The histone deacetylase 9 gene encodes multiple protein isoforms

The Histone Deacetylase 9 Gene Encodes Multiple Protein Isoforms*

Received for publication, December 18, 2002, and in revised form, January 31, 2003Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M212935200

Kevin Petrie‡, Fabien Guidez‡, Louise Howell‡, Lyn Healy‡, Samuel Waxman§, Mel Greaves‡,and Arthur Zelent‡¶

From the ‡Leukemia Research Fund Centre at the Institute of Cancer Research, London SW3 6JB, United Kingdom andthe §Department of Medicine, Mount Sinai Medical Centre, New York, New York 10029

Histone deacetylases (HDACs) perform an importantfunction in transcriptional regulation by modifying thecore histones of the nucleosome. We have now fullycharacterized a new member of the Class II HDAC fam-ily, HDAC9. The enzyme contains a conserved deacety-lase domain, represses reporter activity when recruitedto a promoter, and utilizes histones H3 and H4 as sub-strates in vitro and in vivo. HDAC9 is expressed in atissue-specific pattern that partially overlaps that ofHDAC4. Within the human hematopoietic system, ex-pression of HDAC9 is biased toward cells of monocyticand lymphoid lineages. The HDAC9 gene encodes multi-ple protein isoforms, some of which display distinct cel-lular localization patterns. For example, full-lengthHDAC9 is localized in the nucleus, but the isoform lack-ing the region encoded by exon 7 is in the cytoplasm.HDAC9 interacts and co-localizes in vivo with a numberof transcriptional repressors and co-repressors, includ-ing TEL and N-CoR, whose functions have been impli-cated in the pathogenesis of hematological malignan-cies. These results suggest that HDAC9 plays a role inhematopoiesis; its deregulated expression may be asso-ciated with some human cancers.

In eukaryotes, the ability to dynamically form and maintaindistinct functional domains of chromatin is fundamental tonuclear processes, including regulation of gene transcription(1). The basic repeating unit of chromatin is the nucleosome,which is a complex consisting of 1.75 superhelical turns of DNAwrapped around a core histone octamer comprising two sub-units each of H2A, H2B, H3, and H4 (2). The N-terminal tailsof histones H3 and H4 protrude from the nucleosome andinteract with the negatively charged DNA phosphate backbonewhen in their highly basic, unmodified state (3). These tailscontain specific amino acids that are targets for a variety ofenzymes, producing diverse modifications including acetyla-tion, methylation, and phosphorylation. Acetylation is thus farthe most widely studied and involves substitution of the �-amino group of specific lysines in a process catalyzed by histoneacetyltransferases. This leads to a more acidic residue and anoverall decreased affinity for DNA by the histone octamer. For

the transcriptional machinery, the packaging of DNA into nu-cleosomal arrays presents a major physical obstacle in gainingaccess to the DNA template, and there has long been evidencethat unwinding of nucleosomes because of the acetylation ofhistone tails plays an important role in the activation of tran-scription (4). As expected, enzymes that remove these modifi-cations, histone deacetylases (HDACs),1 are important in genesilencing, and recent studies have implicated abnormal HDACfunction in a number of human cancers (5).

It is therefore important in studies of gene expression toaddress the mechanisms by which HDACs effect specificchanges in chromatin structure. Thus far, 11 histone deacety-lases that share homology through their deacetylase domainshave been characterized, and they may be broadly divided intotwo classes on the basis of homology to the yeast HDACs RPD3(Class I) and HDA1 (Class II) (6–9). Class I HDACs are nuclearproteins that are generally small in size (40–55 kDa) andubiquitously expressed (10–12). In contrast, Class II HDACsare larger (ranging from 100 to 130 kDa), and expression pat-terns tend to be tissue-specific with the exception of HDAC10,which is more widely expressed (8, 9, 13–20). Furthermore,HDACs 4, 5, and 7 may, in response to calcium signalingeffected by Ca2�/calmodulin-dependent kinases, be exportedfrom the nucleus to the cytoplasm upon binding 14-3-3 signal-ing adaptor proteins (21–25). The function of both Class I andII HDACs is, in part, mediated through association with tran-scriptional co-repressors such as SMRT/N-CoR, and mSin3A(15, 26–31), and HDACs have also been found to interactdirectly with some transcription factors and repress their tar-get promoters (32, 33). At least with respect to HDAC3, asso-ciation with the co-repressor appears to play a role in theregulation of its catalytic activity (34).

Here, we report the cloning and characterization of HDAC9,a member of the Class II histone deacetylase family. HDAC9contains 1069 amino acids and functions as deacetylase both invitro and in vivo, and this activity is essential for its associatedrepression of gene expression. Furthermore, HDAC9 is alter-natively spliced to generate multiple protein isoforms that mayharbor distinct biological activities and may be associated withhuman cancer. One of these isoforms consists of the noncata-lytic N-terminal region of HDAC9 (conserved in HDACs 4, 5,and 7) and has been previously identified in Xenopus (MEF2-interacting transcriptional repressor (MITR)) (35) and human(histone deacetylase-related protein (HDRP)) (36, 37).

* This work was supported by funds from the Leukemia ResearchFund of Great Britain, the Institute of Cancer Research Studentship (toK. P.), the Kay Kendall Leukemia Fund (to F. G.), and the SamuelWaxman Cancer Research Foundation. The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AY197371.

¶ To whom correspondence should be addressed: Inst. of Cancer Re-search, 237 Fulham Rd., London SW3 6JB, UK. Tel.: 44-207-3528133;Fax: 44-207-3523299; E-mail: [email protected].

1 The abbreviations used are: HDAC, histone deacetylase; MITR,MEF2-interacting transcriptional repressor; HDRP, histone deacety-lase-related protein; GST, glutathione S-transferase; Luc, luciferase;PBS, phosphate-buffered saline; RT, reverse transcriptase; TRITC, tet-ramethylrhodamine isothiocyanate; DAPI, 4�,6�-diamidino-2-phenylin-dole hydrochloride; contig, group of overlapping clones; ORF, openreading frame; DBD, DNA-binding domain.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 18, Issue of May 2, pp. 16059–16072, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 16059

EXPERIMENTAL PROCEDURES

Isolation of HDAC9 cDNAs and Plasmid Construction—Full-lengthhuman HDAC9 was cloned from Marathon-Ready Human Brain cDNA(Clontech) using the sense primer 9F1 5�-ATGCACAGTATGAT-CAGCTCA-3� and the antisense primer 9R1 5�-GTCACACACAG-GAAATATCAG-3� (see Fig. 2 for the cDNA sequence and translation).HDAC9�CD/MITR/HDRP was cloned from Marathon-Ready HumanBrain cDNA using the sense primer 9F1 and the antisense primer9�CDR1 5�-TCAGATAATGACTTTAATTACAAAT-3�. HDAC9�exon7and HDAC9�exon12 were cloned from the acute monocytic leukemiacell line MONO-MAC-6 using the sense primer 9F1 and the antisenseprimer 9R3 5�-TCTCTAATCCATCCATGCCAA-3�. HDAC9�exon15was cloned from the acute Pre-B ALL cell line REH using the senseprimer 9F2 5�-AGGCTGCTTTTATGCAACAG-3� and the antisenseprimer 9R2 5�-CTGAATGCTTCAAGGTACTCA-3�. See Fig. 1A for aschematic detailing the positions of the above primers. PCR productswere cloned into pCRII (Invitrogen) and sequenced using BigDye (Per-kin Elmer).

