Histone deacetylases in fungi: novel members, new facts

Histone deacetylases in fungi: novel members,new factsPatrick Trojer, Eva M. Brandtner, Gerald Brosch, Peter Loidl, Johannes Galehr,

Roland Linzmaier, Hubertus Haas, Karin Mair, Martin Tribus and Stefan Graessle*

Department of Molecular Biology, University of Innsbruck, Peter-Mayr-Strasse 4b, Innsbruck, A-6020, Austria

Received April 7, 2003; Revised and Accepted May 15, 2003 DDBJ/EMBL/GenBank accession nos+

ABSTRACT

Acetylation is the most prominent modi®cation oncore histones that strongly affects nuclear pro-cesses such as DNA replication, DNA repair andtranscription. Enzymes responsible for the dynamicequilibrium of histone acetylation are histoneacetyltransferases (HATs) and histone deacetylases(HDACs). In this paper we describe the identi®cationof novel HDACs from the ®lamentous fungiAspergillus nidulans and the maize pathogenCochliobolus carbonum. Two of the enzymes arehomologs of Saccharomyces cerevisiae HOS3, anenzyme that has not been identi®ed outside of theestablished yeast systems until now. One of thesehomologs, HosB, showed intrinsic HDAC activityand remarkable resistance against HDAC inhibitorslike trichostatin A (TSA) when recombinant expres-sed in an Escherichia coli host system. Phylo-genetic analysis revealed that HosB, together withother fungal HOS3 orthologs, is a member of aseparate group within the classical HDACs.Immunological investigations with partially puri®edHDAC activities of Aspergillus showed that all clas-sical enzymes are part of high molecular weightcomplexes and that a TSA sensitive class 2 HDACconstitutes the major part of total HDAC activity ofthe fungus. However, further biochemical analysisalso revealed an NAD+-dependent activity that couldbe separated from the other activities by differenttypes of chromatography and obviously representsan enzyme of the sirtuin class.

INTRODUCTION

During the past years it has become clear that chromatinrepresents an important regulatory element that affects nuclearprocesses such as DNA replication, recombination, DNArepair and transcription by tuning the accessibility of DNA forvarious factors. Thereby, cells have elaborated a speci®c

machinery to remodel nucleosomes for speci®c processesoccurring in chromatin (1). In particular, core histones aresusceptible to a wide range of post-translational modi®cations,including phosphorylation, methylation, glycosylation andacetylation. Thereby, acetylation/deacetylation processes atthe e-amino groups of highly conserved N-terminal lysineresidues of H2A, H2B, H3 and H4 are the most prominentmodi®cations (2,3). Recently, the biological signi®cance ofthis modi®cation has started to emerge (4±7). Today a largebody of evidence indicates that acetylation might play a majorrole in the regulation of transcription whereby severalalternative explanations for acetylation effects are discussed(2,8±11). Enzymes responsible for the acetylation process arehistone acetyltransferases (HATs), which usually act as co-activators of transcription and are frequently associated withenhancer-binding proteins or RNA polymerase II (5,12). Thedynamic process of histone acetylation is reversed by histonedeacetylases (HDACs). The ®rst HDAC to be found was thehuman HDAC1, which showed striking sequence homology tothe already known transcriptional regulator RPD3 (reducedpotassium dependency factor) of Saccharomyces cerevisiaecon®rming a link between transcriptional regulation andhistone deacetylation (13). At that time another structurallyrelated protein with HDAC activity, HDA1, was identi®ed inyeast (14). Today RPD3/HDA1 orthologs are reported fromyeast to human and are named in a largely random manner (forreview see 15±17). However, HDACs now are categorizedinto classes according to the yeast proteins RPD3 (class 1) andHDA1 (class 2), respectively. Both classes revealed signi®cantsequence similarities predominantly in a large N-terminaldomain (18). Among RPD3-type enzymes another twoputative members of this class, HOS1 and HOS2 (HDA onesimilar), were identi®ed in yeast but have not yet beendetected as enzymatically active enzymes. However, there arestrong indications that these enzymes preferentially affectribosomal DNA and ribosomal protein genes, respectively(19). Another yeast sequence, HOS3, which is more distantlyrelated to RPD3 and HDA1 was either classi®ed as a class 2HDAC (20) or was placed between the two classes (21), butdoes not actually correlate well with either of the twocategories. Interestingly, HOS3 orthologs have been foundonly in yeast systems so far (1,22).

*To whom correspondence should be addressed. Tel: +43 512 507 3608; Fax: +43 512 507 2866; Email: [email protected]

The authors wish it to be known that, in their opinion, the ®rst two authors should be regarded as joint First Authors

+AF306859, AF307341, AF537125 and AF537126

Nucleic Acids Research, 2003, Vol. 31, No. 14 3971±3981DOI: 10.1093/nar/gkg473

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Recently, a third class of HDACs with homology to yeastSIR2 (silent information regulator) was established. HDACactivity of this enzyme class was demonstrated not only for theyeast enzyme but also for a mouse SIR2 ortholog (23).Furthermore, SIR2 was shown to possess ADP-ribosyltrans-ferase activity (24,25), which is separable from HDACactivity, although deacetylation of histones is dependent onNAD+ (25,26). This NAD+ dependence might provide a linkbetween cellular metabolism and chromatin structure andmaybe is involved in the aging of cells (for review see 27). Incontrast to the activity of class 1 and class 2 HDACs, SIR2-like proteins (sirtuins) cannot be inhibited by known HDACinhibitors, such as trichostatin A (TSA) or HC toxin. However,they are conserved among species from bacteria to human(28), but so far little information is available concerning thefunctional role and the targets of these homologs.

