HDAC1 (1-53)) bound HDAC1 with an ef®ciencycomparable to that of the full-length protein (GST-HDAC1), while the C-terminal construct GST-H,comprising residues 303-482, showed no speci®cbinding. Constructs GST-G, GST-I and GST-Jbound only very weakly, indicating that the HADis located within the extreme N terminus of theHDAC1 protein. Splitting of the HAD into twoparts (amino acid residues 1-24 and amino acidresidues 25-53) resulted in loss of interaction withfull-length HDAC1, suggesting that the centralpart of this domain is crucial for dimerisation (datanot shown). Alternatively, the entire HAD isnecessary for HDAC1 binding.

The N-terminal HDAC association domain isrequired for enzymatic activity and interactionwith RbAp48 and Sin3A/Sin3B

The Rb-binding protein 48 (RbAp48) was co-pur-i®ed with the enzyme during the isolation ofhuman HDAC1 by af®nity chromatography using

Figure 5. Importance of the HDAC association domain (Hbinding proteins. Epitope-tagged HDAC1 wild-type proteinthe Myc-speci®c antibody 9E10 from whole-cell extracts prfrom cells transfected with a GFP plasmid were included astates was analysed for HDAC activity. The result shown issecond half of the immunoprecipitates was analysed on a WMyc epitope, Sin3A, Sin3B, RbAp48 and delta. (c) Myc-taggprotein lacking the HAD were visualised in transiently transand Texas red-conjugated anti-mouse immunoglobulin G bywith DAPI.

a modi®ed form of the deacetylase inhibitor tra-poxin (Taunton et al., 1996). Binding of RbAp48 toHDAC1 is believed to be important for correctfolding of the protein required for catalytic activityand interaction with other proteins (Zhang et al.,1999). To analyse the importance of the HAD forenzymatic activity and formation of the HDAC1-containing complexes, we deleted amino acid resi-dues 1-51 from the N terminus and transientlyexpressed the truncated Myc-tagged protein(HDAC1�N51-Myc) in HeLa cells. Myc-taggedHDAC1 wild-type protein and GFP served ascontrols. Comparable amounts of wild-typeHDAC1-Myc and HDAC1�N51-Myc were immu-noprecipitated and analysed for HDAC activityand association with HDAC1-interacting proteins.As depicted in Figure 5(a) immunoprecipitatedwild-type HDAC1-Myc (wt) displayed robustenzymatic activity, whereas HDAC1�N51-Mychad only background activity comparable to theimmunoprecipitate from green ¯uorescence protein(GFP)-expressing cells. Furthermore, the HDAC1protein lacking the HAD showed strongly reducedinteraction with RbAp48 and complete loss ofassociation with Sin3A and Sin3B (Figure 5(b)). Incontrast, full-length HDAC1-Myc protein wasfound associated with RbAp48, Sin3A and Sin3B.To test the structural integrity of the mutant pro-tein, we probed the same blot with antibodiesspeci®c for a novel HDAC1-interacting proteinnamed delta. This factor was identi®ed recently inour laboratory in a two-hybrid screen and requiresthe C-terminal half of HDAC1 for association (V.K.& C.S., unpublished results). As shown in

AD) for enzymatic activity and interaction with HDAC1-(wt) and HDAC1�N51 were immunoprecipitated with

epared from transiently transfected HeLa cells. Extractsa negative control. (a) One half of the immunoprecipi-

representative of three independent experiments. (b) Theestern blot by sequential probing with antibodies for theed HDAC1 full-length protein (wt) and HDAC1 mutantfected HeLa cells with the epitope-speci®c antibody 9E10indirect immuno¯uorescence. Nuclear DNA was stained


Figure 5(b), both wild-type and mutant HDAC1-Myc interact with delta, suggesting thatHDAC1�N51-Myc has, at least in part, preservedits structure.