Double-stranded oligonucleotide primers were cloned into both endsof the HDAC9 cDNA according to Ausubel et al. (38), utilizing BclI (5�end) and BbsI (3� end) sites to create a N-terminal FLAG-tagged cDNAcontaining 5�-BamHI-XhoI-3� ends. The cDNA was then subcloned intothe pSG5 vector (Stratagene) containing a modified polylinker to createF-HDAC9. FLAG-tagged HDAC9�CD (F-HDAC9�CD) was created bysubcloning a 5�-PstI-XhoI-3� 3� fragment from HDAC9�CD into F-HDAC9. Plasmids containing the various FLAG-tagged alternativelyspliced variants of HDAC9 and HDAC9�CD were constructed by sub-cloning appropriate fragments from the partial cDNAs described aboveinto F-HDAC9 and F-HDAC9�CD.

GST expression vectors were derived from pGEX-5X1 (AmershamBiosciences) by subcloning indicated cDNAs in-frame with the codingregion for glutathione S-transferase. Mammalian two-hybrid expres-sion vectors were derived from pM (Clontech) by subcloning the indi-cated cDNAs in-frame with the coding region for the GAL4 DNA-binding domain. The GAL4uasx5-Tk-Luc reporter was derived from thepT109luc (39) plasmid by inserting five copies of the GAL4 DNA-binding site upstream of the minimal HSV-Tk promoter and luciferase(Luc) gene. The MEF2REx3-Tk-Luc reporter was also derived from thepT109luc plasmid by inserting three copies of the consensus MEF2-binding site. The TELREx3-Tk-Luc reporter, as well as mammalian andin vitro expression vectors for AML1, TEL, and TEL-AML1, have beenpreviously described (40). Mammalian and in vitro expression vectorsfor BCL-6 (41), MEF2D (42), PLZF (43), full-length and partial N-CoR(27, 44), HDAC1 (45), HDAC3 (10), HDAC4 (13), as well as Sin3A andB (30), and SUMO-1 and -2 (46) proteins have been described by others.

Cell Culture—293T, COS-7 and tsCOS cells (47) were maintained inDulbecco’s modified Eagle’s medium with 10% fetal calf serum (Sigma).Hematopoietic cell lines and the colon cancer cell line SW-620 weremaintained in RPMI 1640 medium with 10% fetal calf serum (Sigma).

Isolation of Cell Populations—Adult peripheral blood was taken withinformed consent, and the low density cells (�1.077 g/ml) were sepa-rated using Lymphoprep (Nycomed Pharma AS) and resuspended inphosphate-buffered saline (PBS) containing 1% fetal calf serum (Sig-ma). For sorting, the cells were stained separately with either mousemonoclonal anti-CD19 (Dako) or fluorescein isothiocyanate-conjugatedanti-CD14 (Caltag-Medsystems) followed by enrichment of positivecells using the standard MACS system protocol (Miltenyi Biotec). Ratanti-mouse IgG1 beads were used to isolate CD19�ve cells and anti-fluorescein isothiocyanate beads for CD14�ve cells. Nonviable cells wereexcluded by the addition of the dye To-pro-3 iodide (Molecular Probes)to presorted cells. Enriched cells were analyzed and sorted to �99%purity using a fluorescence-activated cell sorter (FACS Vantage SE;B.D. Biosciences) at two wavelengths (using 530/30- and 660/20-nmband passes).

Histone Deacetylase Assays—Immunoprecipitations for histonedeacetylase assays were essentially performed as described (16). 293Tcells (5 � 107 cells) were transfected with PolyFect (Qiagen). One 10-cmdish was used per four HDAC assays. After 24 h, the cells were har-vested and lysed in low stringency lysis buffer (50 mM Tris-HCl, pH 7.5,120 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet P-40) in the presence of aprotease inhibitor mixture (Roche Molecular Biochemicals). To controlfor expression of different constructs, concentrations of protein extractswere normalized with a modified Lowry assay (Bio-Rad), and Westernblot analysis was performed with the ECL procedure (Amersham Bio-sciences) as described (38). Extracts were precleared by incubation withprotein 10% v/v protein A/G-Sepharose (Sigma) overnight at 4 °C. Pre-cleared lysates were immunoprecipitated by incubation with 10% v/v

M2 anti-FLAG-agarose (Sigma) overnight at 4 °C. Immune complexeswere recovered by washing three times with low stringency lysis buffer,twice with lysis buffer containing 0.5 M NaCl (high stringency), andtwice with HDAC buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 10%glycerol). For inhibition studies, the immunoprecipitated complexeswere preincubated with trichostatin A (400 nM) in HDAC buffer for 30min at 4 °C.

Peptides corresponding to the N-terminal sequences of histone H3(ARTKQTARKSTGGKAPRKQLC) and H4 (SGRGKGGKGLGKG-GAKRHRC) were [3H]acetylated according to the manufacturer’s in-structions (Upstate Biotechnology, Inc.), with 20,000 cpm used as sub-strate per reaction. The beads were resuspended in 200 �l of HDACbuffer containing acetylated peptide and 1 mM phenylmethylsulfonylfluoride. Histone deacetylase activity was determined after incubationfor 4 h at 37 °C, according to the peptide manufacturer’s instructions.

Chromatin Immunoprecipitation Assay—293T cells (5 � 107 cells)were transfected by CaPO4 (Profection, Promega) with 5 �g ofGAL4uasx5-Tk-Luc reporter plasmid plus 10 �g of expression vectorcontaining heterologous fusions of the GAL4 DNA-binding domain withthe indicated cDNAs. Immunoprecipitation of plasmid DNA plus asso-ciated histones was carried out �40 h after transfection according to apreviously published protocol (48), with the following modifications.Histone-DNA complexes were cross-linked by the addition of 1% form-aldehyde to the medium and incubation at 37 °C for 10 min. After lysis,the chromatin was sonicated to 0.2–1.0 kb and diluted 10-fold in IPbuffer (0.01% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH8.0, 150 mM NaCl, plus protease inhibitors). Protein samples for West-ern blotting were taken prior to dilution; control samples for assayinginput DNA were taken after dilution and de-cross-linked. Anti-acety-lated histone H4 polyclonal antibody (Upstate Biotechnology, Inc.) wasused for the immunoprecipitation, and the DNA-histone complexeswere collected overnight with protein A/G-Sepharose beads (SantaCruz). Sequences spanning the GAL4-binding site in the reporter weredetected by semi-quantitative PCR using the forward primer 5�-ATTG-CAGCTTATAATGGTTA-3� and the reverse primer 5�-TTCGAAT-TCGCCAATGACAA-3�. The number of cycles was determined empiri-cally to give results that fall within the linear range of this particularPCR assay, and the reactions were visualized by agarose gel electro-phoresis followed by ethidium bromide staining.

