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Cloning and Characterization of the Murine Histone Deacetylase (HDAC3)
Biochemical and Biophysical Research Communications 263, 482– 490 (1999) Article ID bbrc.1999.1389, available online at http://www.idealibrary.com on
Cloning and Characterization of the Murine Histone Deacetylase (HDAC3) Ulrich Mahlknecht,* ,1 Dieter Hoelzer,† Richard Bucala,* and Eric Verdin‡ *Picower Institute for Medical Research, 350 Community Drive, Manhasset, New York 11030; †Department of Hematology/ Oncology, University of Frankfurt Medical Center, Frankfurt, Germany; and ‡Gladstone Institute of Virology and Immunology, University of California, San Francisco, San Francisco, California 94141
Received August 18, 1999
Histone acetylation modifiers have been described to participate as cofactors in mammalian transcriptional complexes involved in the regulation of cellular proliferation and differentiation. The acetylation of core histone proteins is reversible and regulated by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increasing evidence suggests a connection between histone acetylation and the development of cancer and leukemia. We have recently mapped HDAC3 to mouse chromosome 18B3, a region which is syntenic with human chromosome 5q31, where HDAC3 is imbedded in a group of potential tumor suppressor genes and which has been reported to be the smallest commonly deleted segment in malignant myeloid disease. We report herein the identification and characterization of HDAC3, a yeast RPD3 ortholog in the mouse. Studies on murine HDAC3 may yield important insights on the understanding of myeloproliferative disease in humans. © 1999 Academic Press Key Words: histones; chromatin; histone deacetylase; chromosomes; genes; structural; tumor suppressor.
The posttranslational modification of nucleosomal histones, which converts regions of chromosomes into transcriptionally active or inactive chromatin, represents one of the key events in the regulation of eukaryotic gene expression. The amino-terminal acetylation of e-NH 31 groups on conserved lysine residues is a reversible process and belongs to the most common posttranslational modifications of histones (1, 2). While histone acetyltransferases (HATs) transfer the acetyl Abbreviations: HDAC, histone deacetylase; HAT, histone acetyltransferase. 1 To whom correspondence should be addressed at the University of Frankfurt Medical Center, Department of Hematology/Oncology, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. Fax: 149-696301-7326. E-mail: [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
moiety from acetyl coenzyme A to the e-NH 31 groups of internal lysine residues and hereby neutralize their positive charge, histone deacetylases (HDACs) are responsible for the opposing deacetylation of histones, which removes acetyl groups and therefore reestablishes their positive charge (3, 4). Hyperacetylation of histones is generally correlated with transcriptionally active chromatin, whereas hypoacetylation is generally associated with transcriptional silencing, which in turn is based mainly on the limited access of activation factors (5) and the concurrent binding of transcriptional repressor complexes to DNA, of which HDACs themselves are part (6, 7). So far, three major human and murine histone deacetylases of the RPD3 type, HDAC1, HDAC2 and HDAC3, have been cloned (8 –12). Their enzymatic activity can be inhibited by trichostatin, trapoxin, butyrate and depudecin (11, 13) and is associated with the activation or repression of specific gene products (14, 15). The first human histone deacetylase to be cloned was HDAC1, which was purified by virtue of its interaction with trapoxin, a potent histone deacetylase inhibitor (8) and which was found to be associated with a corepressor complex including mSin3p and N-Cor (7, 16 –19). HDAC2 in turn was found to interact with YY1, a transcription factor that can act both as an activator or a repressor (9, 20). Only little is known on the interaction of the histone deacetylase HDAC3 with other factors and remains still to be investigated. We have recently reported the identification and chartacterization of human HDAC3 an ortholog of the yeast transcriptional modulator Rpd3, which can be detected predominantly in the nuclear compartment and which is expressed in many different cell lines and multiple normal human tissues (10 –12, 21). Herein, we report the identification and characterization of a third murine histone deacetylase HDAC3, which represents a yeast RPD3 ortholog expressing high homology to human HDAC3 (the gene symbol HDAC3 has been approved by the mouse no-
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Exon/Intron Splice-Junctions of the Murine HDAC3 Gene Exon
Exon
59-Splice donor
Intron
39-Splice acceptor
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
55 83 142 82 57 56 134 81 65 65 90 59 80 158 657
TTCCACTATGgtagggctca GAAGATGATCgtgagtgttg GTGATGACTGgtgagggcag AAACAACAAGgtgatgtaac GAAATTTGAGgtaaggttgc AGCTGCTTAAgtaagtatcg CCTGGAACAGgtagggaata GATGACCAGAgtaagttctg GACGTGCATCgtgctccagg GAGGACATGGgtgcgtgcct CCCGGTGTTGgtgagtgttc CCCTATAGTGgtaagatcac CTCACGCCAGgttagcagct ACTACAGCAGgtctggggca
94 2045 .2.6 kb 126 115 314 677 376 166 1484 111 248 267 3270
ccaccctcagGAGCTGGACA ccactcccagGTCTTCAAGC tgccctgcagCCCAGTGTTT tatctttcagATCTGTGATA ctccccacagGCCTCTGGCT ctcacaccagGTACCACCCT ttttctgtagGTGACATGTA cctctcccagGTTACAAGCA ctttttgcagTGTGGCGCTG tttctaacagGGAATGTGTT ctggtttcagGACATATGAA catctctaagAATACTTCGA tctaaactagTATCTGGACC tgttttacagGCCAGAAGCA
Note. Exon sequences are given in uppercase and intron sequences are given in lowercase. The sizes of the single exons and introns are indicated. Consensus splice donor and splice acceptor sequences are given in bold.