Another separate class of HDACs, the HD2 enzymes arenamed after the founding member, the maize HD2 (29,30).Silencing of AtHD2A expression, a HD2 ortholog inArabidopsis thaliana, resulted in aborted seed developmentin transgenic Arabidopsis plants, suggesting that the AtHD2Agene product is important in the reproductive development ofA.thaliana (31). However, enzymes of the HD2 class havebeen found only in plants so far.

Recently, we have characterized RpdA and HosA, twoclass 1 enzymes of Aspergillus nidulans, which were the ®rstHDACs identi®ed in a ®lamentous fungus (32). Shortlythereafter we also found orthologs of these enzymes in theplant pathogenic fungus Cochliobolus carbonum (33,34).Interestingly, engineered Cochliobolus mutants of one of thesegenes had smaller conidia, exhibited a ~50% reduction in totalHDAC activity, and showed a reduction in growth on certaincarbohydrates (33). Very recently, another fungal class 1HDAC was identi®ed in the corn smut fungus Ustilagomaydis, which can substitute for RPD3 in S.cerevisiae. In anU.maydis knock out strain the proliferation to matureteliospores was blocked upon karyogamy (35).

Now we report the identi®cation and molecular character-ization of another four fungal HDAC sequences of A.nidulansand C.carbonum. With high sequence similarity to yeastHDA1, two of the deduced enzymes, the A.nidulans HdaA andthe C.carbonum HDC3, are typical class 2 enzymes. The othertwo, HosB and HDC4, are the ®rst HOS3 orthologs found inmulticellular eukaryotes. Comprehensive alignments of thesefungal HDAC sequences together with the well known yeastsequences resulted in a novel phylogenetic HDAC tree.Further investigations of the Aspergillus enzymes revealedthat HosB has intrinsic HDAC activity and is resistant againstHDAC inhibitors even when expressed in a prokaryoticexpression system. However, it is not HosB but HdaA thatseems to play the major role in the deacetylation process ofhistones in A.nidulans.

MATERIALS AND METHODS

Strains and growth media

Generally, A.nidulans strain A4 (Glasgow wild type) providedfrom the Fungal Genetic Stock Center (FGSC, Kansas City,KS) and C.carbonum strain SB111 provided from theAmerican Type Culture Collection (90305) were used.

Aspergillus was grown in minimal medium according to(36) for 24 h at 37°C in shake culture; Cochliobolus wasgrown in HMT medium according to (37) for 2 days at 25°C inshake culture.

Identi®cation and characterization of the HDA1orthologs hdaA and HDC3

Total A.nidulans and C.carbonum RNA was used to preparecDNA employing Superscript reverse transcriptase (LifeTechnologies, Bethesda, MD). cDNA was used as templatefor the ampli®cation of speci®c fragments of hdaA and HDC3.Two mixed degenerated 17mer oligodeoxynucleotides wereused as primers for polymerase chain reaction (PCR). Forwardprimer HDA1 was 5¢-CCNCCNGGNCAYCAYGC-3¢ basedon the amino acid sequence PPGHHA, and reversed primerHDA2 was 5¢-GTNCCRTTNCCRTGRTG-3¢ based onHHGNGT. Both amino acid sequences descend from highlyconserved regions of class 2 HDACs (Fig. 1). Ampli®cationproducts of ~150 bp were isolated, cloned into a pGEMT-Easy vector (Promega) and sequenced.

Full-length genomic sequences of hdaA and HDC3 wereobtained by using the ampli®ed fragments as probes for thescreening of an A.nidulans chromosome-speci®c cosmidlibrary constructed in pWE15 (38) and pLORIST2 (39) anda lEMBL3 genomic library of C.carbonum DNA (40),respectively. To determine number and location of prospectedintrons, genomic sequences were compared with the corres-ponding cDNA sequences generated by reverse transcribed(RT)±PCR. The nucleotide and deduced amino acid sequencesof HdaA and HDC3 have been deposited in the GenBankdatabase (accession numbers AF306859 and AF307341,respectively).

Identi®cation and characterization of the HOS3orthologs hosB and HDC4

Blast searches with the chromosome IV-speci®c genomicdatabase (http://aspergillus-genomics.org/) of A.nidulans andthe HOS3 sequence of S.cerevisiae revealed a sequence of 45amino acids with 39% identity to the query. Based on theaccording contig (g2000Jan142006_4209) two speci®c oligo-deoxynucleotides, HOSa (5¢-CCATCTCCTGTTTCCCCC-3¢,a non-coding sequence of the promoter region of hosB), andreversed primer HOSb (5¢-GGAACGGCGGAACAGGG-3¢)based on amino acid sequence PVPPFQ (Fig. 1) were designedand used for the ampli®cation of a ~900 bp hosB fragment.The ampli®cation product subsequently was used to screen thechromosome-speci®c A.nidulans cosmid library (as describedabove) reduced to clones representing sequences of chromo-some IV (41) that led to the full-length sequence of hosB.Sequence information of hosB and yeast HOS3 allowed theconstruction of another two degenerated forward primersHDCa (5¢-GAYTTYGAYYTNCAYCA-3¢) and HDCb(5¢-GAYYTNCAYCAYGGNGA-3¢) based on amino acidsequences DFDLHH and DLHHGD and two degeneratedreversed primers HDCc (5¢-SWNGCRTCRAANCCNGC-3¢)and HDCd (5¢-ACRTTNACYTTRTGNCKYTGCAT-3¢)based on sequences AGFDAS and MQRHKVNVP (Fig. 1).Primers were used in different combinations for the ampli®-cation of a 400 bp HDC4 cDNA fragment of C.carbonum,which was used for the screening of the lEMBL3 genomiclibrary of C.carbonum (as described above) and led to the

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full-length sequence of HDC4. The nucleotide and deducedamino acid sequences of HosB and HDC4 have been depositedin the GenBank database (accession numbers AF537125 andAF537126, respectively).