Previous reports have demonstrated thatHDAC1 is a nuclear protein (Taunton et al., 1996;Bartl et al., 1997). To exclude the possibility thatthe lack of interaction with Sin3A and Sin3B is dueto mistargeting of HDAC1�N51-Myc, we analysedthe intracellular localisation of the mutant proteinby indirect immuno¯uorescence analysis of transi-ently transfected HeLa cells. As shown inFigure 5(c), HDAC1�N51-Myc is localised in thenucleus, indicating that removal of the N-terminalHAD was without consequence for the recognitionof HDAC1 by the nuclear import machinery (com-pare to Figure 7(c), upper panel).

Nuclear localisation of HDAC1 depends on alysine-rich motif within the C terminus

Next, we intended to identify the HDAC1domain required for nuclear localisation of theenzyme. The import of proteins into the nucleus isusually linked to the presence of nuclear localis-

ation signals that are recognised by the importincomplex (Nigg, 1997). Recently, the C terminus ofXenopus HDAC1 was found to be important fornuclear localisation of the enzyme (Ryan et al.,1999; Vermaak et al., 1999). The lysine-rich C-term-inal region of mammalian HDAC1 comprises amotif with homology to part of the c-myc NLS(Figure 6(a); Dang & Lee, 1988). A similar sequenceis present in the C terminus of HDAC2, whileHDAC3 is lacking most of the correspondingC-terminal domain (see Figure 1). To test the func-tion of this sequence as nuclear localisation signal(NLS), we examined the intracellular localisation ofN-terminally HA-tagged HDAC1 full-lengthprotein (HA-HDAC1) and the corresponding epi-tope-tagged protein lacking the last 45 C-terminalamino acid residues (HA-HDAC1�C45) in retrovi-rally infected Swiss 3T3 cells. As shown inFigure 6(b), HA-HDAC1 was predominantly loca-lised in the nucleus while deletion of the C termi-nus of HA-HDAC1 resulted in a cytoplasmicdistribution of HA-HDAC1�C45. This result indi-cates that the C-terminal domain is required forthe nuclear localisation of HA-HDAC1.

Figure 6. Characterisation of theC-terminal nuclear localisationdomain of HDAC1. (a) Alignmentof the nuclear localisation domainof HDAC1 (residues 438-482) con-taining the core motif (residues438-445, underlined) with hom-ology to the human c-myc NLS(residues 335-343). (b) Deletion ofthe C-terminal 45 residues resultsin cytosolic localisation of HA-HDAC1. Full-length HA-HDAC1and HA-HDAC1�45 wereexpressed in Swiss 3T3 cells by ret-roviral infection and visualisedwith anti-HA antibody and Texasred-conjugated anti-mouse immu-noglobulin G. The nuclear DNAwas stained with DAPI. (c) The Cterminus of HDAC1 is suf®cient fornuclear localisation of the cytosolicMyc-tagged mouse thymidinekinase (TK). Myc-TK and Myc-TK�45 were detected in retrovi-rally infected Swiss 3T3 cells withmonoclonal antibody 9E10 andTexas red-conjugated anti-mouseimmunoglobulin G, respectively.(d) The HDAC1 core NLS(KKAKRVKT) mediates nuclearlocalisation of green ¯uorescenceprotein (GFP). GFP or GFP-NLSwere transiently expressed in Swiss3T3 cells and visualised by ¯uor-escence microscopy (Zeiss Axiovert135TV).


To test whether this sequence is suf®cient fornuclear import, we fused residues 438-482 ofHDAC1 to the cytosolic protein thymidine kinase(TK). As shown in Figure 6(c) the TK-HDAC1fusion protein (Myc-TK�45) was localised inthe nucleus, while wild-type thymidine kinase(Myc-TK) was found in the cytosol of transfectedSwiss 3T3 ®broblasts. These data indicate that theC terminus (amino acid residues 438-482) ofmammalian HDAC1 is suf®cient for nuclearlocalisation.