menclature committee at the Jackson Laboratory, Bar Harbor, ME). MATERIALS AND METHODS DNA preparation. BAC DNA was prepared from clone 478F3 according to established protocols (22). Mouse genomic DNA was purchased from Clontech (Palo Alto, CA). Identification of the murine HDAC3 cDNA. A homology search of the EST database at NCBI (National Center for Biotechnology Information) yielded seventeen promising I.M.A.G.E. consortium cDNA clones, which were obtained from Genome Systems (St. Louis, MO) and fully sequenced. Identification of BAC genomic clone 478F3. BAC genomic clones were isolated by screening of an arrayed BAC genomic filter library (Genome Systems) with the [a 32P]dCTP-labeled murine HDAC3 cDNA. Six BAC clones with an average insert size of 120 kb were isolated. Their authenticity was confirmed by DNA sequence analysis. Southern blot. 10 mg of murine genomic DNA or 3 mg BAC DNA were digested with 20 U of the restriction enzymes HindIII, PstI, NcoI, Bgl2, NotI, PmeI (GIBCO BRL, Gaithersburg, MD), and separated by 0.8% agarose gel electrophoresis in 13 TBE (0.1 M Tris, 0.09 M Boric Acid, 0.001 M EDTA) at 1.5 V/cm. Agarose gels were incubated twice for 20 min in denaturing solution (1.5 M NaCl, 0.5 M NaOH) and twice for 20 min in neutralizing solution (1.5 M NaCl, 0.5 M Tris (pH 7.2), 1 mM EDTA), and transferred onto a nylon membrane (Hybond-N, Amersham, Arlington Heights, IL) overnight by capillarity in 203 SSPE (3 M NaCl, 0.2 M NaH 2PO 4, 20 mM EDTA, pH 7.4). DNA was cross-linked to the nylon membranes by exposure to UV light (Biorad GS Gene Linker, Hercules, CA), washed for 20 min in 23 SSPE and prehybridized for 1 h at 55°C in hybridization buffer (Digene, Beltsville, MD). The purified HDAC3 cDNA probe was labeled with [a- 32P]dCTP (DuPont,Wilmington, DE) using the Multiprime DNA labelling system (Amersham), denatured, and added to the prehybridization buffer and allowed to hybridize for at least 16 h at 55°C. Membranes were washed twice for 20 min in 23 SSPE-0.1% SDS, twice for 20 min in 0.23 SSPE-0.1% SDS at room temperature and once for 1 h at 65°C in 0.23 SSPE-0.1% SDS.
Autoradiographic exposures with two intensifying screens were carried out for 1–7 days at 270°C. Instrumental methods. Dye terminator cycle sequencing was performed using the ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA polymerase (Perkin Elmer, Branchburg, NJ) and analyzed with an ABI PRISM 377 DNA sequencer which utilizes the four-color, single-lane sequencing chemistry. PCR methods. The HDAC3 sequence was determined by primer walking on both strands using a direct sequencing strategy (23). Sequencing reactions were performed using 1 mg BAC DNA and 20-30mer oligonucleotide primers (GENSET, La Jolla, CA). Sequencing reactions were set up in a volume of 20 ml containing 10 pmol of the sequencing primer, 8 ml Terminator Ready Reaction Mix (ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit, Perkin Elmer), DNA as indicated and ddH 2 O added up to a final volume of 20 ml. The thermal cycling profile for the sequencing of genomic DNA was as follows: denaturation 10 min at 95°C (1 cycle) followed by 45 cycles alternating between 30 s at 60°C and 30 sec at 95°C and a final storage of the samples at 4°C. The thermal cycling profile for the sequencing of the cDNA-clones was as follows: denaturation at 95°C for 30 s, annealing at 50°C for 15 s, extension at 60°C for 4 min (25 cycles), and storage at 4°C. Sequence analysis and computer database searches. The exon/ intron junctions (Table 1) were determined by comparison of the mouse HDAC3 cDNA (GenBank, Accession No. AF074881) sequence with the murine HDAC3 genomic sequence (GenBank, Accession No. AF079309 and AF079310). Sequences were analyzed with the MacVector sofware (Oxford Molecular Group PLC). Sequence comparisons were performed with the BLAST algorithm of the GenBank and EMBL databases. Protein Motifs were identified online at http:// expasy.hcuge.ch with the program PROSITE and double-checked using the MotifFinder program at http://www.genome.ad.jp, but still remain to be experimentally confirmed (Fig. 2). Expression analysis. A multiple tissue Northern blot filter carrying 2 mg of poly(A) 1 RNA from different adult mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis was purchased from Clontech. Hybridization was carried out in ExpressHyb hybrid-
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FIG. 1. The complete sequence of HDAC3 cDNA together with the predicted amino acid sequence is shown with the location of each intron with respect to the cDNA sequence. The HDAC3 cDNA is 1975 bp long and yields a protein of 424 amino acids. A splice variant of murine HDAC3 did not include nucleotides 205 to 777 (boxed) of the consensus sequence and was predicted to translate into an open reading frame of 233 amino acids. CpG-rich elements in the 59 upstream promoter region are italicized and putative transcription factor binding sites in the promoter region are underlined. The translational start (ATG) and stop codons (TGA) are double underlined, the polyadenylation signal (AATAAA) is boxed.
ization solution (Clontech) at 65°C for 1 h using 10 6 cpm/ml of a 32P random-primed labeled mouse HDAC3 cDNA probe. The filter was washed in 23 SSC, 0.5% SDS at room temperature for 30 min and
then in 0.13 SSC, 0.1% SDS at 50°C for 40 min. The filter was then exposed to BioMax films (Kodak) with intensifying screens for 24 h in the short exposure or for 7 days in the long exposure.
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FIG. 2. Genomic organization of the murine HDAC3 gene. The genomic organization of the HDAC3 gene, which includes the relative position of exons and introns is shown. Two potential phosphorylation sites (PKC phosphorylation site in exon VII, TK phosphorylation site in exon VIII) and a N-glycosylation site in exon X are conserved among all members of the family of RPD3 histone deacetylases and are indicated with their relative position. Repetitive sequences, known as short and long interspersed nuclear elements (SINEs and LINE), are also indicated.
Phylogenetic analysis. Phylogenetic trees were constructed from sequences of different species which were obtained from a protein sequence similarity search with the HDAC3 protein and nucleotide sequences using the BLAST 2.0 program at NCBI database (24). Progressive multiple sequence alignments were performed with the CLUSTAL W Multiple Alignment Program Version 1.7 (25). Trees were calculated using the PUZZLE Software, which is a computer program to reconstruct phylogenetic trees from molecular sequence data by maximum likelihood (26). In maximum likelihood methods one searches for the maximum likelihood value for the character state configurations among the sequences under study for each possible tree and chooses the one with the largest likelihood value as the preferred tree. The obtained data were then drawn with the TREEVIEW software, which is a program for displaying and printing phylogenies (27). The numbers along branches represent percentage values for bootstrap statistical support. The scale bar indicates the genetic distance of the branch lengths.