Northern blot analysis

The expression of hdaA and hosB was analyzed in northernhybridization experiments according to (42). Fungi weregrown for 24 h, northern analysis was done as described earlier(32). Hybridizations were performed with 32P-labeled PCRfragments of hdaA and hosB. Results were corrected forloading and blotting of mRNA by using the g-actin gene ofA.nidulans (43) as an internal control.

Expression and puri®cation of recombinant HDACs ofA.nidulans in E.coli, production of polyclonal antibodies

The coding sequences of HdaA and HosB, but also thesequences of already identi®ed class 1 HDACs RpdA and

HosA (32) were ampli®ed from fungal cDNA. Recognitionsites for restriction enzymes were fused to the PCR primersin order to clone the ampli®ed products into a pQE 60 orpQE9 expression vector (Qiagen), respectively. Subsequently,Escherichia coli M15 cells were transformed with thegenerated recombinant expression constructs. Induction withIPTG led to the recombinant proteins with a 63 His af®nitytag on the C-(pQE 60) or the N-terminus (pQE 9) allowingpuri®cation of the fragments on an immobilized metal-ionaf®nity resin. Eluted products were detected at a wavelengthof 280 nm and puri®ed fractions were analyzed by SDS±PAGE and Coomassie brilliant blue staining (Fig. 2A and B).100 mg of puri®ed recombinant proteins were used for each ofa total of four subcutaneous injections in rabbit. After the ®nalbleed, blood was allowed to clot and serum was separatedby centrifugation at 10 000 g for 10 min at 4°C. Antibodieswere puri®ed from antiserum by protein G±Sepharoseaf®nity chromatography (prepacked HiTrap af®nity columns,

Figure 1. Multiple sequence alignment of HDA1-type HDACs and HOS3-type HDACs of A.nidulans, C.carbonum and S.cerevisiae. The alignment showsonly the conserved N-terminal parts of the enzymes. Identical amino acids within a class are boxed in pink (HDA1 class) or blue (HOS3 class), identicalamino acids between both classes are boxed in yellow. Gaps are indicated by dashes introduced for alignment purpose. Numbers indicate the amino acidposition of each protein. Small transparent circles represent amino acids that are invariant in all known HDACs of C.carbonum, A.nidulans and S.cerevisiae.Red circles indicate invariant amino acids identi®ed to play an essential role in HDAC activity. Selected primers used for PCR approaches and cloning of theenzymes derived from amino acids sequences are marked by arrows.

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Amersham Pharmacia Biotech) following the manufacturersinstructions. Speci®city of the antisera was tested byimmunoblotting using crude protein extracts of inducedE.coli expression strains. Each antiserum reacted with thecorresponding recombinant fragment; no cross reactivitybetween antisera and E.coli proteins or the other recombinantHDAC fragment was detected.

For the production of active recombinant HosB, the codingsequence of hosB was cloned into a pGEX-5X-1 expressionvector (Amersham Pharmacia Biotech). Escherichia coli

BL21 were used as host cells for the expression of HosB.Induction with 1 mM IPTG led to a soluble, active HosB fusedto the C-terminus of glutathione S-transferase (GST) ofSchistosoma japonicum (44) (Fig. 3A). Aliquots of thesupernatant of brie¯y sonicated host cells were assayed forHDAC activity with and without different concentrations ofTSA (Fig. 3B and C). A strain expressing the GST tag onlyserved as negative control.

Protein extraction

Mycelia of Aspergillus were collected and washed in asintered glass ®lter, thoroughly dried with ®lter paper and

Figure 3. Expression of recombinant HosB and analysis of HDAC activity.(A) Enzymatically active HosB was expressed as a GST-fusion protein.Coomassie staining of IPTG-induced E.coli GST-HosB cell extractsrevealed the presence of the recombinant HosB fusion protein in theexpected length (+). The recombinant protein is marked with an asterisk.No recombinant product could be detected under non-induced growth con-ditions (±). A strain expressing the GST-tag served as control for theactivity assay (Co). (B) HDAC activity in the supernatant of the correspond-ing lysed E.coli strains. Supernatant of the induced strain (+) versus notinduced cells (±) and the induced control strain (Co). (C) Inhibition ofrecombinant HosB and puri®ed HdaA obtained from the peak fraction ofFigure 4B. Inhibition of both enzymes was tested under various concentra-tions of TSA. Inhibition was calculated as a ratio of enzyme reactionscontaining the corresponding inhibitor versus no inhibitor.

Figure 2. Recombinant expression of HDACs for the production of poly-clonal antibodies in rabbit. (A) Schematic representation of HDC3, HDC4,hdaA, hosB, rpdA and hosA. Bars illustrate the open reading frames inter-rupted by introns (i). The positions of primers used for ampli®cation ofgene fragments are shown by triangles. Lines above the bars indicate thefragments with the predicted molecular weight used for over-expression ofthe Aspergillus enzymes in E.coli (see Fig. 2B and 3A). The numbers inparentheses give the molecular mass of recombinant RpdA-fragment as itactually migrates in SDS±PAGE. (B) Fifteen microliters of E.coli extractscontaining recombinant fragments of RpdA and HosB or the full-lengthproteins HdaA and HosA (1), or aliquots of the corresponding puri®edproteins (2) were subjected to SDS±10% PAGE and stained with Coomassieblue. Molecular mass of marker proteins is indicated; corresponding recom-binant products are marked by asterisks. Puri®ed recombinant products sub-sequently were used for the immunization of rabbits for the production ofspeci®c antibodies. The HdaA expression strain grown under non-inducedconditions served as negative control (Co).