RNA Isolation and Reverse Transcriptase-PCR Analysis—Total RNAwas extracted from purified cells and cell lines using an RNeasy kit(Qiagen). cDNA was made from total RNA by reverse transcriptionusing Moloney murine leukemia virus (Invitrogen) or Omniscript (Qia-gen) reverse transcriptases in accordance with the manufacturers’ in-structions. HDAC9 cDNA was primed with 5�-TAAAACAGAGGCAG-CAGGGGAAGAGTGAGT-3� and HDAC9�CD/MITR cDNA was primedwith 5�-TCAGATAATGACTTTAATTACAAATCCTGG-3�. Glyceralde-hyde-3-phosphate dehydrogenase cDNA was primed with random hex-amers (Roche Diagnostics). 2 �g of total RNA was used per 20-�lreaction, and 2 �l of cDNA was used per subsequent PCR. The primerpairs were used for semi-quantitative PCR are shown in Fig. 5A. PCRswere visualized by agarose gel electrophoresis and ethidium bromidestaining.

Preparation of an HDAC9-specific Antiserum—A rabbit antiserumspecific to the C terminus of human HDAC9 was raised using a syn-thetic peptide corresponding to amino acids 1046–1060 of the protein(DVEQPFAQEDSRTAG), which was conjugated to diptheria toxoid(Mimotopes). This C-terminal region is not conserved in any otherHDACs closely related to the HDAC9 protein.

Antibodies to BCL-6 (D-9), GAL4 (DBD) (RK5C1), N-CoR (C-20), TEL(C-20), TEL (N-19), HDAC4 (H-92), HDAC5 (P-16), HDAC6 (H-300),and HDRP/MITR (G-15) (this antibody also recognizes full-lengthHDAC9 protein) were obtained from Santa Cruz Biotechnology. Anti-bodies to Myc tag (9B11), HDAC1, HDAC3, and HDAC7 were obtainedfrom Cell Signaling Technology, and anti-FLAG M2 antibody was ob-tained from Sigma. Anti-mouse and anti-rabbit horseradish peroxidase-linked secondary antibodies used in chemiluminescent detection ofWestern blots were purchased from Amersham Biosciences.

GST Pull-down Assays—All of the GST fusion proteins were pre-pared using standard procedures (43). [35S]Methionine-labeled proteinswere synthesized in vitro using a rabbit reticulocyte-coupled transcrip-tion-translation system (Promega), following the supplier’s directions.35S-Labeled proteins were incubated with 1 �g of GST or a given fusionprotein. The assays were performed in NETN buffer (20 mmol/liter Tris,pH 8.0, 100 mmol/liter NaCl, 1 mmol/liter EDTA, 0.5% Nonidet P-40) at4 °C for 1 h with gentle rocking. Glutathione-Sepharose beads werewashed five times with H buffer (20 mmol/liter HEPES, pH 7.7, 50mmol/liter KCl, 20% glycerol, 0.1% Nonidet P-40). The bound proteins

Alternatively Spliced HDAC9 Isoforms16060

were eluted in Laemmli loading buffer and separated by SDS-PAGE.The gels were fixed in 25% isopropanol and 10% acetic acid, dried, andexposed to Biomax film (Kodak).

Co-immunoprecipitation Assays—10-cm dishes containing tsCOScells were transfected with 2 �g each of expression constructs encodingN-CoR, TEL, and FLAG-tagged HDAC9 isoforms using PolyFect (Qia-gen). After 36 h the cells were washed in PBS and harvested, and theimmunocomplexes were isolated using the Catch and Release immuno-precipitation system (Upstate Biotechnology, Inc.) according to themanufacturer’s instructions. Co-immunoprecipitated HDAC9 isoformswere resolved by SDS-PAGE, blotted, and detected with anti-FLAG M2antibody. Endogenously expressed proteins were co-immunoprecipi-tated as above and detected with affinity-purified anti-HDAC9 rabbitpolyclonal antibody.

Double Immunofluorescence Microscopy—COS-7 cells growing oncoverslips were transfected using PolyFect (Qiagen). After 36 h, thecells were fixed with 100% methanol at �20 °C for 10 min and washedthree times for 5 min in PBS. The cells were permeabilized with 0.5%Triton X-100 in PBS and washed three times for 5 min in PBS. The cellswere blocked for 30 min at room temperature in 2% bovine serumalbumin (Vector Laboratories) and 5% relevant serum (Jackson Immu-noresearch Laboratories, Ltd.) for the secondary antibody to be used.The primary antibodies were diluted to the appropriate concentrationsin blocking buffer, centrifuged at 4 °C for 20 min, and then incubated at4 °C overnight. The cells were washed three times for 15 min in PBSand incubated with the appropriate fluorescein isothiocyanate- orTRITC-conjugated secondary antibody (Jackson Immunoresearch Lab-oratories, Ltd.) for 2 h at room temperature. The cells were washedthree times for 15 min in PBS and mounted on slides using Vectashieldwith DAPI (Vector Laboratories). Imaging was performed on a ZeissAxioplan2 microscope with a cooled CCD camera (Photometrics Quan-tix) using Smartcapture 2 software (Digital Scientific).

Confocal Immunofluorescence Microscopy—104 REH cells in logphase growth were cytospun and fixed in 4% paraformaldehyde for 15min at room temperature. The samples were subsequently processed asdescribed above. The cell nuclei were stained with To-pro-3 iodide(Molecular Probes), and slides were mounted with Vectashield (VectorLaboratories). Affinity-purified anti-HDAC9 rabbit polyclonal antibodywas used for detection, and imaging was performed on a Leica TCS-SP2system.

In Vivo Sumoylation Assays—In vivo sumoylation assays were car-ried out using the nickel affinity pull-down technique (49). Briefly,tsCOS cells in 10-cm dishes were transfected with 2 �g each of expres-sion constructs encoding F-HDAC9, F-HDAC9�exon12, and polyhisti-dine-tagged SUMO-1 or -2 using PolyFect (Qiagen). After 36 h the cellswere washed in PBS and harvested directly in 1 ml of guanidine lysisbuffer (6 M guanidine HCl, 100 mM NaCl, 10 mM Tris, 50 mM NaH2PO4,pH 8.0), sonicated, and centrifuged. The cleared samples were incu-bated for 2 h with 20 �l (packed volume) of Talon nickel affinity beads(Clontech). The bound proteins were washed twice in lysis buffer, threetimes in urea buffer (8 M urea, 100 mM NaCl, 50 mM NaH2PO4, pH 6.5),and once in cold PBS before being eluted by boiling in Laemmli loadingbuffer. 20% of each guanidine lysis sample was removed and precipi-tated for 15 min on ice with 5% trichloroacetic acid. Samples werecentrifuged, washed in 100% ethanol, and resuspended in Laemmliloading buffer. The proteins were separated by SDS-PAGE and detectedwith affinity-purified anti-HDAC9 rabbit polyclonal antibody.