RESULTS Cloning of murine cDNAs encoding a novel member of the histone deacetylase family. Homology searches of the dbEST at NCBI (National Center for Biotechnology Information) (28) using the 428-amino-acid human HDAC3 sequence identified seventeen murine ESTs as potential novel HDAC3 homologs. Sequence analysis of the corresponding cDNA clones, which were obtained from Genome Systems (St. Louis, MO) found EST clone 1122558 (Accession AA638123) to contain the full length 1975 bp murine HDAC3 cDNA (GenBank AF074881) with an open reading frame of 1272 bp that would be predicted to produce a 424 aa protein with a molecular weight of 48.4 kD, and EST clone 554310 (Accession AA102998) to contain a 1402 bp long variant of murine HDAC3, which did not include nucleotides 205 to 777 of the consensus sequence (GenBank AF074882) and which would translate into an open reading frame of 233 aa with a predicted molecular weight of 26.9 kD, suggesting the presence of alternative splicing. When aligned with sequences of previ-
ously identified HDAC3 homologs derived from the NCBI databases and from unpublished studies, human and murine HDAC3 are 92% identical on the DNA level, while the translated human and murine HDAC3 sequences are 99% identical. The ORF was preceded by translational stop codons in all reading frames and the context of the predicted initiating methionine exhibited agreement with the Kozak consensus (ACCATGG) for the initiation of translation (29). The transcriptional START site for murine HDAC3 is most likely located at or around 222 bp upstream of the ATG translational initiation codon, since this is the uppermost position which could be sequenced from the murine HDAC3 transcript. This however, awaits to be confirmed by ribonuclease protection analysis. Since murine HDAC3 contains a pinpoint mutation in position 1273 (insertion of a thymine), a frame shift changes the asparagine in position 424 (GAT) to a termination signal TGA(T). Thus, murine HDAC3 is prematurely terminated, being 4 aa shorter than human HDAC3. The basis for the use of splice donors and acceptor sites differing from those predicted by the GT-AG rule at these positions in our alternatively spliced clone is unclear so far. The polyadenylation consensus sequence AAUAAA (30) was identified 15 bp upstream of the polyadenylation site (Fig. 1). Identification and structure of the murine HDAC3 genomic locus. BAC genomic clones were isolated by screening of an arrayed BAC genomic filter library (Genome Systems) by hybridization with the [a 32P]dCTP-labeled murine HDAC3 cDNA. Six BAC clones with an average insert size of 120 kb were isolated. Their authenticity was confirmed by DNA sequence analysis. One of the clones (BAC-478F3) was found to contain the complete HDAC3 gene and was therefore used for sequencing. The entire sequence of
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FIG. 3—Continued
the gene has been determined, except for one gap in intron III where direct sequencing was not feasible due to repeated primer mispairing to long-stretched regions of multiple repetitive elements on both strands (Fig. 2) (GenBank AF079309 and AF079310). Murine HDAC3 was found to encompass at least 14 kb in length (exclusive of the promoter region) consisting of 15 coding exons ranging between 55 bp and 692 bp in size. The last exon (XV) however, is only partially translated (93 bp out of 692 bp). We have also sequenced approximately 1 kb of the 59 flanking promoter region, which was found to be rich in CpG elements, while it was lacking the canonical TATA- and CAAT-boxes. The pattern of putative transcription factor binding sites was similar to what we had found within the human HDAC3 promoter (unpublished observation). The genomic structure of murine HDAC3 was determined by comparison of the HDAC3 genomic and cDNA sequences and is shown schematically in Fig. 2, while the sequences and locations of intron– exon borders are given in Table 1. This information will be useful for identifying HDAC3 mutations and eventually for evaluating the role of such mutations in human disease. Gene copy number analysis. Southern blot analysis using the full-length mouse HDAC3 cDNA as a probe revealed multiple bands in HindIII, PstI, NcoI, Bgl2, NotI, and PmeI-digested mouse genomic and BAC478F3 DNAs (data not shown). A slight difference in migration between the BAC and murine genomic DNAs was due to load effects. Linear regression analysis of the density of one of the bands from BAC-478F3 was used to determine the HDAC3 copy number, which was found to correspond to one copy of BAC-478F3. This indicated that HDAC3 is present as a single-copy
gene. This observation was further supported by recently reported fluorescence in situ hybridization (FISH) studies, which showed one single site of hybridization on murine metaphase chromosomes for mouse HDAC3 (31). Northern blot analysis of HDAC3 expression. The expression of mouse HDAC3 was examined by Northern blot analysis. A 1975-bp HDAC3 cDNA labeled with [a- 32P]dCTP was hybridized to a filter blotted with approximately 2 mg of poly(A) 1 RNA from eight mouse tissues. Because of some inconsistencies in the preparation, the actual amount of RNA which has been loaded onto each lane is somewhat different. Therefore the same blot was stripped and rehybridized with the b-actin cDNA. If the same amount of RNA was loaded, the b-actin-band would have been expected to be the same for each lane. However, the amount of RNA loaded onto each lane is different (Fig. 4 lower panel), and needs to be taken into consideration when the tissue-specific expression of HDAC3 is interpreted (Fig. 4, upper panel). A transcript of ;2 kb was detected in all the samples except on the lane containing mRNA from mouse spleen tissue. An additional transcript of ;3 kb was detected on the lanes containing mRNA from mouse brain, liver and testicular tissues. Interestingly, the tissue-specific expression of murine HDAC3 is completely different from the pattern of ubiquitous expression in all tissues which had been tested (12, 11). Murine HDAC3 is significantly overexpressed in testicular tissue, while it is practically absent in the spleen. The expression in the heart seems similar to that seen in the liver, the brain and the kidneys, while the expression in lung and skeletal muscle tissues seems rather to be decreased.
FIG. 3. (a) Amino acid sequence alignment of HDAC3 homologues: (Accession numbers of the sequences used in this alignment: yeast RPD3 (P32561), human HDAC1 (Q13547), human HDAC2 (Q92769), human HDAC3 (AF039703), mouse HDAC1 (O09106), mouse HDAC2 (P70288), mouse HDAC3 (AF074881), chicken HDAC1 (AF043328), chicken HDAC2 (P56519), chicken HDAC3 (P56520), Xenopus laevis HDAC1 (O42227), Strongylocentrotus purpuratus HDAC1 (P56518), Drosophila melanogaster HDAC1 (AF026949), C. elegans HDAC1 (O17695), A. thaliana HDAC1 (O22446), Zea mays HDAC1 (P56521). (b) Comparison of the protein sequences of HDAC3 homologues. The numbers represent the percentages of identity/similarity with the upper right indicating sequence identity and the lower left indicating sequence similarity.