An eight amino acid residue HDAC1 corenuclear localisation signal targets GFP proteinto the nucleus

Next, we asked whether the short peptidesequence with homology to part of the c-myc NLScould serve as a core NLS. To this end, we fusedthe peptide KKAKRVKT to the C terminus of theCFP to form GFP-NLS. Indeed, GFP-NLS was con-centrated in the nucleus of transiently transfectedSwiss 3T3 cells compared to cells transfected withGFP alone (Figure 6(d)). Therefore the amino acidsequence KKAKRVKT represents the core signalfor nuclear localisation of HDAC1.

The N-terminal HA-epitope interferes withenzymatic activity and oligomerisationof HDAC1

An HDAC1 full-length protein with a C-terminalMyc-tag displays enzymatic activity (Bartl et al.,1997) and is able to oligomerise (Figure 3).However, assays with N-terminally HA-taggedHDAC1 protein (HA-HDAC1) yielded completelydivergent results, as summarised in Figure 7(d).Immunoprecipitated HA-HDAC1 displayed onlybackground activity, while comparable amounts ofHDAC1-Myc (see Figure 7(b)) had signi®cantHDAC activity (Figure 7(a)). We concluded thatthe N-terminal HA-tag interferes with deacetylaseactivity.

Since HDAC1 point mutants (H28A, H68A,D174H) were still associated with residual activitydue to the interaction with endogenous HDAC1(see Figure 3(a)), we next asked if the N-terminalepitope also affects the ability of HDAC1 to oligo-merise. To this end, we immunoprecipitated HA-HDAC1 with HA-speci®c antibody and analysedthe immunoprecipitate on Western blots. Indeed,HA-HDAC1 failed to interact with endogenousHDAC1, as indicated by the absence of the faster-migrating endogenous HDAC1 in the HA-immu-noprecipitate probed with the HDAC1-speci®cantiserum (Figure 7(b)). In contrast, Myc-immuno-precipitates of HDAC1-Myc contained endogenousHDAC1, as indicated by the slightly faster-migrating band (indicated by an asterisk) inWestern blot analysis with the HDAC1-speci®cantiserum (Figure 7(c), and see Figure 3(b)). Thesedata suggest that the N-terminal HA-epitope inter-feres with enzymatic activity and oligomerisation.

HDAC1 can be imported into the nucleus inthe absence of its nuclear localisation domain

As shown in Figure 3, HDAC1 oligomerisationleads to association of active endogenous enzymewith inactive HDAC1 mutants. Consequently, weasked wether HDAC1 self-association could con-tribute to the nuclear import of HDAC1. To answerthis question, we chose HDAC1-Myc as a model.As shown here, HDAC1-Myc is enzymaticallyactive (Figure 3(a)), able to oligomerise (Figure 3(b))and localised exclusively in the nucleus of trans-fected Swiss 3T3 cells (Figure 7(c), upper panel).Deletion of the nuclear localisation domain (aminoacid residues 438-482) from HDAC1-Myc(HDAC1�C45-Myc) had only a mild effect on itsintracellular localisation. As shown in the middlepanel of Figure 7(c), a large fraction ofHDAC1�C45-Myc remained transported into thenucleus. Only a relatively small fraction of theprotein was found in the cytosol. This ®ndingis in contrast to the results obtained for theHA-HDAC1�C45 protein, which was foundpredominantly in the cytosol (Figure 6(b)).

We rationalised that association ofHDAC1�C45-Myc with endogenous HDACsmight allow the ef®cient nuclear translocation inthe absence of the NLS. Along this line, the corre-sponding HA-tagged mutant HA-HDAC1�C45remained in the cytosol due to its inability to oligo-merise. To test this hypothesis, we deleted the N-terminal domain from HDAC1�C45-Myc andexpressed the resulting polypeptide (HDAC1(131-437)-Myc) in Swiss 3T3 ®broblasts. Indeed,HDAC1(131-437)-Myc, lacking both the nuclearlocalisation domain and the HAD, was localised inthe cytosol (Figure 7(c), lower panel). Our dataindicate that nuclear import of HDAC1 can beaccomplished by two structural features: by aC-terminal nuclear localisation signal or by theN-terminal HAD, which enables the protein tointeract with another HDAC1 containing such anNLS.