RESULTS

Cloning of Full-length HDAC9 cDNA—The GenBankTM (50)high throughput genomic sequence data base (htgs) wassearched with the amino acid sequences corresponding to thedeacetylase domains of various Class I and II HDACs. SeveralDNA sequences encoding peptides with significant homology toHDAC5 were found on a human BAC clone RP11–8I15 (acces-sion number AC016186), which contains 70 unordered contigsand maps to chromosome 18. When the GenBankTM nucleotidedata base was searched with a composite of the novel sequencesshowing homology to HDAC5, it aligned exactly with BACclone CTB-13P7 (accession number AC002088), which mappedto chromosome 7p21.1, indicating that clone RP11–8I15 hadbeen submitted incorrectly to the htgs data base. A search ofthe expressed sequence tag data base (dbEST) revealed thatcDNA corresponding to the putative histone deacetylase hadbeen identified in germinal center B cells (accession number

AA287983); therefore several hematopoietic cell lines were an-alyzed by RT-PCR with oligonucleotide primers specific for theputative HDAC sequence and also HDRP/MITR, which hadbeen mapped to 7p15–p21 (35). In addition to the amplifyingthe putative HDAC sequence itself; it was found that thisprimer set also amplified cDNA from HDRP/MITR through tothe putative HDAC domain. The Ensembl Genome Server (51)indicated an open reading frame (ORF) containing 22 exons(�2700 bp of sequence), and RT-PCR was performed usingoligonucleotide primers from within the known sequence of theputative HDAC9 cDNA established by us and the expected stopcodon of the ORF, but no products were observed. These data,together with the observed conservation of C-terminal aminoacid residues among other Class II HDACs indicated the ab-sence of the entire ORF. 3�-Rapid amplification of cDNA endswas performed but failed to yield the remaining sequence, soGenBankTM was searched for overlapping expressed sequencetags to “walk” along the cDNA (Fig. 1B). This analysis revealedthat the sequence corresponding to the final five exons of theputative HDAC gene contained in BAC clones RP5–1194E15and GS1–465N13 (accession numbers AC004994 and AC004744,respectively) had been submitted in the antisense direction inrelation to the rest of the gene (Fig. 1C). These clones accountedfor almost 160 kb of DNA, and subsequent analysis with themin the correct orientation provided a genomic sequence thatmatched the ORF that we had generated from overlappingexpressed sequence tags and, when translated, showed signif-icant homology with the amino acid sequences for HDACs 4, 5and 7 (see Fig. 3A). We designated this novel gene HDAC9. AnORF mapping to the HDAC9 locus has been independentlycloned by another group (7); however, it does not encode thefull-length protein (see further below).

The HDAC9 Gene Is Differentially Spliced to Encode MultipleIsoforms—The full-length product of the HDAC9 gene com-prises 1069 amino acids as shown in Fig. 2 and is encoded byexons 2–26 (exon1 is untranslated). The 26 exons that form theHDAC9 cDNA span �500 kb of genomic sequence on 7p21.1.The isoform that lacks the catalytic domain, HDAC9�CD(HDRP/MITR), is 593 amino acids in length and contains 16residues of unique sequence encoded by a region of exon 12,which is 3� to the splice donor site used to generate the HDAC9ORF (Fig. 2). There are several exon deletions that may occurthat naturally preserve the open reading frame of HDAC9 (Fig.1C), and two have been identified and cloned. It has beenpreviously suggested that HDAC9 is potentially alternativelyspliced at exon7 (7), and this is indeed the case. HDAC9�exon7is 1025 amino acids long and contains an Ala 3 Glu substitu-tion at position 222 as the result of the deletion of exon 7. Thisisoform lacks two serines (Ser223 and Ser253), which when phos-phorylated have been implicated in 14-3-3 protein-dependentshuttling of HDAC4 and 5 from the nucleus to the cytoplasm,and also a tripartite nuclear localization signal (21, 23, 52).Exon 12 may also be deleted in-frame to generate an alternateprotein isoform that is 981 amino acids long. The region en-coded by exon 12 contains a conserved sumoylation site iden-tified in HDAC4 (53) and a potential leucine zipper motif thatmay mediate interactions between HDAC9 and other proteins.cDNA that encodes an isoform possessing neither exon 7 norexon 12 has also been cloned. HDAC9�exon15 is 1027 aminoacids long and lacks a region within the catalytic domain ad-jacent to the active site. This isoform does not naturally con-serve the ORF of HDAC9 and may have undergone RNAediting.

Phylogenetic analysis of HDAC9 shows that it is a member ofthe Class II histone deacetylases and is most closely related toHDAC5. The recent discovery of HDACs 10 and 11 indicate

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that, based on analysis of HDAC catalytic domain (Fig. 3B) andwhole protein (Fig. 3C), there is a subdivision of the Class IIhistone deacetylase group consisting of HDACs 6, 10, and 11.

HDAC9 Possesses Deacetylase Activity—To determinewhether HDAC9 possesses histone deacetylase activity, an invitro assay was performed using anti-FLAG immunoprecipi-

tated HDAC9. As shown in Fig. 4A, HDAC9 catalyzes thedeacetylation of peptides corresponding to the N-terminal tailsof both histone H3 and H4 with overall activity comparablewith that of HDAC4. Additionally, the Class II HDACs 4 and 9appear to deacetylate the histone H4 peptide substrate lesseffectively when compared with HDAC1. The value for deacety-

FIG. 1. Isolation of HDAC9 cDNA and analysis of the HDAC9 gene. A, schematic representation of the two major HDAC9 isoforms togetherwith the oligonucleotide primers (indicated as arrows) used in cloning of their respective cDNAs. B, identification of the 3� sequence of the HDAC9open reading frame. The shaded letters represent the open reading frame derived by using overlapping expressed sequence tags (indicated byGenBankTM accession number) to walk along chromosome 7p21.1. C, genomic organization of HDAC9. The accession numbers of the clones thatcomprise the HDAC9 gene are shown together with the exon positions on chromosome 7, which were established using the BLAT alignment tool(84). HDAC9 exon/intron splice junctions are also detailed with consensus splice donor and acceptor sequences between exons (uppercase letters)and introns (lowercase letters) underlined. Exons highlighted in gray have the potential to be spliced out in-frame. �exon7 and �exon12 cDNAshave been detected by RT-PCR.


lation of histone H4 peptide is 44% for HDAC4 and 36% forHDAC9, respectively, of the value observed for histone H3peptide. This contrasts with HDAC1, which deacetylated his-tone H4 peptide with 70% of the activity seen for histone H3peptide. These data are specific for a given HDAC, because ahigh stringency wash removes endogenous Class I and II

HDACs present in the lysate that are not directly bound byanti-FLAG M2 agarose beads (Fig. 4B). Moreover, isoforms ofHDAC9 that either lack (HDAC9�CD) or possess a deletion(HDAC9�exon15) in the catalytic domain do not deacetylatehistone H3 peptide effectively (Fig. 4A). This indicates thatco-immunoprecipitation of uncharacterized proteins possessing

FIG. 2. HDAC9 cDNA and predicted amino acid sequence. HDAC9 full-length cDNA has been cloned using human brain mRNA. It containsan open reading frame of 3210 bp, which yields a 1069-amino acid protein. The amino acids are indicated in single-letter code and are numberedon the right. The positions of 25 introns and their sizes in bp are also indicated. The HDAC9�exon7 isoform results in an Ala3 Glu substitutionat position 222. The sequence specific to HDAC9�CD exon 12 is shown in italics.


histone deacetylase activities or known HDACs at levels notdetected by our analysis is not likely to account for the observedin vitro activity of HDAC9. The in vitro assay results arecorroborated in vivo by chromatin immunoprecipitation analy-sis (Fig. 4C). There is a difference in the amount of target DNAsequence that may be co-immunoprecipitated with acetylatedhistone H3, compared with acetylated histone H4, when pro-teins containing a heterologous fusion of GAL4 DNA-bindingdomain to the catalytic domain of HDAC9 or to a lesser extentthe whole HDAC9 protein are tested. This result is not ob-served for HDAC9�CD and is most likely a reflection of theability of this N-terminal isoform of HDAC9 lacking the cata-lytic domain to bind Class I and II HDACs in vivo (54).