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Phylogenetic analysis. We have screened the expressed sequence tag database (NCBI) with the yeast RPD3 protein sequence and identified several yeast RPD3 histone deacetylase orthologs, which were sharing a significant degree of sequence identity with RPD3, indicating a high degree of phylogenetic conservation of protein structure and associated function throughout evolution. A consensus evolutionary tree was obtained using an alignment of RPD3 and orthologs from 15 species. The tree was constructed after bootstrapping as outlined under Materials and Methods (Fig. 5). DISCUSSION Histone acetylation modifiers have been described to participate as cofactors in mammalian transcriptional complexes involved in the regulation of cellular proliferation and differentiation. In addition, increasing evidence implicates a connection between cancer and transcriptional control by histone acetylation and deacetylation (32, 33). HDAC1, HDAC2 and HDAC3 are orthologs of the yeast RPD3 family of histone deacetylases and are highly conserved throughout evolution. We have recently identified and characterized human HDAC3 (11), which is located on chromosome 5q31 (21) in a region, which is known to be the smallest commonly deleted region of chromosome 5 in malignant myeloid disease (34). Since we became interested in studying HDAC3 in the mouse model, we recently localized the murine HDAC3 gene to chromosome 18B3 (31), within a group of growth factors and putative tumor suppressor genes, which are syntenic with the distal portion of human chromosome 5q (35). In the present study, we report both the identification, cloning and determination of two murine HDAC3 cDNAs and of the HDAC3 murine genomic sequence. Murine HDAC3 is a single-copy gene spanning a region of at least 14 kb. The gene was fully sequenced, except for one gap, within intron III, which could not be determined due to repeated primer mispairing to longstretched regions of multiple repetitive elements on both strands. The structure of the gene is similar to the previously characterized human HDAC3 gene with 15 exons ranging between 55 bp and 692 bp in size and conserved splice junction positions. The gene is controlled by a promoter which is rich in CpG elements and lacks the canonical TATA- and CAAT-boxes. HDAC3 is highly homologous to members of the RPD3 and HDA1 families of histone deacetylases at both the DNA and the protein levels (Fig. 3). This homology includes a cluster of exons (IV-X), which has been reported to contain the catalytic core of HDACs (36) and which seems to be the most conserved domain among all known RPD3-related proteins, including species having branched off early during evolution, like Caenorhabditis elegans and Arabidopsis thaliana. Inter-
FIG. 4. (Upper panel) Mouse multiple tissue Northern blot analysis showing an overexpression of HDAC3 in testicular tissue, while the gene seems not to be significantly expressed in the spleen. (Lower panel) Taking the expression of b-ACTIN into consideration, the relative amount of loaded RNA seems highest for skeletal muscle tissue and lowest for kidney tissue.
estingly, the alternatively spliced variant of murine HDAC3 is missing exactly this highly conserved cluster ranging from exons IV to X, which seems to be critical for the enzymatic activity of histone deacetylases, therefore suggesting that HDAC3 might also have functions other then the deacetylation of histones. In addition, several repetitive sequences, known as short (SINES) and long (LINES) interspersed nuclear elements, which essentially consist of sequentially aligned Alu (AGCT) restriction elements (SINES) on one hand and sets of KpnI and BamHI segments on the other hand (37), were found (Fig. 2). In evolutionary terms, they have probably contributed to genetic differences between species and individuals by playing a role in retrotransposition events and in promoting unequal crossing-over. While human HDAC3 can be detected predominantly in the nuclear compartment and is ubiquitously expressed in many different cell lines and multiple normal human tissues (10 –12), the mRNA-expression pattern of murine HDAC3 is surprisingly different and shows an overexperssion of HDAC3 in testicular tissue, while it is not significantly expressed in the spleen (Fig. 4). An extensive exploration of the 59 flanking promoter region may help to explain this phenomenon and awaits to be further studied experimentally. Since the steady-state of histone acetylation and deacetylation plays a key role in the regulation of transcription, the loss of genetic material, resulting from chromosomal loss or deletion or from other mechanisms, with the consequence of the development of HDAC3 hemizygosity, might lead to a gene dosage effect or the unmasking of a recessive allele, such as a tumor-suppressor gene, on the cytogenetically “normal” homolog. As a consequence, the steady-state of
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FIG. 5. HDAC phylogenetic tree. This tree is based on the yeast RPD3 protein sequence and includes only orthologs of the RPD3 family of histone deacetylases. The numbers along branches represent percentage values for bootstrap statistical support. The scale bar indicates the genetic distance of the branch lengths (Accession numbers of the sequences used in this tree: yeast RPD3 (P32561), human HDAC1 (Q13547), human HDAC2 (Q92769), human HDAC3 (AF039703), mouse HDAC1 (O09106), mouse HDAC2 (P70288), mouse HDAC3 (AF074881), chicken HDAC1 (AF043328), chicken HDAC2 (P56519), chicken HDAC3 (P56520), Xenopus laevis HDAC1 (O42227), Strongylocentrotus purpuratus HDAC1 (P56518), Drosophila melanogaster HDAC1 (AF026949), C. elegans HDAC1 (O17695), A. thaliana HDAC1 (O22446), Zea mays HDAC1 (P56521).
histone acetylation may be shifted toward acetylation at the level of specific genes targeted by HDAC3 and either upregulate or downregulate transcriptional events (14, 15). Therefore, the identification of a consistent loss of HDAC3 (band 18B3 in the mouse, band 5q31 in humans) implies that loss of genes critical for
normal myeloid growth and differentiation may uncover inactivating mutations on the remaining allele (monoallelic expression, loss of heterozygosity). However, limited available material, the paucity of highly polymorphic loci both on the distal arm of chromsome 5 and mouse chromosome 18B3, and the technical limi-
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tations in separating the affected cells from the normal cell population, have rendered heterozygosity analyses particularly arduous. Concluding, the mouse HDAC3 gene displays striking similarity to the previously characterized human HDAC3 gene both on the genomic and the protein levels, which suggests also similarly close functional properties. Studies on HDAC3 in the mouse model may therefore help to improve the understanding of the pathogenesis of myeloproliferative disease in humans. ACKNOWLEDGMENTS This work was supported by the German National Science Foundation (Deutsche Forschungsgemeinschaft, MA 2057/1-1) and institutional funds from the Picower Institute for Medical Research.