Discussion

In this work, we have characterised two func-tional domains of the murine histone deacetylaseHDAC1: the N-terminal HDAC association domain(HAD) and the C-terminal nuclear localisationdomain containing a core NLS.

Most of the mammalian class I and class IIHDACs were identi®ed as histone deacetylatingenzymes due to their homology with humanHDAC1 or the related yeast protein HDA1,respectively. Therefore it is not surprising that allthese proteins share a highly conserved catalyticdomain. Nearly two-thirds of the HDAC1 proteinsequence has signi®cant similarity with otherHDACs. Strikingly, this homology is shared alsowith prokaryotic enzymes, strongly suggestingthat a large part of the enzyme, the acuC/APChomology domain (HDAC1, residues 25-303), is

Figure 7. Divergent effects of an N-terminal HA-tag and a C-terminal Myc-tag on histone deacetylase activity,oligomerisation and nuclear shuttling of HDAC1. (a) Speci®c activity of immunoprecipitated HA-HDAC1 andHDAC1-Myc. Epitope-tagged HDAC1 was immunoprecipitated from 500 mg of total protein with the respective anti-bodies. Immunoprecipitates (IP) and whole-cell extracts (inputs) were analysed in HDAC enzyme assays. (b) HA-HDAC1 fails to interact with endogenous HDAC1. HA-HDAC1 and HDAC1-Myc immunoprecipitates with the epi-tope-speci®c antibodies and input extracts were analysed on Western blots. Epitope-tagged protein and co-immuno-precipitated endogenous HDAC were detected with the respective epitope-speci®c antibody or with HDAC1antiserum as indicated (W). Endogenous HDAC1 protein is indicated by an asterisk (*). HA-tagged HDAC1 migratesslightly slower than HDAC1 protein with the Myc-epitope. (c) Nuclear shuttling of HDAC1-Myc occurs in theabsence of the nuclear localisation domain, but requires the presence of the HDAC association domain (HAD) ofHDAC1. Myc-tagged HDAC1 proteins were visualised in stably infected Swiss 3T3 cells with antibody 9E10 andTexas red-conjugated anti-mouse immunoglobulin G by indirect immuno¯uorescence. Nuclear DNA was stainedwith DAPI. (d) Schematic overview on properties of HDAC1 proteins used in this work. The HDAC associationdomain (HAD) and the nuclear localisation domain (NLD) are highlighted in light grey. The data for enzymaticactivity and oligomerisation of HDAC1�45-Myc, HDAC1(131-435)-Myc and HA-HDAC1�45 are not shown.


essential for enzymatic function (Khochbin &Wolffe, 1997; Ladomery et al., 1997; Leipe &Landsman, 1997). In agreement with this hypoth-esis, mutations of the highly conserved residuesH28 and H68 of HDAC1 outside the previouslydescribed deacetylase consensus motif (HDAC1,residues 140-208; Hassig et al., 1998) lead to signi®-cant loss of enzymatic activity (Figure 2). However,HDAC proteins mutated in conserved residues arefrequently found to display residual enzymaticactivity (Hassig et al., 1998; Miska et al., 1999; andthis study). This observation has now been clari-®ed by the ®nding that HDACs can oligomerise.

We show here that in addition to HDAC2(Hassig et al., 1998), HDAC1 itself and HDAC3 canassociate with HDAC1. The binding is most prob-ably direct and requires the presence of a con-

served HDAC association domain (HAD) withinthe N terminus of HDAC1. The HAD seems tohave a more general function, since its deletionabolishes both enzymatic activity and associationwith the HDAC1-interacting proteins RbAp48 andSin3A/B. The idea that the N-terminal domain ofHDAC1 is of special importance is supported bythe recent ®nding that the adenovirus proteinGAM1 can interfere with HDAC activity bybinding to the HAD (S. Chiocca, V.K., C.S. &M. Cotten, unpublished results).