Distribution of HDAC9 mRNA Is Tissue-specific and May BeDeregulated in Human Cancers—Analysis of HDAC4, HDAC9,and HDAC9�CD expression in various human tissues and celllines reveals that not only are there differences in the pattern

of expression between HDAC4 and HDAC9 but also betweenthe transcripts encoding the two major isoforms of HDAC9(Fig. 5B). For example, all three transcripts were expressed inskeletal muscle and the adult and fetal brain (Fig. 5B, lanes 3and 12), although HDAC9 and HDAC9�CD were considerablymore abundant in fetal tissue. HDAC4 alone is expressed inkidney, liver, and the myeloid leukemic cell line HL60 (Fig. 5B,lanes 7, 8, and 16). Although expression of HDAC9 was verylow or absent in heart, T cell leukemia cell line MOLT-3, andearly myeloid leukemia cell line KG-1, both HDAC4 andHDAC9�CD were co-expressed in these tissues (Fig. 5B, lanes2, 14, and 17). To examine this further, we have carried outRT-PCR analysis for multiple HDAC9 isoforms in a large num-ber of hematopoietic cell lines.

When HDAC9 and HDAC9�CD transcripts are analyzed forexpression of exon 7 and 12 deletion splice variants in hema-topoietic tissues and cell lines (Fig. 5C), mRNA encoding the

FIG. 3. Phylogenetic analysis of HDAC9. A, amino acid sequence alignment of HDAC9, HDAC4, HDAC5, HDAC7, and a bacterialdeacetylase, HDLP. The indicated sequences were aligned using Clustal W. Identical residues are boxed and highlighted in dark gray; similarresidues are shaded in light gray. B, evaluation of amino acid identities and similarities of HDAC9 deacetylase domain compared with those ofHDAC1 and other Class II histone deacetylases. The histone deacetylase domains are shown in light gray. The percentage values were obtainedby comparing the deacetylase domain of HDAC9 with the indicated protein sequences on the BioEdit Sequence Alignment Editor using theBlosum62 matrix. C, phylogenetic tree of HDAC1 through to HDAC11. The sequences were aligned using the Clustal W server at the Center forMolecular and Biomolecular Informatics (University of Nijmegen, Nijmegen, Holland). The PHYLIP notation output was used to construct anunrooted tree with Unrooted (Manolo Gouy, University Claude Bernard, Lyon, France).


isoform lacking the catalytic domain appears to be expressed inmany cell types. Although where co-expressed with full-lengthHDAC9, HDAC9�CD is considerably more abundant, we can-not exclude the possibility that this may be due to the efficiencyof amplification. In normal tissues, HDAC9 transcripts encod-ing the catalytic domain were found at low levels in the bone

marrow, spleen, and thymus (Fig. 5C, lanes 2–4). The highestlevels, however, were observed in cells expressing CD14�ve

(monocyte/macrophage) and, to a lesser extent, CD19�ve (Bcell) surface markers (Fig. 5C, lanes 5 and 6). Further, inspec-tion of the RT-PCR data reveal that HDAC9 is generally ex-pressed in pre-B cell acute lymphoblastic leukemia cell lines

FIG. 4. HDAC9 possesses histone deacetylase activity. A, HDAC9 deacetylates histone H3 and H4 peptide in vitro. 293T cells weretransfected with FLAG-tagged HDACs as indicated. Whole cell lysates were produced, and the HDACs were precipitated with anti-FLAG agarose.The precipitates were thoroughly washed and assayed for their ability to deacetylate [3H]acetyl-labeled peptides corresponding to the N terminusof histones H3 or H4. Free acetate was extracted, and the number of cpm were measured by liquid scintillation. The HDAC inhibitor trichostatinA was added to control reactions as indicated. The cells were transfected with pSG5 empty vector (Stratagene) as a negative control. This is takento be the background level of deacetylase activity measured (dark gray). B, under high stringency conditions, HDAC9 does not co-immunopre-cipitate with other previously characterized members of the histone deacetylase family. 293T cells were transfected with FLAG-tagged HDAC9 andimmunoprecipitated as described above. The precipitates were then immunoblotted with antibodies raised against the indicated HDACs. Asidefrom HDAC9, input values for the indicated HDACs show levels of the endogenous enzyme. C, HDAC9 causes hypoacetylation of target genes invivo. 293T cells were transfected with GAL4uasx5-Tk-Luc reporter together with GAL4 DNA-binding domain fusions as indicated. GAL4DBD-HDAC9CD contains only the catalytic domain of HDAC9. The GAL4 reporter is derived from the pT109luc plasmid (39). This plasmid contains aSV40 origin of replication, which has been shown to induce chromatinization of plasmid DNA in cell lines expressing large T antigen (85). Solublechromatin preparations from the transfections were immunoprecipitated with antibodies against acetylated histone H3 (�AcHistone H3) or H4(�AcHistone H4) (Upstate Biotechnology, Inc.) and analyzed by semi-quantitative PCR. As negative and positive controls, soluble chromatinpreparations were immunoprecipitated with IgG or anti-GAL4 (DBD) (RK5C1) antibody, respectively. Aliquots of the chromatin were also analyzedbefore immunoprecipitation (DNA input). The numerical values, which reflect relative abundance of acetylated chromatin, were obtained bydensitometric analysis using Labworks analysis software (Ultra-violet Products).


(Fig. 5C, lanes 7–9), B cell lymphoma cell lines (Fig. 5C, lanes10–13), and also the plasma cell line U-266 (Fig. 5C, lane 14).HDAC9 is also expressed in some T cell lines (Fig. 5C, lanes15–18). HDAC9 is not expressed in various acute myeloid leu-kemia cell lines (Fig. 5C, lanes 19–22), with the exception ofKG1 (a multilineage lymphomyelocytic cell line) (55), whichexpresses HDAC9�CD. Two of four acute monocytic leukemiacell lines analyzed expressed isoforms of HDAC9, and lowlevels are also found in the erythroleukemia cell line, HEL (Fig.5C, lane 27). Overall, the expression data suggest a selectivealthough not absolute bias of HDAC9 expression towardlymphoid and monocytic cells within the hematopoietic system.