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Cloning and Characterization of the Murine Histone Deacetylase (HDAC3) Ulrich Mahlknecht,* ,1 Dieter Hoelzer,† Richard Bucala,* and Eric Verdin‡ *Picower Institute for Medical Research, 350 Community Drive, Manhasset, New York 11030; †Department of Hematology/ Oncology, University of Frankfurt Medical Center, Frankfurt, Germany; and ‡Gladstone Institute of Virology and Immunology, University of California, San Francisco, San Francisco, California 94141
Received August 18, 1999
Histone acetylation modifiers have been described to participate as cofactors in mammalian transcriptional complexes involved in the regulation of cellular proliferation and differentiation. The acetylation of core histone proteins is reversible and regulated by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increasing evidence suggests a connection between histone acetylation and the development of cancer and leukemia. We have recently mapped HDAC3 to mouse chromosome 18B3, a region which is syntenic with human chromosome 5q31, where HDAC3 is imbedded in a group of potential tumor suppressor genes and which has been reported to be the smallest commonly deleted segment in malignant myeloid disease. We report herein the identification and characterization of HDAC3, a yeast RPD3 ortholog in the mouse. Studies on murine HDAC3 may yield important insights on the understanding of myeloproliferative disease in humans. © 1999 Academic Press Key Words: histones; chromatin; histone deacetylase; chromosomes; genes; structural; tumor suppressor.
The posttranslational modification of nucleosomal histones, which converts regions of chromosomes into transcriptionally active or inactive chromatin, represents one of the key events in the regulation of eukaryotic gene expression. The amino-terminal acetylation of e-NH 31 groups on conserved lysine residues is a reversible process and belongs to the most common posttranslational modifications of histones (1, 2). While histone acetyltransferases (HATs) transfer the acetyl Abbreviations: HDAC, histone deacetylase; HAT, histone acetyltransferase. 1 To whom correspondence should be addressed at the University of Frankfurt Medical Center, Department of Hematology/Oncology, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. Fax: 149-696301-7326. E-mail: [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
moiety from acetyl coenzyme A to the e-NH 31 groups of internal lysine residues and hereby neutralize their positive charge, histone deacetylases (HDACs) are responsible for the opposing deacetylation of histones, which removes acetyl groups and therefore reestablishes their positive charge (3, 4). Hyperacetylation of histones is generally correlated with transcriptionally active chromatin, whereas hypoacetylation is generally associated with transcriptional silencing, which in turn is based mainly on the limited access of activation factors (5) and the concurrent binding of transcriptional repressor complexes to DNA, of which HDACs themselves are part (6, 7). So far, three major human and murine histone deacetylases of the RPD3 type, HDAC1, HDAC2 and HDAC3, have been cloned (8 –12). Their enzymatic activity can be inhibited by trichostatin, trapoxin, butyrate and depudecin (11, 13) and is associated with the activation or repression of specific gene products (14, 15). The first human histone deacetylase to be cloned was HDAC1, which was purified by virtue of its interaction with trapoxin, a potent histone deacetylase inhibitor (8) and which was found to be associated with a corepressor complex including mSin3p and N-Cor (7, 16 –19). HDAC2 in turn was found to interact with YY1, a transcription factor that can act both as an activator or a repressor (9, 20). Only little is known on the interaction of the histone deacetylase HDAC3 with other factors and remains still to be investigated. We have recently reported the identification and chartacterization of human HDAC3 an ortholog of the yeast transcriptional modulator Rpd3, which can be detected predominantly in the nuclear compartment and which is expressed in many different cell lines and multiple normal human tissues (10 –12, 21). Herein, we report the identification and characterization of a third murine histone deacetylase HDAC3, which represents a yeast RPD3 ortholog expressing high homology to human HDAC3 (the gene symbol HDAC3 has been approved by the mouse no-
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Exon/Intron Splice-Junctions of the Murine HDAC3 Gene Exon
Exon
59-Splice donor
Intron
39-Splice acceptor
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
55 83 142 82 57 56 134 81 65 65 90 59 80 158 657
TTCCACTATGgtagggctca GAAGATGATCgtgagtgttg GTGATGACTGgtgagggcag AAACAACAAGgtgatgtaac GAAATTTGAGgtaaggttgc AGCTGCTTAAgtaagtatcg CCTGGAACAGgtagggaata GATGACCAGAgtaagttctg GACGTGCATCgtgctccagg GAGGACATGGgtgcgtgcct CCCGGTGTTGgtgagtgttc CCCTATAGTGgtaagatcac CTCACGCCAGgttagcagct ACTACAGCAGgtctggggca
94 2045 .2.6 kb 126 115 314 677 376 166 1484 111 248 267 3270
ccaccctcagGAGCTGGACA ccactcccagGTCTTCAAGC tgccctgcagCCCAGTGTTT tatctttcagATCTGTGATA ctccccacagGCCTCTGGCT ctcacaccagGTACCACCCT ttttctgtagGTGACATGTA cctctcccagGTTACAAGCA ctttttgcagTGTGGCGCTG tttctaacagGGAATGTGTT ctggtttcagGACATATGAA catctctaagAATACTTCGA tctaaactagTATCTGGACC tgttttacagGCCAGAAGCA
Note. Exon sequences are given in uppercase and intron sequences are given in lowercase. The sizes of the single exons and introns are indicated. Consensus splice donor and splice acceptor sequences are given in bold.