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immediately frozen in liquid nitrogen. Fifteen grams of frozenmycelia were ground to powder in an IKA grinding machineand the powder was suspended in 60 ml of buffer B [15 mMTris±HCl, pH 8.0, 0.25 mM EDTA, 1 mM 2-mercaptoethanol,10% (v/v) glycerol]. The mixture was stirred on ice for 10 minand centrifuged for 30 min at 37 000 g at 4°C. The supernatantwas used for SourceQ-anion exchange-chromatography.

SourceQ-chromatography

The supernatant was incubated batchwise with 10 ml ofSource 15Q anion exchange medium (Amersham PharmaciaBiotech) for 5 h at 4°C, equilibrated with buffer B. The matrixwas pelleted by centrifugation and transferred to an FPLCcolumn (1.6 3 15 cm). Elution of proteins was performed with50 ml of a linear gradient from 10 to 500 mM NaCl in buffer Bat a ¯ow rate of 1 ml/min. Fractions of 1.5 ml were collectedand assayed for HDAC activity. Fractions with HDACactivity were pooled and concentrated to a ®nal volume of1 ml by centrifugation (2500 g, 4°C) using Centriprep YM-10(Amicon).

Size exclusion chromatography (Superdex 200)

Pooled and concentrated fractions from SourceQ-chromato-graphy were applied onto a Superdex 200 FPLC column (1.6 360 cm, 120 ml, Amersham Pharmacia Biotech), equilibratedwith 200 mM NaCl in buffer B. The ¯ow rate was maintainedat 1 ml/min, and fractions of 1.5 ml were collected and assayedfor HDAC activity. For estimation of the molecular weight ofthe native enzymes the Superdex 200 column was calibratedwith proteins of known molecular weight.

Histone deacetylase assay

HDAC activity was determined as described (45) using[3H]acetate pre-labeled chicken reticulocyte histones as sub-strate. Fifty microliter samples of the chromatography frac-tions were mixed with 10 ml of total [3H]acetate pre-labeledchicken reticulocyte histones (4 mg/ml). After incubation for1 h at 25°C the reaction was stopped by addition of 50 ml of1 M HCl/0.4 M acetate and 0.8 ml ethylacetate. Aftercentrifugation at 10 000 g for 5 min an aliquot of 600 ml of theupper phase was counted for radioactivity in 3 ml liquidscintillation cocktail. For the detection of NAD+-dependentHDAC activity, NAD+ was added to the mixture to a ®nalconcentration of 0.5 mM. The incubation temperature was30°C for 1 h.

Immunoprecipitation

For immunoprecipitation (IP) experiments 50 ml of partiallypuri®ed HDAC fractions of A.nidulans (S200-chromato-graphy) were mixed with 2 mg of af®nity puri®ed HDACantibodies, 30 ml of protein A±Sepharose, equilibrated withbuffer B, and were incubated for 2 h by permanent shaking at4°C. To avoid unspeci®c binding the mixture was adjusted to300 mM NaCl. After centrifugation for 2 min at 3000 g thesupernatant was saved for HDAC assay and protein blotting.Pellets were washed three times with 500 ml of buffer B +300 mM NaCl and ®nally resuspended in 20 ml of buffer B.Entire pellets and 50 ml of supernatants were used for standardHDAC assay. Immunoblotting precipitates were mixed withSDS sample buffer, boiled, centrifuged for 5 min at 10 000 gand the resulting supernatant was used for SDS±PAGE.

Immunoblotting

Enzyme fractions of IP experiments and fractions from theS200-chromatography were electrophoresed in precast 10%SDS Tris±Glycin polyacrylamide gels (Invitrogen, USA) at35 mA for 1.5 h at room temperature. Gels were blotted ontoHybond nitrocellulose membrane (Amersham PharmaciaBiotech) at 25 V for 2 h and membranes were blocked with2% (w/v) skim milk in phosphate-buffered saline (PBS, 20 mMat pH 7.4) for 2 h. Membrane strips were incubated with1:1000 diluted af®nity puri®ed antibodies against RpdA,HdaA, HosA and HosB in 2% skim milk in PBS at 4°Covernight. After washing, strips were incubated for 2 h withalkaline phosphatase conjugated secondary anti-rabbit Ig(Sigma) and immunodetection was performed using theBCIP/NBT Color Substrate (Promega) following themanufacturer's instructions.

Computational methods

WUBLASTP and BLASTX were used for the identi®cation ofprotein coding regions by database similarity search. Multiplealignments were produced using CLUSTALW 1.8 (46), thegraphic representation of the alignment was done with SeqVu1.1 (Garven Institute of Medical Research, Sydney, Australia)for the Macintosh Computer. Phylogenetic trees were gener-ated based on the alignment in CLUSTALW using PAUP*(version 4.0b10, Swofford/2002; Sinauer Associates, Inc.,Sunderland, UK). Preliminary sequence data from Aspergillusfumigatus were obtained from the Institute for GenomicResearch website at http://www.tigr.org. Sequence data ofNeurospora crassa were obtained from the NeurosporaSequencing Project, Whitehead Institute/MIT Center forGenome Research website at http://www-genome.wi.mit.edu.

RESULTS

Identi®cation and characterization of new HDACs ofA.nidulans and C.carbonum

Fragments of hdaA and HDC3, both members of the HDA1class, and hosB and HDC4, two HOS3 homologs, wereampli®ed from cDNA of A.nidulans and C.carbonum asdescribed in Materials and Methods.

Comparison of genomic and cDNA sequences revealedopen reading frames of 2346 (hdaA), 2541 (HDC3), 3375(hosB) and 3579 bp (HDC4), equivalent to enzymes of apredicted molecular mass of 87, 95, 121 and 128 kDa,respectively. Multiple sequence alignment of the deducedamino acid sequences with HDACs of S.cerevisiae (Fig. 1)and Schizosaccharomyces pombe (data not shown) con®rmedHdaA and HDC3 as class 2 enzymes and HosB and HDC4 asmembers of the HOS3 group, respectively.