HDAC1 and HDAC2 are frequently found in thesame complexes (reviewed by Ayer, 1999; Cress &Seto, 2000; Ng & Bird, 2000), while HDAC3 isusually associated with HDAC4 and HDAC5(Grozinger et al., 1999). In accordance with thesereports, we found HDAC2, but not HDAC3 in


HDAC1-immunoprecipitates from HeLa cellextracts (data not shown). However, HDAC3 wasreported to associate with HDAC1 in some celllines, providing additional biological support forthe HAD-dependent in vitro interaction of the twoproteins described here (discussed by Grozingeret al., 1999). In contrast, HDAC6 contains two cata-lytic domains and does not associate with otherclass I or class II HDACs (Grozinger et al., 1999).These data suggest that in vivo HDAC proteincomplexes contain at least two catalytic domains.It is possible that the number of active centres percomplex is even higher, since chicken HDAC waspuri®ed as a tetramer (Li et al., 1996). The compo-sition of homo and hetero-oligomeric HDAC com-plexes seems to be determined, at least in part, bycell type-speci®c expression patterns of theenzymes but could be a potential target for theregulation of HDAC function. Recently, HDAC1/2complexes have been characterized in HeLa cells(Humphrey et al., 2001). The presence of distinct aswell as common HDAC1 and HDAC2 high mol-ecular mass complexes suggests distinct regulatoryfunctions of these enzymes.

Like all known mammalian class I histone deace-tylases, HDAC1 shows predominantly nuclearlocalisation. The C terminus is rich in chargedamino acids, and is necessary and suf®cient fornuclear import of the protein in the absence of theHAD. The lysine-rich, eight amino acid residuemotif KKAKRVKT with similarity to the c-mycNLS (Dang & Lee, 1988) is suf®cient for the trans-port of GFP into the nucleus. The HDAC1 NLS isnot conserved among all class I histone deacety-lases. While HDAC2 has a basic amino acid stretchwithin the corresponding protein region, HDAC3lacks most of the C terminus (Figure 1). Therecently identi®ed new class I family memberHDAC8 seems to have a different NLS in the cen-tral domain of the protein (Hu et al., 2000). How-ever, our data show that a different mechanism,the interaction with another HDAC molecule, canbe employed for the nuclear import of HDAC1.Deletion of the nuclear localisation domain reducesbut does not fully abolish nuclear import ofHDAC1. Removal of both the HAD and the NLSin HDAC1(131-435)-Myc retains the enzyme in thecytosol. The fact that HDAC1 (and probably otherrelated HDACs) is capable of shuttling an HDAC1protein lacking the nuclear localisation domain ef®-ciently into the nucleus suggests a potential mech-anism for nuclear import of HDAC1-associatedproteins and HDAC molecules lacking an NLSsequence.

In contrast to the class I enzymes, class IIHDACs are located either in the cytoplasm or inthe nucleus, indicating their ability to shuttlebetween these compartments (Grozinger &Schreiber, 2000; Miska et al., 1999; Verdel et al.,2000). HDAC4 and HDAC5 were found to interactwith members of the MEF2 transcription factorfamily, suggesting a role of class II enzymes duringmuscle differentiation (Lemercier et al., 2000; Miska

et al., 1999; Wang et al., 1999). Phosphorylation-dependent association with the cytosolic anchorprotein 14-3-3 results in sequestration of HDAC4and HDAC5 to the cytosol (Grozinger & Schreiber,2000). Recently, the HDAC6 protein was shown tocontain a nuclear export signal that is essential forexclusion of the enzyme from the nucleus (Verdelet al., 2000). The shuttling of HDAC enzymes couldprovide an additional mechanism for the regu-lation of speci®c nuclear HDAC activities. On theother hand, the presence of HDAC enzymes in thecytosol might have a physiological role in targetingacetylated proteins outside the nucleus.