menclature committee at the Jackson Laboratory, Bar Harbor, ME). MATERIALS AND METHODS DNA preparation. BAC DNA was prepared from clone 478F3 according to established protocols (22). Mouse genomic DNA was purchased from Clontech (Palo Alto, CA). Identification of the murine HDAC3 cDNA. A homology search of the EST database at NCBI (National Center for Biotechnology Information) yielded seventeen promising I.M.A.G.E. consortium cDNA clones, which were obtained from Genome Systems (St. Louis, MO) and fully sequenced. Identification of BAC genomic clone 478F3. BAC genomic clones were isolated by screening of an arrayed BAC genomic filter library (Genome Systems) with the [a 32P]dCTP-labeled murine HDAC3 cDNA. Six BAC clones with an average insert size of 120 kb were isolated. Their authenticity was confirmed by DNA sequence analysis. Southern blot. 10 mg of murine genomic DNA or 3 mg BAC DNA were digested with 20 U of the restriction enzymes HindIII, PstI, NcoI, Bgl2, NotI, PmeI (GIBCO BRL, Gaithersburg, MD), and separated by 0.8% agarose gel electrophoresis in 13 TBE (0.1 M Tris, 0.09 M Boric Acid, 0.001 M EDTA) at 1.5 V/cm. Agarose gels were incubated twice for 20 min in denaturing solution (1.5 M NaCl, 0.5 M NaOH) and twice for 20 min in neutralizing solution (1.5 M NaCl, 0.5 M Tris (pH 7.2), 1 mM EDTA), and transferred onto a nylon membrane (Hybond-N, Amersham, Arlington Heights, IL) overnight by capillarity in 203 SSPE (3 M NaCl, 0.2 M NaH 2PO 4, 20 mM EDTA, pH 7.4). DNA was cross-linked to the nylon membranes by exposure to UV light (Biorad GS Gene Linker, Hercules, CA), washed for 20 min in 23 SSPE and prehybridized for 1 h at 55°C in hybridization buffer (Digene, Beltsville, MD). The purified HDAC3 cDNA probe was labeled with [a- 32P]dCTP (DuPont,Wilmington, DE) using the Multiprime DNA labelling system (Amersham), denatured, and added to the prehybridization buffer and allowed to hybridize for at least 16 h at 55°C. Membranes were washed twice for 20 min in 23 SSPE-0.1% SDS, twice for 20 min in 0.23 SSPE-0.1% SDS at room temperature and once for 1 h at 65°C in 0.23 SSPE-0.1% SDS.
Autoradiographic exposures with two intensifying screens were carried out for 1–7 days at 270°C. Instrumental methods. Dye terminator cycle sequencing was performed using the ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA polymerase (Perkin Elmer, Branchburg, NJ) and analyzed with an ABI PRISM 377 DNA sequencer which utilizes the four-color, single-lane sequencing chemistry. PCR methods. The HDAC3 sequence was determined by primer walking on both strands using a direct sequencing strategy (23). Sequencing reactions were performed using 1 mg BAC DNA and 20-30mer oligonucleotide primers (GENSET, La Jolla, CA). Sequencing reactions were set up in a volume of 20 ml containing 10 pmol of the sequencing primer, 8 ml Terminator Ready Reaction Mix (ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit, Perkin Elmer), DNA as indicated and ddH 2 O added up to a final volume of 20 ml. The thermal cycling profile for the sequencing of genomic DNA was as follows: denaturation 10 min at 95°C (1 cycle) followed by 45 cycles alternating between 30 s at 60°C and 30 sec at 95°C and a final storage of the samples at 4°C. The thermal cycling profile for the sequencing of the cDNA-clones was as follows: denaturation at 95°C for 30 s, annealing at 50°C for 15 s, extension at 60°C for 4 min (25 cycles), and storage at 4°C. Sequence analysis and computer database searches. The exon/ intron junctions (Table 1) were determined by comparison of the mouse HDAC3 cDNA (GenBank, Accession No. AF074881) sequence with the murine HDAC3 genomic sequence (GenBank, Accession No. AF079309 and AF079310). Sequences were analyzed with the MacVector sofware (Oxford Molecular Group PLC). Sequence comparisons were performed with the BLAST algorithm of the GenBank and EMBL databases. Protein Motifs were identified online at http:// expasy.hcuge.ch with the program PROSITE and double-checked using the MotifFinder program at http://www.genome.ad.jp, but still remain to be experimentally confirmed (Fig. 2). Expression analysis. A multiple tissue Northern blot filter carrying 2 mg of poly(A) 1 RNA from different adult mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis was purchased from Clontech. Hybridization was carried out in ExpressHyb hybrid-
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FIG. 1. The complete sequence of HDAC3 cDNA together with the predicted amino acid sequence is shown with the location of each intron with respect to the cDNA sequence. The HDAC3 cDNA is 1975 bp long and yields a protein of 424 amino acids. A splice variant of murine HDAC3 did not include nucleotides 205 to 777 (boxed) of the consensus sequence and was predicted to translate into an open reading frame of 233 amino acids. CpG-rich elements in the 59 upstream promoter region are italicized and putative transcription factor binding sites in the promoter region are underlined. The translational start (ATG) and stop codons (TGA) are double underlined, the polyadenylation signal (AATAAA) is boxed.
ization solution (Clontech) at 65°C for 1 h using 10 6 cpm/ml of a 32P random-primed labeled mouse HDAC3 cDNA probe. The filter was washed in 23 SSC, 0.5% SDS at room temperature for 30 min and
then in 0.13 SSC, 0.1% SDS at 50°C for 40 min. The filter was then exposed to BioMax films (Kodak) with intensifying screens for 24 h in the short exposure or for 7 days in the long exposure.
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FIG. 2. Genomic organization of the murine HDAC3 gene. The genomic organization of the HDAC3 gene, which includes the relative position of exons and introns is shown. Two potential phosphorylation sites (PKC phosphorylation site in exon VII, TK phosphorylation site in exon VIII) and a N-glycosylation site in exon X are conserved among all members of the family of RPD3 histone deacetylases and are indicated with their relative position. Repetitive sequences, known as short and long interspersed nuclear elements (SINEs and LINE), are also indicated.
Phylogenetic analysis. Phylogenetic trees were constructed from sequences of different species which were obtained from a protein sequence similarity search with the HDAC3 protein and nucleotide sequences using the BLAST 2.0 program at NCBI database (24). Progressive multiple sequence alignments were performed with the CLUSTAL W Multiple Alignment Program Version 1.7 (25). Trees were calculated using the PUZZLE Software, which is a computer program to reconstruct phylogenetic trees from molecular sequence data by maximum likelihood (26). In maximum likelihood methods one searches for the maximum likelihood value for the character state configurations among the sequences under study for each possible tree and chooses the one with the largest likelihood value as the preferred tree. The obtained data were then drawn with the TREEVIEW software, which is a program for displaying and printing phylogenies (27). The numbers along branches represent percentage values for bootstrap statistical support. The scale bar indicates the genetic distance of the branch lengths.