The coding sequences of the HDA1 orthologs HDC3 andhdaA are interrupted by 7 and 14 introns (Fig. 2A),respectively, with exon/intron splice sites and intron sizestypical for ®lamentous fungi. Comparison between thecDNA and the genomic sequence revealed that the shortestexon of hdaA encodes for not more than four amino acids. Incontrast, no introns are present in the HOS3 orthologs hosBand HDC4. Sequences have been deposited in the GenBankdatabase (accession numbers are speci®ed in Table 1). Thechromosome speci®city of the Aspergillus cosmid library




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allowed the chromosomal localization of hdaA on chromo-some II, and hosB on chromosome IV, respectively.

To determine the copy number of the genes, Southern blotanalyses were performed with genomic DNA of the fungi.Blotting results showed that all genes are single copysequences (data not shown). This ®nding was supported bythe fact that sequence searches in the N.crassa genomedatabase revealed just one single contig of high similarity toeach of the Aspergillus/Cochliobolus genes (Table 1). HDACsequences of A.nidulans and C.carbonum were alignedtogether with the corresponding sequences of S.cerevisiaeand the putative orthologs of N.crassa and A.fumigatus. Anunrooted phylogenetic tree was generated based on thealignment in CLUSTALW using PAUP* version 4.0b10(Fig. 7). Sequence analysis suggests that the fungal HDACenzymes have diverged into three distinct (sub)classes. TheHOS3-type enzymes thereby show a clear distance to theRPD3 and the HDA1 group.

Transcription analysis of hdaA and hosB

To determine the transcription levels of the novel HDACgenes hdaA and hosB in A.nidulans, northern blot analyseswere performed using poly A RNA of the fungus. The detectedtranscripts corresponded to the length of the coding sequencesincluding the UTRs of the genes (data not shown). However,expression of HosB was low under used growth conditions ofthe fungus.

Recombinant expression of Aspergillus HDACs

Recombinant proteins were used to raise polyclonal antibodiesfor the identi®cation of HDACs of A.nidulans by immuno-blotting and IP assays. Recombinant HdaA and HosA, arecombinant fragment of the C-terminus of RpdA (32), and theconserved core domain of HosB (Fig. 2A) were expressed inE.coli M15 cells and puri®ed via a 63 His af®nity tag on thetermini of the fragments (Fig. 2B) as described above. Anextraordinary shift of ~20 kDa of the recombinant RpdAfragment relative to its predicted molecular weight might bedue to a speci®c property of the C-terminal part of fungalRpd3-like proteins. A similar shift was observed for HDC2,the RpdA homolog of C.carbonum (34). Finally, the puri®edrecombinant proteins were used for the production ofpolyclonal antibodies in rabbits. Since all recombinant His-tagged HDACs form inclusion bodies in the host cells, theywere solubilized for the immunization procedure under

denaturing conditions in 8 M urea. In order to attain activesoluble HosB, the coding sequence of HosB was expressed infusion with a GST tag (Fig. 2A). After induction with IPTG,signi®cant enzyme activity was detected in the supernatant ofthe cell lysate but not in the lysate of an induced control strainexpressing the GST protein only (Fig. 3A and B). Todetermine the sensitivity of this activity against HDACinhibitors, we repeated the assay under various concentrationsof TSA and HC toxin, respectively. Inhibition of recombinantHosB activity was compared with that of puri®ed HdaA of thefungus. HdaA was highly sensitive against TSA treatment(Fig. 3C), whereas HosB turned out to be remarkably resistant(84 versus 4% reduction of activity at a concentration of 30 nMTSA and 98.5 versus 33% at 300nM TSA, respectively). Evenat a TSA concentration of 1 mM HosB showed an activity of>40%. A similar resistance of the enzyme was obtained whenHC toxin was used as inhibitory reagent (data not shown).

Partial puri®cation of the Aspergillus HDACs

Protein extracts of A.nidulans shake culture mycelium weresubjected to SourceQ-anion exchange chromatography. Themain HDAC activity peak eluted at a salt concentration of270 mM NaCl (Fig. 4A). However, an additional minoractivity peak could be detected at ~180 mM when the enzymeassay was performed in the presence of 0.5 mM NAD+. ThisNAD+-dependent HDAC activity is likely to represent a SIR2homologous enzyme of Aspergillus. For further puri®cationand improved separation of the detected activities, peakfractions 14±34 of the SourceQ-chromatography were pooled,concentrated and loaded onto an S200 gel ®ltration column.The major HDAC activity eluted at ~450 kDa (Fig. 4B)indicating that the corresponding enzyme(s) obviously act(s)as high molecular mass complex(es). When the HDACactivity assay was performed in the presence of 0.5 mMNAD+, a second minor peak was detected at ~60 kDa. Tocon®rm the assumption that the main activity is due to thepresence of a class 1 or class 2 HDAC and the small peakrepresents a SIR2 homolog of Aspergillus, enzyme activityassays were repeated in the presence of TSA; HOS3 or NAD+-dependent SIR2-related enzymes are known to be largelyresistant to TSA. As expected, the HDAC activity of the majorpeak was sensitive to TSA. An inhibition of >80% wasobtained at a concentration of 30 nM TSA; 300 nMTSA resulted in complete inhibition of enzymatic activity.