Samples from colon cancer cell lines showed increased ex-pression of HDAC9 relative to normal colon tissue HDAC9mRNA (Fig. 5C, lanes 31–34), especially variants lacking exons7 and 12 in SW-620. In general, there is greater variety ofexpression of relative amounts of differentially spliced HDAC9transcripts in samples containing the catalytic domain ofHDAC9 than those lacking it. One exception is bone marrow(Fig. 5C, lane 2), where there is very little expression of

HDAC9�CD transcripts containing exon 7.HDAC9 Isoforms Interact Differentially with Co-repressors

and Proteins Implicated in Hematalogical Malignancies—Pre-viously, we and others have demonstrated involvement of his-tone deacetylases in the pathogenesis of both lymphoid andmyeloid neoplasms (40, 56, 57). Given the expression of HDAC9in cell lines and samples derived from B cell tumors, we exam-ined whether it could interact with any oncogene productsknown to recruit HDAC-containing complexes and to be in-volved in B cell malignances. As anticipated, HDAC9 wasfound to associate in vitro with the TEL protein, which is fusedto AML1 in pre-B cell childhood acute lymphoblastic leukemia(58, 59), and also BCL-6, which is frequently associated with Bcell neoplasias (60) (Fig. 6A, lanes 3 and 4). HDAC9 also inter-acted with HDACs 3 and 4 (Fig. 6A, lanes 1 and 2) and HDAC1and PLZF (data not shown). In addition, HDAC9 was found tointeract with the co-repressors mSin3A, mSin3B, and N-CoR,whose activities have been implicated in the mechanism ofaction of several human cancers (61) (Fig. 6A, lanes 5–8). Thesedata indicate that N-CoR contacts HDAC9 at multiple points,

FIG. 5. Tissue distribution of HDAC9 transcripts. A, the oligonucleotide primer sequences used for RT-PCR analysis are detailed togetherwith a schematic representation of the primer positions in HDAC9 and HDAC9�CD (arrows). B, differential expression of Class II HDACs innormal tissues and leukemic cell lines. RT-PCR was performed on cDNA derived from normal human tissue (1–13) and cell line (14–19) total RNAas indicated. PCR products were resolved by agarose gel electrophoresis and visualized by ethidium bromide under UV light. C, HDAC9 isoformsare differentially expressed in the B cell lineage and cell lines derived from B cell malignancies. RT-PCR was performed on RNAs derived fromnormal human tissues (1–8), primary CLL samples (9–12), primary childhood acute lymphoblastic leukemia samples (13–16), or hematopoietic celllines (17–41) total RNA, as indicated. NLB (7–8) refers to normal B cell. Where indicated (SAC/IL2), the cells were stimulated with Staphylococcusaureus Cowan I strain (1/5000) and interleukin 2 (50 units/ml). The identity of different isoforms (indicated on the left) was confirmed byisoform-specific and general oligonucleotide probes (data not shown).


with the C-terminal region of N-CoR also interacting with thecatalytic domain of HDAC9. Additionally, the catalytic domainof HDAC9 was found to associate with mSin3A but notmSin3B.

The ability of HDAC9 expressed in vivo to interact withN-CoR or TEL was also examined. Consistent with the in vitrodata, full-length HDAC9 isoforms were found to interact withN-CoR and TEL (Fig. 6B, lanes 12 and 17). However, TELco-precipitated poorly with HDAC9�exon7 compared with N-CoR (Fig. 6B, lanes 13 and 18), indicating that the major site ofinteraction between TEL and HDAC9 lies within exon 7. Co-transfection of N-CoR or TEL with isoforms of HDAC9 lackingthe catalytic domain produced a markedly weaker interactionrelative to that observed for full-length HDAC9 when com-pared with the in vitro data (Fig. 6B, lanes 14, 15, 19, and 20).This suggests that the catalytic domain may be interactingwith other proteins in vivo that act to stabilize the proteincomplex. Finally, the interactions between endogenously ex-pressed HDAC9 and BCL-6, N-CoR, and TEL were examined incell lines known to express these proteins (40, 62, 63) (Fig. 6C).

The results demonstrate that HDAC9 and the above factors canassociate under physiological levels.

The in vitro and in vivo interaction data between HDAC9and N-CoR and TEL are corroborated by specific patterns ofcellular localization between various isoforms of HDAC9 andits interacting partners. Fig. 7A shows the cellular localizationof various isoforms of FLAG-tagged HDAC9 when visualizedeither with anti-FLAG antibody or an antibody to an N-termi-nal epitope of HDAC9. Note that HDAC9�exon7 is completelyexcluded from the nucleus. As previously reported for themouse homologue, HDAC9�CD punctate nuclear localization(64). The cellular distribution of HDAC9 isoforms containingthe catalytic domain was also investigated (Fig. 7B) using anantibody raised against a peptide corresponding to a C-termi-nal region of HDAC9 (see “Experimental Procedures”). HDAC9displayed a diffuse pattern of distribution within the nuclei ofthe REH cells and was also found in the cytoplasm, whichcorroborates RT-PCR data for this cell line showing expressionof HDAC9�exon7 mRNA (Fig. 5C, lane 8).

To further analyze the interactions of HDAC9 and its iso-

FIG. 6. HDAC9 interacts in vivo and in vitro with other histone deacetylases, co-repressors, and transcription factors implicatedin hematological malignancies. A, GST fusion proteins were incubated with 35S-radiolabeled proteins as indicated, precipitated with gluta-thione-Sepharose, and visualized by autoradiography. GST-HDAC9, GST-HDAC9CD, and GST-HDAC9�CD were produced from Escherichia coliDH5�. [35S]Methionine-labeled proteins were synthesized in vitro using a rabbit reticulocyte lysate-coupled transcription-translation system. B,the indicated FLAG-tagged isoforms of HDAC9 were co-transfected with N-CoR and TEL expression vectors. Total protein levels were normalized,and protein complexes were immunoprecipitated with anti-N-CoR (C-20) and anti-TEL (C-20) antibodies as indicated. Co-immunoprecipitatedHDAC9 isoforms were resolved on a 5% SDS-polyacrylamide gel and detected by immunoblotting with anti-FLAG M2 antibody. The arrowheadindicates suspected degradation or incompletely translated products that appear with the FLAG-tagged HDAC9 expression vectors containing thecatalytic domain. The cells transfected with pSG5 empty vector (Stratagene) were included as a negative control (lanes 1 and 10). C, endogenousHDAC9 was immunoprecipitated from NALM-6, RAJI, and REH cell lines with the indicated antibodies. A specific blocking peptide for anti-N-CoR(C-20) and an irrelevant antibody, anti-GAL4 (DBD) (RK5C1) were included as negative controls. Samples containing co-immunoprecipitatedHDAC9 were resolved on a 7.5% SDS-polyacrylamide gel and detected by immunoblotting with affinity-purified rabbit serum containing antibodiesraised against the C-terminal region of HDAC9.


forms in the context of the cell, we performed double immuno-fluorescence assays. When HDAC9 was co-expressed with N-CoR (Fig. 7C), they co-localize within the nucleus, and N-CoR

assumes a more diffuse nuclear distribution that is identical tothat seen with HDAC9 alone. However, when N-CoR was co-expressed with HDAC9�exon7, there was a dramatic change in