RESULTS Cloning of murine cDNAs encoding a novel member of the histone deacetylase family. Homology searches of the dbEST at NCBI (National Center for Biotechnology Information) (28) using the 428-amino-acid human HDAC3 sequence identified seventeen murine ESTs as potential novel HDAC3 homologs. Sequence analysis of the corresponding cDNA clones, which were obtained from Genome Systems (St. Louis, MO) found EST clone 1122558 (Accession AA638123) to contain the full length 1975 bp murine HDAC3 cDNA (GenBank AF074881) with an open reading frame of 1272 bp that would be predicted to produce a 424 aa protein with a molecular weight of 48.4 kD, and EST clone 554310 (Accession AA102998) to contain a 1402 bp long variant of murine HDAC3, which did not include nucleotides 205 to 777 of the consensus sequence (GenBank AF074882) and which would translate into an open reading frame of 233 aa with a predicted molecular weight of 26.9 kD, suggesting the presence of alternative splicing. When aligned with sequences of previ-
ously identified HDAC3 homologs derived from the NCBI databases and from unpublished studies, human and murine HDAC3 are 92% identical on the DNA level, while the translated human and murine HDAC3 sequences are 99% identical. The ORF was preceded by translational stop codons in all reading frames and the context of the predicted initiating methionine exhibited agreement with the Kozak consensus (ACCATGG) for the initiation of translation (29). The transcriptional START site for murine HDAC3 is most likely located at or around 222 bp upstream of the ATG translational initiation codon, since this is the uppermost position which could be sequenced from the murine HDAC3 transcript. This however, awaits to be confirmed by ribonuclease protection analysis. Since murine HDAC3 contains a pinpoint mutation in position 1273 (insertion of a thymine), a frame shift changes the asparagine in position 424 (GAT) to a termination signal TGA(T). Thus, murine HDAC3 is prematurely terminated, being 4 aa shorter than human HDAC3. The basis for the use of splice donors and acceptor sites differing from those predicted by the GT-AG rule at these positions in our alternatively spliced clone is unclear so far. The polyadenylation consensus sequence AAUAAA (30) was identified 15 bp upstream of the polyadenylation site (Fig. 1). Identification and structure of the murine HDAC3 genomic locus. BAC genomic clones were isolated by screening of an arrayed BAC genomic filter library (Genome Systems) by hybridization with the [a 32P]dCTP-labeled murine HDAC3 cDNA. Six BAC clones with an average insert size of 120 kb were isolated. Their authenticity was confirmed by DNA sequence analysis. One of the clones (BAC-478F3) was found to contain the complete HDAC3 gene and was therefore used for sequencing. The entire sequence of
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FIG. 3—Continued
the gene has been determined, except for one gap in intron III where direct sequencing was not feasible due to repeated primer mispairing to long-stretched regions of multiple repetitive elements on both strands (Fig. 2) (GenBank AF079309 and AF079310). Murine HDAC3 was found to encompass at least 14 kb in length (exclusive of the promoter region) consisting of 15 coding exons ranging between 55 bp and 692 bp in size. The last exon (XV) however, is only partially translated (93 bp out of 692 bp). We have also sequenced approximately 1 kb of the 59 flanking promoter region, which was found to be rich in CpG elements, while it was lacking the canonical TATA- and CAAT-boxes. The pattern of putative transcription factor binding sites was similar to what we had found within the human HDAC3 promoter (unpublished observation). The genomic structure of murine HDAC3 was determined by comparison of the HDAC3 genomic and cDNA sequences and is shown schematically in Fig. 2, while the sequences and locations of intron– exon borders are given in Table 1. This information will be useful for identifying HDAC3 mutations and eventually for evaluating the role of such mutations in human disease. Gene copy number analysis. Southern blot analysis using the full-length mouse HDAC3 cDNA as a probe revealed multiple bands in HindIII, PstI, NcoI, Bgl2, NotI, and PmeI-digested mouse genomic and BAC478F3 DNAs (data not shown). A slight difference in migration between the BAC and murine genomic DNAs was due to load effects. Linear regression analysis of the density of one of the bands from BAC-478F3 was used to determine the HDAC3 copy number, which was found to correspond to one copy of BAC-478F3. This indicated that HDAC3 is present as a single-copy
gene. This observation was further supported by recently reported fluorescence in situ hybridization (FISH) studies, which showed one single site of hybridization on murine metaphase chromosomes for mouse HDAC3 (31). Northern blot analysis of HDAC3 expression. The expression of mouse HDAC3 was examined by Northern blot analysis. A 1975-bp HDAC3 cDNA labeled with [a- 32P]dCTP was hybridized to a filter blotted with approximately 2 mg of poly(A) 1 RNA from eight mouse tissues. Because of some inconsistencies in the preparation, the actual amount of RNA which has been loaded onto each lane is somewhat different. Therefore the same blot was stripped and rehybridized with the b-actin cDNA. If the same amount of RNA was loaded, the b-actin-band would have been expected to be the same for each lane. However, the amount of RNA loaded onto each lane is different (Fig. 4 lower panel), and needs to be taken into consideration when the tissue-specific expression of HDAC3 is interpreted (Fig. 4, upper panel). A transcript of ;2 kb was detected in all the samples except on the lane containing mRNA from mouse spleen tissue. An additional transcript of ;3 kb was detected on the lanes containing mRNA from mouse brain, liver and testicular tissues. Interestingly, the tissue-specific expression of murine HDAC3 is completely different from the pattern of ubiquitous expression in all tissues which had been tested (12, 11). Murine HDAC3 is significantly overexpressed in testicular tissue, while it is practically absent in the spleen. The expression in the heart seems similar to that seen in the liver, the brain and the kidneys, while the expression in lung and skeletal muscle tissues seems rather to be decreased.
FIG. 3. (a) Amino acid sequence alignment of HDAC3 homologues: (Accession numbers of the sequences used in this alignment: yeast RPD3 (P32561), human HDAC1 (Q13547), human HDAC2 (Q92769), human HDAC3 (AF039703), mouse HDAC1 (O09106), mouse HDAC2 (P70288), mouse HDAC3 (AF074881), chicken HDAC1 (AF043328), chicken HDAC2 (P56519), chicken HDAC3 (P56520), Xenopus laevis HDAC1 (O42227), Strongylocentrotus purpuratus HDAC1 (P56518), Drosophila melanogaster HDAC1 (AF026949), C. elegans HDAC1 (O17695), A. thaliana HDAC1 (O22446), Zea mays HDAC1 (P56521). (b) Comparison of the protein sequences of HDAC3 homologues. The numbers represent the percentages of identity/similarity with the upper right indicating sequence identity and the lower left indicating sequence similarity.