Table 1. Histone deacetylases of A.nidulans and C.carbonum

Name Class Chromosome Introns GenBankAccession number

Reference Highest identity to

S.cerevisiae A.fumigatus N.crassa

RpdA 1 III 3 AF163862 (32) RPD3 Contig 322 Contig 1.91HosA 1 II 0 AF164342 (32) HOS2 Contig 22 Contig 1.1059HdaA 2 II 14 AF306859 This paper HDA1 Contig 610 Contig 1.17HosB ± IV 0 AF537125 This paper HOS3 Contig 143 Contig 1.451HDC1 1 ND 0 AF306507 (33) HOS2 Contig 22 Contig 1.1059HDC2 1 ND 1 AF349677 [J.D. Walton, unpublished] RPD3 Contig 322 Contig 1.91HDC3 2 ND 7 AF307341 This paper HDA1 Contig 610 Contig 1.17HDC4 ± ND 0 AF537126 This paper HOS3 Contig 143 Contig 1.451

Chromosomal location and intron information of the corresponding genes are given. Sequence information of the deduced proteins and the identity to HDACsof S.cerevisiae and putative HDAC sequences of N.crassa and A.fumigatus are shown on the right columns of the table. The ®rst four enzymes are fromA.nidulans, the latter are from C.carbonum. (ND) not determined.




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However, no inhibition by TSA was observed for the lowmolecular weight NAD+-dependent enzyme activity (Fig. 4B).

Immunological identi®cation of the enzymes

In order to further characterize the partially puri®ed enzymecomplex(es) of the major HDAC peak of Aspergillus, fractionsof the S200 chromatography were subjected to immuno-blotting with antibodies against RpdA, HosA, HdaA and

HosB. Whereas anti-HosB antibodies did not reveal animmunosignal, anti-HdaA antibodies detected a protein bandat an apparent molecular weight of ~98 000, which is inaccordance with the predicted molecular weight. The intensityof the immunosignal exactly corresponded with the pattern ofHDAC activity in the S200 fractions (Fig. 5A). Incubation ofthe same blots with anti-RpdA antibodies yielded signals atmolecular weights of ~120 000 and 100 000. Although thepredicted molecular weight of RpdA is 75 000 the enzymemigrates at a much higher apparent molecular weight inSDS±PAGE. This ®nding was substantiated by the unusualelectrophoretic mobility of the C-terminal recombinant RpdAfragment (Fig. 2). Furthermore, we recently observed a similarshift for the recombinant HDC2, the RpdA ortholog ofC.carbonum (34). However, the maximum intensity of theimmunosignal in fraction 31 did not exactly correspond to theHDAC activity with its maximum in fraction 35 but wasshifted to the left end (higher molecular weight) of the activitypeak (Fig. 5C). When the blots were probed with antibodiesagainst HosA, a faint band at a molecular weight of ~55 000could be detected, which corresponds to the predictedmolecular weight of the HOS2 ortholog. In the case ofHosA, the maximum of intensity was shifted to the right end(lower molecular weight) of the activity peak (Fig. 5B).

These ®ndings suggest that HDACs of A.nidulans elute ascomponents of high molecular mass complexes of ~400±500 kDa and that the major part of total enzyme activity is dueto HdaA.

Imunoprecipitation experiments

To rule out the possibility that further (still unidenti®ed)HDACs signi®cantly contribute to HDAC activity, IP experi-ments were performed. Peak fraction 35 was incubatedwith puri®ed HdaA antibodies and immunocomplexes were

Figure 4. Puri®cation of HDAC complexes of A.nidulans. (A) Elution pro-®le of a Source 15Q anion exchange column of fungal protein extract.Elution was performed with 60 ml of a linear gradient from 10 to 500 mMNaCl in buffer B at a ¯ow rate of 1 ml/min. Fractions of 1.5 ml were col-lected and assayed for HDAC activity with (triangles) and without (squares)0.5 mM NAD+. Salt concentrations of eluting peak fractions are indicated.Fractions used for size exclusion chromatography are bold. (B) Size exclu-sion chromatography (Superdex 200). SourceQ chromatography peak frac-tions were concentrated and applied to an FPLC column equilibrated with0.2 M NaCl in buffer B. The ¯ow rate was 1 ml/min, fractions of 1.5 mlwere collected and assayed for HDAC activity with (triangles) and without(squares) 0.5 mM NAD+ and 300 nM TSA, respectively. Estimated nativesize of complexes are indicated. Fractions used for IP experiments withspeci®c antibodies against RpdA (fraction 31), HdaA (fraction 35) andHosA (fraction 39) are bold.

Figure 5. Immunological identi®cation of HDACs of A.nidulans. Aliquotsof peak fractions 27±43 of the size exclusion chromatography were sub-jected to SDS±PAGE with subsequent blotting onto nitrocellulose mem-branes. Speci®c antibodies against HdaA, HosA and RpdA were used forimmunodetection. Fraction of the activity peak is underlined, fractions withthe maximum amount of each HDAC used for IP are bold. Molecularweights of the detected proteins are indicated.




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precipitated using protein A±Sepharose. Supernatant andpellet were tested for HDAC activity and subjected toimmunoblotting. HDAC activity was quantitatively precipit-ated by HdaA antibodies with a concomitant depletion of theprotein from the supernatant (Fig. 6A). In a control reactionusing protein A±Sepharose without antibody, neither deple-tion of HDAC activity from the supernatant nor HdaAprecipitation was observed. Obviously, HdaA contributes thevast majority of total HDAC peak activity.

IP experiments were also performed with fraction 31containing the major portion of RpdA and fraction 39containing the major portion of HosA using the correspondingRpdA and HosA antibodies. In the case of fraction 31, 45% oftotal HDAC activity was precipitated (Fig. 6B). Immunoblotswith anti-RpdA antibody revealed that RpdA was quantita-tively precipitated and removed from the supernatant.Additionally, subsequent immunoblotting of the same IPwith anti-HdaA antibody revealed a HdaA signal in thesupernatant but not in the pellet (data not shown), indicatingthat the remaining enzyme activity in the supernatant derivesfrom the HDA1 ortholog HdaA. This assumption is supportedby an HdaA immunosignal in fraction 31 of the S200Superdex chromatography (Fig. 5A).