In this context, it is interesting to note that Swiss3T3 cells stably expressing HDAC1 lacking thenuclear localisation domain (HDAC1�C45) showdelayed cell-cycle progression (J. T. & C. S., unpub-lished results). This phenotype is different fromthat observed in ®broblasts overexpressing full-length HDAC1. As described (Bartl et al., 1997),HDAC1 overexpressing ®broblasts show increased4C DNA content, suggesting cell-cycle delayduring the G2/M phases. In contrast, expression ofan enzymatically active HDAC�C45 protein inSwiss 3T3 cells results in a prolonged S phase. Cur-rently, we are investigating potential targets ofHDAC�C45 and the phenotype of HDAC�45expressing ®broblasts in more detail.

Materials and Methods

Cell culture and cell transfection/infection

Swiss 3T3 ®broblasts, HeLa cells and the packagingcell line BOSC23 were grown in Dulbecco's modi®edEagle's medium supplemented with 10 % (v/v) foetalcalf serum. Swiss 3T3 cells were infected with retroviralsupernatants obtained from BOSC23 cells transientlytransfected with pBABE vectors as described (Bartl et al.,1997). Cells were selected for ®ve days in medium sup-plemented with 5 mg/ml puromycin and puromycin-resistant clones (50-100 per dish) were pooled for furtheranalysis. Swiss 3T3 ®broblasts and HeLa cells were tran-siently transfected using DAC-30 transfection reagent(Eurogentech, Seraing, Belgium) according to the manu-facturer's instructions.

Plasmid construction

Deletions and mutations within the HDAC1 codingregion were generated by standard PCR methods.Speci®c information concerning the oligonucleotidesequences is available upon request. The resulting PCRfragments were cloned into the retroviral vectorspBABE-Puro or pBABE-Hygro (Morgenstern & Land,1990) for mammalian expression, the pBluescriptSK vec-tor (Stratagene) for in vitro transcription/translation inreticulocyte lysates and the pGEX-4T1 plasmid forexpression as GST-fusion proteins in E. coli. To constructthe thymidine kinase-HDAC1 fusion protein Myc-TK � 45, a PCR fragment encoding the C-terminal partof HDAC1 (residues 438-482) was cloned in-frame intothe vector pSVLTK�30 (Sutterluety et al., 1996), whichencodes a C-terminal thymidine kinase deletion mutantwith an N-terminal Myc-tag (Myc-TK). The resultingprotein was named Myc-TK�45. The GFP-NLS protein


was obtained by fusing the peptide KKAKRVKT (resi-dues 438-445 of mouse HDAC1) to the C terminus ofGFP. To this end, a corresponding double-stranded oli-gonucleotide was ligated into the BglII/BamHI-digestedpEGFP-C vector (Clontech), giving pEGFP-NLS.

Glutathione S-transferase pull down assays

For generation of GST-HDAC fusion proteins, the cod-ing sequences for murine HDAC1, HDAC2 and humanHDAC3 were ligated into BamHI/EcoRI-cut pGEX-2TK.Lysis of the bacteria, puri®cation of the recombinant pro-teins and binding to the glutathione beads was done asdescribed (Doetzlhofer et al., 1999). Beads coated withGST-HDAC1, GST-HDAC2, GST-HDAC3 or GST alonewere incubated in lysis buffer (20 mM Tris-HCl (pH 8.0),100 mM NaCl, 1 mM EDTA, 0.5 % (v/v) Nonidet P-40,1 mM PMSF, 2 mM DTT, Complete Protease InhibitorCocktail) with in vitro translated [35S]methionine-labelledHDAC1 fragments for two hours. After three washes inlysis buffer and one wash in RIPA buffer (150 mM NaCl,1 % (v/v) Nonidet P-40, 0.5 % (w/v) sodium deoxycho-late, 0.1 % (w/v) SDS, 50 mM Tris-HCl, pH 8.0), boundproteins were eluted by boiling in SDS-PAGE loadingbuffer, resolved by electrophoresis, and visualised byexposure to X-ray ®lms.