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Phylogenetic analysis. We have screened the expressed sequence tag database (NCBI) with the yeast RPD3 protein sequence and identified several yeast RPD3 histone deacetylase orthologs, which were sharing a significant degree of sequence identity with RPD3, indicating a high degree of phylogenetic conservation of protein structure and associated function throughout evolution. A consensus evolutionary tree was obtained using an alignment of RPD3 and orthologs from 15 species. The tree was constructed after bootstrapping as outlined under Materials and Methods (Fig. 5). DISCUSSION Histone acetylation modifiers have been described to participate as cofactors in mammalian transcriptional complexes involved in the regulation of cellular proliferation and differentiation. In addition, increasing evidence implicates a connection between cancer and transcriptional control by histone acetylation and deacetylation (32, 33). HDAC1, HDAC2 and HDAC3 are orthologs of the yeast RPD3 family of histone deacetylases and are highly conserved throughout evolution. We have recently identified and characterized human HDAC3 (11), which is located on chromosome 5q31 (21) in a region, which is known to be the smallest commonly deleted region of chromosome 5 in malignant myeloid disease (34). Since we became interested in studying HDAC3 in the mouse model, we recently localized the murine HDAC3 gene to chromosome 18B3 (31), within a group of growth factors and putative tumor suppressor genes, which are syntenic with the distal portion of human chromosome 5q (35). In the present study, we report both the identification, cloning and determination of two murine HDAC3 cDNAs and of the HDAC3 murine genomic sequence. Murine HDAC3 is a single-copy gene spanning a region of at least 14 kb. The gene was fully sequenced, except for one gap, within intron III, which could not be determined due to repeated primer mispairing to longstretched regions of multiple repetitive elements on both strands. The structure of the gene is similar to the previously characterized human HDAC3 gene with 15 exons ranging between 55 bp and 692 bp in size and conserved splice junction positions. The gene is controlled by a promoter which is rich in CpG elements and lacks the canonical TATA- and CAAT-boxes. HDAC3 is highly homologous to members of the RPD3 and HDA1 families of histone deacetylases at both the DNA and the protein levels (Fig. 3). This homology includes a cluster of exons (IV-X), which has been reported to contain the catalytic core of HDACs (36) and which seems to be the most conserved domain among all known RPD3-related proteins, including species having branched off early during evolution, like Caenorhabditis elegans and Arabidopsis thaliana. Inter-
FIG. 4. (Upper panel) Mouse multiple tissue Northern blot analysis showing an overexpression of HDAC3 in testicular tissue, while the gene seems not to be significantly expressed in the spleen. (Lower panel) Taking the expression of b-ACTIN into consideration, the relative amount of loaded RNA seems highest for skeletal muscle tissue and lowest for kidney tissue.
estingly, the alternatively spliced variant of murine HDAC3 is missing exactly this highly conserved cluster ranging from exons IV to X, which seems to be critical for the enzymatic activity of histone deacetylases, therefore suggesting that HDAC3 might also have functions other then the deacetylation of histones. In addition, several repetitive sequences, known as short (SINES) and long (LINES) interspersed nuclear elements, which essentially consist of sequentially aligned Alu (AGCT) restriction elements (SINES) on one hand and sets of KpnI and BamHI segments on the other hand (37), were found (Fig. 2). In evolutionary terms, they have probably contributed to genetic differences between species and individuals by playing a role in retrotransposition events and in promoting unequal crossing-over. While human HDAC3 can be detected predominantly in the nuclear compartment and is ubiquitously expressed in many different cell lines and multiple normal human tissues (10 –12), the mRNA-expression pattern of murine HDAC3 is surprisingly different and shows an overexperssion of HDAC3 in testicular tissue, while it is not significantly expressed in the spleen (Fig. 4). An extensive exploration of the 59 flanking promoter region may help to explain this phenomenon and awaits to be further studied experimentally. Since the steady-state of histone acetylation and deacetylation plays a key role in the regulation of transcription, the loss of genetic material, resulting from chromosomal loss or deletion or from other mechanisms, with the consequence of the development of HDAC3 hemizygosity, might lead to a gene dosage effect or the unmasking of a recessive allele, such as a tumor-suppressor gene, on the cytogenetically “normal” homolog. As a consequence, the steady-state of
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FIG. 5. HDAC phylogenetic tree. This tree is based on the yeast RPD3 protein sequence and includes only orthologs of the RPD3 family of histone deacetylases. The numbers along branches represent percentage values for bootstrap statistical support. The scale bar indicates the genetic distance of the branch lengths (Accession numbers of the sequences used in this tree: yeast RPD3 (P32561), human HDAC1 (Q13547), human HDAC2 (Q92769), human HDAC3 (AF039703), mouse HDAC1 (O09106), mouse HDAC2 (P70288), mouse HDAC3 (AF074881), chicken HDAC1 (AF043328), chicken HDAC2 (P56519), chicken HDAC3 (P56520), Xenopus laevis HDAC1 (O42227), Strongylocentrotus purpuratus HDAC1 (P56518), Drosophila melanogaster HDAC1 (AF026949), C. elegans HDAC1 (O17695), A. thaliana HDAC1 (O22446), Zea mays HDAC1 (P56521).
histone acetylation may be shifted toward acetylation at the level of specific genes targeted by HDAC3 and either upregulate or downregulate transcriptional events (14, 15). Therefore, the identification of a consistent loss of HDAC3 (band 18B3 in the mouse, band 5q31 in humans) implies that loss of genes critical for
normal myeloid growth and differentiation may uncover inactivating mutations on the remaining allele (monoallelic expression, loss of heterozygosity). However, limited available material, the paucity of highly polymorphic loci both on the distal arm of chromsome 5 and mouse chromosome 18B3, and the technical limi-
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tations in separating the affected cells from the normal cell population, have rendered heterozygosity analyses particularly arduous. Concluding, the mouse HDAC3 gene displays striking similarity to the previously characterized human HDAC3 gene both on the genomic and the protein levels, which suggests also similarly close functional properties. Studies on HDAC3 in the mouse model may therefore help to improve the understanding of the pathogenesis of myeloproliferative disease in humans. ACKNOWLEDGMENTS This work was supported by the German National Science Foundation (Deutsche Forschungsgemeinschaft, MA 2057/1-1) and institutional funds from the Picower Institute for Medical Research.
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