In the case of fraction 39, no signi®cant HDAC activitycould be precipitated with the anti-HosA antibody (data notshown). Immunoblotting with anti-HdaA antibodies revealedHdaA in the supernatant; this portion of HdaA together withthe corresponding enzyme activity could be precipitated withanti-HdaA antibody (data not shown). Obviously, the bulk ofHDAC activity of fractions 37±41 is due to the activity ofHdaA and, under the employed experimental conditions,HosA plays a minor role as an active enzyme in the fungus.

DISCUSSION

The enzymes

Since the ®rst HDAC was identi®ed in 1996 (13) the numberof newly identi®ed HDAC enzymes in a wide spectrum oforganisms has rapidly increased. Within the past 2 years thenumber of human class 1 and class 2 HDACs rose from 5 to 11(47). In addition to these classical enzymes, two classes ofstructurally unrelated HDACs have been identi®ed: theNAD+-dependent sirtuins (48) and the plant speci®c HD2class (16). HOS3 of S.cerevisiae was the founding member ofanother group of classical enzymes that have been character-ized only in yeast systems as yet (22,49). Here we report the®rst identi®cation of HOS3 members from multicellulareukaryotes, from the ®lamentous fungi A.nidulans andC.carbonum. Furthermore, we found putative HOS3 orthologsin the genomic sequence of the fungi A.fumigatus andN.crassa but not in other eukaryotic organisms. These ®ndingsled to the conclusion that HOS3-type enzymes are not uniquefor yeast (as assumed) but may be a peculiarity of fungi,similar to the HD2 class in plants. Understanding the functionof HOS3 orthologs in fungi will require further studies.However, a HOS3-de®cient yeast mutant displayed elevatedacetylation at lysines 5, 8 and 12 of histone H4 (49).Furthermore, yeast HOS3 has intrinsic enzymatic activityand is largely insensitive towards known HDAC inhibitors. Inparticular, the latter represents a remarkable feature that is

very unusual for class 1 and class 2 HDACs. Here we coulddemonstrate intrinsic activity and resistance against inhibitorsfor HosB, which con®rms its af®liation to the HOS3 group.Despite conspicuous sequence similarities and the analogicalbiochemical properties between HOS3 and the orthologspresented in this paper some differences exist as well. HDC4and HosB are among the largest HDACs identi®ed so far. Withmolecular weights of 128 000 and 121 000 HDC4 and HosBare considerably larger than HOS3 (79 000). The extra-ordinary size of these enzymes was substantiated by the searchfor putative HOS3 homologs in A.fumigatus and N.crassa

Figure 6. IP of HDACs of A.nidulans. Fifty microliters of fraction 35 or 31of the S200 size exclusion chromatography was incubated with 2 mg ofimmunopuri®ed antibodies against (A) HdaA or (B) RpdA together with30 ml of protein A±Sepharose, equilibrated with buffer B. Incubation wasperformed under continuous shaking at 4°C for 2 h. After centrifugation,the supernatant (S) was used for the HDAC assay and immunoblotting.Pellets (P) were washed three times and ®nally resuspended in 20 ml ofbuffer B. The entire pellet and 50 ml of supernatant was used for theHDAC-assay. (Co), Control: incubation was performed without speci®cantibody but with protein A±Sepharose, (In) input. For immunoblotting,20 ml of supernatant and the entire pellet were applied to SDS±PAGE withsubsequent immunoblotting using anti-HdaA and anti-RpdA antibodies.Molecular weight of detected proteins is indicated. Data represent results ofthree independent experiments (A + B).




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(Fig. 7) that also revealed predicted molecular weightsbetween 120 000 and 130 000. In contrast to its yeastortholog, HosB apparently did not contribute signi®cantly tothe total HDAC activity of A.nidulans. From our point of viewthere are several possible explanations for the apparent lack ofin vivo activity of the enzyme. (i) HosB may, when associatedwith speci®c protein complexes in vivo, only be active withintact chromatin but may be enzymatically inactive in anin vitro assay using isolated core histones as substrate. (ii) Avery low expression rate of the enzyme may prevent thedetection of the enzyme and its activity. This assumption issupported by the fact that northern analysis revealed a ratherweak hosB transcript under the used growth conditions.HOS3-type proteins of ®lamentous fungi may represent a kindof resistant safeguard enzymes that are upregulated and/orbecome catalytical active in cases when other HDAC com-plexes are disturbed or inhibited. Very recently, this wassuggested for a putative inhibitor-insensitive HDAC activityof C.carbonum (34,50). (iii) HosB may be more susceptible todegradation processes than other HDACs and thereby mightbe lost under the used chromatographically puri®cationprocedures.

In contrast to HosB and HDC4, HdaA and HDC3 havemolecular weights that correspond well to the yeast counter-part HDA1 (80 000±95 000). HDA1 proteins are characterizedby a large proportion of identical regions in the middle part ofthe sequence with an absolute conservation of amino acids thatare essential for catalytic activity (see Fig. 1). The exon/intron

structure of the HDC3 and hdaA gene is remarkable. HDC3has 7 introns, hdaA has 14, which is, to our knowledge, thehighest number of introns that ever has been reported for afungal gene. Interestingly, the shortest exon in the hdaA geneencodes only four amino acids.