FIG. 7. Immunofluorescence analysis of HDAC9. A, HDAC9 is alternatively spliced to generate multiple isoforms. COS-7 cells weretransiently transfected with F-HDAC9, F-HDAC9�exon7, F-HDAC9�exon12, F-HDAC9�exon15, F-HDAC9�CD, or F-HDAC9�CD�exon7 asindicated. After methanol fixation, the cells were stained with DAPI (blue) and FLAG antibody (green). B, HDAC9 isoforms containing the catalyticdomain are expressed endogenously in the childhood acute lymphoblastic leukemia cell line, REH. After fixation with 4% paraformaldehyde, thecells were stained with To-pro-3 iodide (blue) and affinity-purified anti-HDAC9 antibody. C, HDAC9 interacts with the co-repressor N-CoR. COS-7cells were transiently transfected with N-CoR and F-HDAC9, F-HDAC9�exon7, F-HDAC9�CD, or F-HDAC9�CD�exon7 as indicated. Aftermethanol fixation, the cells were stained with DAPI (blue) and anti-FLAG M2 (green) or N-CoR (C-20) (red) antibodies. D, HDAC9 interacts withTEL. The COS-7 cells were transiently transfected TEL and F-HDAC9, F-HDAC9�exon7, F-HDAC9�CD, or F-HDAC9�CD�exon7 as indicated.After methanol fixation, the cells were stained with DAPI (blue) and anti-FLAG M2 (green) or TEL (N-19) (red) antibodies.


the distribution of the co-repressor to the cytoplasmic locationof the HDAC9�exon7 isoform. Recruitment or retention of N-CoR to the cytoplasm was not observed upon co-expression withHDAC9�CD�exon7, indicating that the domains of interactionbetween HDAC9 and N-CoR lay within both exon 7 and thecatalytic domain. This is supported by the in vitro data showingthat the catalytic domain of HDAC9 interacts with the C-terminal region of N-CoR (Fig. 6A, lane 8). Similar results werealso observed for BCL-6 (data not shown). In contrast to N-CoR,TEL was not excluded from the nucleus when co-expressedwith either HDAC9�CD or the full-length HDAC9 lacking exon7 (Fig. 7D), further indicating that the main domain of inter-action between HDAC9 and TEL is mediated through exon 7. Itappears from the above results that different HDAC9 isoformsdisplay individual interaction profiles with various proteins,and this is reinforced by the observation that AML1 co-localizesexclusively with HDAC9�CD (data not shown), suggesting thatthe domain of interaction lies within the C-terminal sequenceunique to this isoform.

HDAC9 Represses Transcriptional Activity in Vivo—It hasbeen previously established that HDACs repress transcriptionwhen tethered to DNA as Gal4 fusion proteins (11). As expectedfrom earlier experiments (Fig. 4, A and C), this effect is alsoobserved with HDAC9, HDAC9�CD, and HDAC9CD alone(Fig. 8A). A GAL4uasx5-TK-Luc reporter gene was transientlytransfected into 293T cells together with the expression vectorsfor the indicated GAL4 fusion proteins. Although HDAC9�CDlacks an HDAC domain, it associates with other HDACs andco-repressors (Figs. 6 and 7), and this may be reflected in itsability to repress reporter gene expression.

The ability of HDAC9 to associate with transcriptional re-pressors, such as TEL for example, suggested that HDAC9could play a role in repression of promoter activities by theseproteins. To evaluate this for the TEL protein, HDAC9 and justthe HDAC9 catalytic domain were co-transfected with TELexpression vector and a luciferase reporter gene containingthree copies of the TEL consensus binding site (5�-TAAACAG-GAAGT-3�). As expected, the addition of HDAC9 potentiatedrepression by TEL, and the degree of repression observed wasgreater for TEL plus full-length HDAC9 than for TEL plusHDAC9�CD (Fig. 8B).

When Sparrow et al. (35) first identified the Xenopus homo-logue of HDAC9�CD, they reported that it associated withmyocyte enhancer factor 2D (xMEF2D) and repressedxMEF2D-mediated transcription. In the human hematopoieticsystem, MEF2D is found in both B and T cells (65), and HDAC9was tested for its ability to affect MEF2D-mediated transcrip-tion. As expected, co-transfection of HDAC9 with MEF2Dabolished MEF2D-mediated transcriptional activation andrepressed a MEF2REx3-Tk-Luc reporter. Co-transfection ofHDAC9�CD with MEF2D produces a similar result, but thelevel of MEF2D-mediated activation was only returned to thebasal level (Fig. 8C). This reflects the results observed withTEL promoter activity and indicates that full-length HDAC9forms a more powerful repressor complex in this context.

Deletion of HDAC9 Exon 12 Results in the Loss of a SiteModified by Sumoylation—To test whether the differentHDAC9 isoforms containing the catalytic domain are endog-enously expressed protein, Western blot analysis was per-formed on whole cell lysates from tsCOS cells transfected withdifferent isoforms of HDAC9 (Fig. 9, lanes 1–3) and selectedhematopoietic and colon cancer cell lines that were used inRT-PCR analysis, plus the T-cell leukemia cell line JM-1 (Fig.9, lanes 4–9). The anti-HDAC9 rabbit polyclonal antibody usedspecifically recognizes endogenous proteins expressed in cellspositive for HDAC9 mRNA isoforms (Fig. 5C). Although overall

protein and mRNA levels are comparable between the differentsamples examined, the full-length protein isoform is the mostabundant species. This could be due to decreased efficiency oftranslation and/or protein isoform stability. The Western anal-ysis shows that HDAC9 has an apparent molecular mass ofaround 160 kDa, which is greater than the predicted molecularmass of 117.5 kDa and indicates that HDAC9 has undergonepost-translational modifications. Interestingly, the HDAC9�-

FIG. 8. HDAC9 represses transcriptional activity in vivo. A,GAL4-HDAC9 represses basal transcription. GAL4uasx5-TK-Luc (400ng) was transfected into 293T cells along with GAL4DBD-HDAC9,-HDAC9�CD, or -HDAC9CD (200 ng each) as indicated. Luciferaseactivity was normalized by co-transfection of �-galactosidase. The par-ent GAL4 fusion vector, pM (Clontech), was used to equalize theamount of GAL4DBD in each transfection. The basal value was set to 1.B, HDAC9 represses MEF2D-mediated activation. MEF2REx3-Tk-Luc(200 ng) was transfected into 293T cells along with pSG5 empty vector,pCMV-MEF2D, F-HDAC9, or F-HDAC9�CD/MITR (200 ng each).pSG5 empty vector (Stratagene) was used to equalize the amount ofDNA in each transfection. The value for MEF2REx3-Tk-Luc alone wasset to 1. C, HDAC9 enhances TEL-mediated repression. TELREx3-Tk-Luc (400 ng) was transfected into 293T cells along with pSG5 emptyvector, pSG5-TEL, F-HDAC9, or F-HDAC9�CD/MITR (200 ng each).The value for TELREx3-Tk-Luc alone was set to 1. The values are shownas fold repression.


exon12 isoform, which only encodes 80 residues, shows anunexpectedly large decrease in apparent size (relative to �7and �15 deletions), suggesting the presence of a site of post-translational modification. This was thought most likely to besumoylation, which has been detected on the correspondingregion of HDAC4 (46, 53). To examine this possibility, tsCOScells were transfected with HDAC9 and HDAC9�exon12 to-gether with polyhistidine-tagged SUMO-1 and -2. Upon nickelaffinity precipitation of His-tagged protein complexes, only thefull-length HDAC9 isoform containing exon 12 was visible as aSUMO-1 or -2 conjugate (Fig. 10, lanes 8 and 9). SUMO-2 mayitself be sumoylated to form polymeric chains (53), and themultiple bands observed in lane 8 are due to endogenousSUMO-2 present in the cells. This is illustrated by the fact thatthe polymeric chains containing His-tagged SUMO-2 in lane 9migrate slightly more slowly than those observed in lane 8,where only the terminal SUMO-1 may be His-tagged. Note thatmono-sumoylated HDAC9 precipitates with markedly less ef-ficiency compared with di- and tri-sumoylated HDAC9. This ismost likely due to a combination the presence of multiple Histags and/or improved access to the tag.