Protein isolation and immunoprecipitation

Whole-cell protein extraction and immunoprecipita-tion experiments were performed as described(Adamczewski et al., 1993; Bartl et al., 1997). Brie¯y,equal amounts of protein were incubated in 200 ml ofextraction buffer (20 mM Tris-HCl (pH 8.0), 100 mMNaCl, 1 mM EDTA, 0.5 % (v/v) Nonidet P-40, 1 mMPMSF, 2 mM DTT, Complete Protease Inhibitor Cocktail)with the respective antibody for one hour at 4 �C. Afterthe addition of 20 ml of protein A/protein G-Sepharosebead suspension (10 % v/v, Pharmacia) the mixture wasfurther incubated with gentle agitation for 12 hours at4 �C. Immunoprecipitation of HA-tagged proteins wasperformed with HA-antibody cross-linked to protein A-Sepharose. After three washes with extraction buffer, thebeads were resuspended in 60 ml of extraction buffer. A20 ml aliquot of the suspension was examined for proteinexpression on Western blots and the remaining 40 mlwas assayed for histone deacetylase activity (see below).

Immunoprecipition and detection of HA-tagged andMyc-tagged proteins were performed with the mono-clonal sera for the hemagglutinin epitope (12CA5 and16B12) and for the Myc-epitope (9E10), respectively.HDAC1 was immunoprecipitated and visualised onWestern blots with a polyclonal rabbit antibody raisedagainst a recombinant mouse HDAC1 polypeptide(Upstate Biotechnology).

Histone deacetylase assays

Histone deacetylase assays were done as described(Bartl et al., 1997; Lechner et al., 1996). To measure enzy-matic HDAC activity, equal amounts of protein (10 mgwhole-cell extract) or immunoprecipitated HDAC1 pro-tein (40 ml of a total volume of 60 ml, see above) wereincubated with 10 ml of [3H]acetate-labelled chickenerythrocyte histones in a total volume of 50 ml for onehour at 30 �C. The reaction was stopped by the additionof 36 ml of 1 M HCl, 0.4 M acetate and extracted with800 ml of ethyl acetate. After centrifugation at 8400 g for

®ve minutes, a 600 ml aliquot of the organic phase wascounted in 3 ml of liquid scintillation cocktail.

Western blot analysis andindirect immunofluorescence

The following antibodies were used for Western blotanalysis: HDAC1 (Upstate Biotechnology), RbAp48(Upstate Biotechnology), Sin3A and Sin3B (both SantaCruz), Myc (9E10), HA (16B12) and delta (V. K. & C. S.,unpublished results). Proteins were resolved on SDS/10 % polyacrylamide gels. Gels were blotted onto nitro-cellulose membranes and HDAC1 was detected by Wes-tern blot analysis using an ECL kit (NEN). Subcellularlocalisation of HDAC1 protein was determined by indir-ect immuno¯uorescence. Cells were ®xed in 1 % (v/v)paraformaldehyde for ®ve minutes and permeabilised in2 % paraformaldehyde, 0.1 % (v/v) Triton for 15 minutes.Cells were incubated overnight with the monoclonalanti-HA antibody 16B12 or anti-Myc 9E10 antibody, andthe epitope-tagged protein was detected with a TexasRed-conjugated anti-mouse IgG secondary antibody(Accu-Specs) in a Zeiss Axiovert 135TV microscope.Nuclear DNA was visualised with 4',6-diamidino-2-phenylindole (DAPI).

Acknowledgements

We are grateful to E. Seto for providing HDAC2 andHDAC3 expression vectors, to W. Stockinger for theGST-LR8 protein, to A. Matejowics for valuable technicalhelp and to E. Ogris for the HA and Myc antibodies. Wethank M. Rembold, B. Schuettengruber, G. Lagger andan anynomous reviewer for helpful comments, and M.Cotten for critical reading of this manuscript. This workwas supported by FWF project P13638-GEN and AntonDreher-Gedaechtnisschenkung grant 308-1997.

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Edited by J. Karn

(Received 14 August 2000; received in revised form 22 February 2001; accepted 22 February 2001)

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Homo-oligomerisation and nuclear localisation of mouse histone deacetylase 1