The activity

One central claim of this paper is that HdaA and RpdA aremajor contributors to total HDAC activity of A.nidulans. Theclass 2 enzyme HdaA accounts for the main part of the HDACactivity and exists as a part of a multiprotein complex of~450 kDa (Fig. 5A). However, IP experiments showed that theclass 1 enzyme RpdA also contributes to the HDAC activityand is part of an enzyme complex that is even larger in size.These results are in line with ®ndings in C.carbonum where ananti-HDC2 (RpdA homolog) antibody could immunopre-cipitate only a minor part of the HDAC activity (34).Furthermore, our results also correspond with biochemicalevidence of yeast HDAC complexes (14) where the 350 kDaHDA complex (containing HDA1) was shown to contributesigni®cantly more to the overall HDAC activity than the HDBcomplex (containing RPD3). Although HosA, another class 1enzyme of Aspergillus, could be detected on immunoblots ofenzymatically active fractions, IP yielded only negligibleactivity; we therefore conclude that HosA plays only a minorrole if any for total Aspergillus HDAC activity. Thisassumption is con®rmed by the lack of enzymatic activity ofthe corresponding yeast HOS2. Although several lines ofgenetic evidence indicate that HOS2 posesses HDAC activity(51±53), it never has been characterized as a functionalenzyme. However, discrepant results exist with respect toHOS2-de®cient mutants as well. Whereas in yeast HOS2deletion has no apparent effect on growth or phenotype, thecorresponding HDC1 deletion strain of C.carbonum displayssigni®cantly altered capabilities of utilizing different carbonsources and also exhibits a change in the pathogenicity againstits host maize (33).

Apart from the well known class 1 and class 2 HDACs, it isevident that, as in higher eukaryotes, an NAD+-dependentSIR2-like HDAC also accounts for a part of the total HDACactivity in ®lamentous fungi. This was con®rmed just recentlyby the identi®cation and cloning of a SIR2 homologous genein Aspergillus and Cochliobolus (unpublished data of our lab).However, it is quite clear from our chromatographic analysisthat SIR2-type HDACs contribute a minor part of the totalHDAC activity and are well separated from the other HDACsin different types of chromatography (Fig. 4).

The classi®cation

Currently we refer to four different classes of HDACs: RPD3homologs, HDA1 homologs, sirtuins and the plant HD2-speci®c enzymes. Unfortunately, nomenclature of (putative)HDACs is confusing (15) and classi®cation is unsatisfactoryfor a number of reasons. On one hand RPD3-related class 1enzymes and HDA1-related class 2 HDACs are characterizedby considerable sequence homologies/similarities (21), on theother hand two additional, totally different classes of HDACsexist (plant HD2; sirtuins) that have neither any relation toeach other nor to the class 1 and class 2 enzymes. Furthermore,a growing number of HOS3-type enzymes like HosB andHDC4 have conserved domains with both, class 1 and class 2

Figure 7. Unrooted phylogenetic tree of fungal HDACs. anHosA, anRpdA,anHdaA (bold) and anHosB (bold) are from A.nidulans (32 and this paper).ccHDC1, ccHDC2, ccHDC3 (bold) and ccHDC4 (bold) are fromC.carbonum (33,34 and this paper). Predicted HDACs of N.crassa are fromcontigs 1.1059 (ncHOS2), 1.91 (ncRPD3), 1.17 (ncHDA1) and 1.451(ncHOS3) of the eponymous genomic DNA sequences of N.crassa (http://www.tigr.org). Predicted HDACs of A.fumigatus are from contigs 22(afHOS2), 322 (afRPD3), 610 (afHDA1) and 143 (afHOS3) of the genomicDNA sequence of A.fumigatus (http://www-genome.wi.mit.edu). scHOS2,scRPD3, scHDA1, scHOS1 and scHOS3 are from S.cerevisiae (14,54).Sequences were aligned using ClustalW 1.8 (46) and trees were calculatedusing PAUP*4.0b10. Numbers indicate the bootstrap probability values ofobserving the branch topology shown (1000 re-sampling; a value of 100indicates the maximum of probability that the split is certain). The giventree was calculated with the `neighbor joining' (NJ) algorithm.




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enzymes (Fig. 1) but do not really ®t in either class (Fig. 7). Inaddition, these proteins share several unique sequence motivesthat may be important domains responsible for the insensi-tivity towards HDAC inhibitors and other speci®c biochemicalproperties of this enzyme group. Consequently, they aremembers of an additional HDAC class (class 5). Moreover itshould be mentioned that the RPD3 group is divided into twobranches (namely the RPD3/HOS2 enzymes and yeast HOS1).However, there are no further HOS1 orthologs identi®ed sofar, which at present would justify a separate HOS1 group.

To make HDACs with pronounced sequence similaritiesclearly distinguishable from enzymes of other groups, wesuggest a reconsideration of the current nomenclature ofHDAC enzymes. One suggestion is to distinguish three mainclasses of HDACs: (i) a class 1, the `classical' HDACs thatcontains subclasses with strong sequence similarities to RPD3,HDA1 and HOS3; (ii) a class 2 that contains the NAD+-dependent sirtuines with the subclasses SIR2 and HST1-4 and(iii) a class 3 that contains plant speci®c HD2-relatedenzymes.

ACKNOWLEDGEMENTS

The authors thank Jonathan D. Walton (East Lansing) forcritical comments and discussion. This work was supported bygrants from the Austrian Science Foundation to S.G. (P15439)and G.B. (P13209), and a grant from the Austrian NationalBank to P.L. (P-9000). Sequencing of A.fumigatus wasaccomplished with support from National Institute ofAllergy and Infectious Diseases (NIAID) to Dr DavidDenning, University of Manchester, UK, and Dr WilliamNierman at TIGR. The genomic sequence of N.crassa wasobtained from the Neurospora Sequencing Project, WhiteheadInstitute/MIT Center for Genome Research.

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Histone deacetylases in fungi: novel members, new facts

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