DISCUSSION

In this manuscript, we describe the full and complete cloningand characterization of the ninth member of the histonedeacetylase family, HDAC9. The HDAC9 gene is located at7p21.1, a region implicated in neurological disorders (66–69)and a variety of cancers including colorectal cancer (70), fibro-sarcoma (71), childhood acute lymphoblastic leukemia (72),Wilms’ tumor (65), and peripheral nerve sheath tumors (73).The 3� end of the HDAC9 ORF is located only approximately150 kb from the TWIST gene, which is implicated craniosyn-ostosis-associated Saethre-Chotzen syndrome (74–76). Investi-gation of the genomic neighborhood of the TWIST gene showedthat deletions of DNA that encompassed the HDAC9 locus ledto a more severe phenotype with significant learning diffi-culties (68). The potential involvement of HDAC9 in centralnervous system development and the pathogenesis of Saethre-Chotzen syndrome is corroborated by its expression in thedeveloping brain.

The possibility that gene dosage effects involving HDAC9 areimportant is also underlined by the correlation of increasedHDAC9 expression in colon cancer cell lines with the findingthat around 37% of colon cancers possessed gains of DNAsequence corresponding to 7p21 (70). The HDAC9 gene com-prises 26 exons and spans �500 kb (more than 12 times the sizeof HDAC5 (77) and almost 40 times that of HDAC3 (78)), andthis may render it more susceptible to the effects of genomicinstability. Comparison of the mouse open reading frame withhuman HDAC9 shows a high degree of conservation (data not

shown), including the potential for the same in-frame alterna-tive splicing. This point is reinforced when a comparison ismade of the noncoding genomic DNA contained within themouse and human HDAC9 genes. There are zones of highhomology present, especially in the regions adjacent to theregulated exons 7 and 12.

A sequence representing an incomplete open reading frameof full-length HDAC9 has been recently reported by Zhou et al.(7). It is possible that this sequence is a truncated isoform ofHDAC9 lacking exons 25 and 26. Because sequences encodedby exons 25 and 26 are largely conserved among Class IIHDACs and comprise the very C terminus of the catalyticdomain, it is not surprising that the truncated HDAC9 productreported by Zhou et al. (7) possessed very little deacetylaseactivity (only 10% of the activity of HDAC4). This clearly con-trasts with the activity level of the full-length HDAC9 proteinreported here, which is close to that of HDAC4 (Fig. 4A).

Until recently, expression analyses indicated that both ClassI and II HDACs existed as single isoforms (11–13, 16). This isno longer the case with respect to Class II HDACs becausemajor variants have been detected for HDAC7 and 10 (8, 15,18–20). It should be noted that Zhou et al. (7) also reported thepresence of an alternate isoform of HDAC9 that consisted of atranscript lacking exons 21–26 and encoding 19 residues ofunique sequence, in much the same way as HDAC9�CD. Re-cent studies on HDAC4 have shown that the region encoded byexon 7 contains residues that form a powerful nuclear localiza-tion signal and two serines, which when phosphorylated facil-itate the binding of 14-3-3 signaling proteins (52). These resi-dues are conserved in HDAC9, and proteins lacking the exon7region may have a dramatic effect on the cellular localization ofinteracting factors such as N-CoR (Fig. 7C). The interactionsbetween HDAC9 isoforms and interacting proteins such as

FIG. 9. Expression of endogenous HDAC9 proteins. Whole celllysates were prepared from tsCOS cells transfected with F-HDAC9,F-HDAC9�exon7, F-HDAC9�exon12, and selected cell lines as indi-cated. Total protein levels were normalized, and the samples wereresolved on a 5% SDS-polyacrylamide gel. Western blot analysis wasperformed with affinity-purified rabbit serum containing antibodiesraised against the C-terminal region of HDAC9. Enhanced chemilumi-nescence was used for detection.

FIG. 10. SUMO-1 and -2 are conjugated to HDAC9 but notHDAC9�exon12 in vivo. Whole cell lysates were prepared from ts-COS cells co-transfected with F-HDAC9, F- HDAC9�exon12, and His6-tagged SUMO-1 or -2 as indicated. pSG5 empty vector (Stratagene) wasused as a control plasmid. Samples precipitated with trichloroaceticacid to show input protein levels (lanes 1–6) and nickel pull-downs(lanes 7–12) were analyzed by Western blotting using anti-HDAC9rabbit polyclonal antibody. The open arrowheads indicate mono-, di-,and tri-sumoylated HDAC9. The solid arrowhead refers to a nonspecificband observed with the trichloroacetic acid precipitates.


BCl-6, TEL, or N-CoR merit more detailed investigation, but itseems likely that many of the interactions are mediatedthrough multiple regions in HDAC9 that are specific for a givenpartner protein. This introduces a level of complexity withregard to HDAC9 association with partner proteins not thusfar reported for other family members. It has been suggestedthat HDACs may act as recruiting centers for specific proteincomplexes as well as being functional enzymes (79), and thesedata would tend to be in line with this notion.

The other differentially spliced isoform investigated wasHDAC9�exon12, which also contains potential functional do-mains such as a leucine zipper motif and a sumoylation site.The HDAC9�exon12 isoform cannot be sumoylated and mayprove to exert an important and separate function becauseSUMO is believed to alter the interaction properties of itstargets, often affecting their localization within the cell (80).Moreover, recent studies have shown that sumoylation canaffect HDAC1 and HDAC4 catalytic activity (46, 81).

In summary, the HDAC9 gene encodes multiple, functionallydistinct, and differentially expressed protein isoforms. Generegulation arising from alternative splicing is recognized as anincreasingly important factor in the creation of proteome diver-sity (82, 83). The genomic size and degree to which HDAC9 isregulated is unprecedented among other HDACs, and it per-haps reflects the complexity of a system controlling its activi-ties and may indicate a wider role for its function than justhistone and/or protein modification.

Acknowledgments—We thank Eric Verdin for HDAC3 cDNA, StuartSchreiber and Tony Kouzarides for HDAC4 cDNAs, Eric Oslon forMEF2D cDNA, Riccardo Dalla-Favera for BCL-6 cDNA, and AnneDejean for SUMO-1 and -2 cDNA.

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The histone deacetylase 9 gene encodes multiple protein isoforms

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