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POZ Domain
Biochemical and Biophysical Research Communications 273, 991–996 (2000) doi:10.1006/bbrc.2000.3053, available online at http://www.idealibrary.com on
Molecular Cloning and Characterization of a Novel Human Gene Containing Ankyrin Repeat and Double BTB/POZ Domain Ken-Shwo Dai, Wei Wei, and Choong-Chin Liew Institute of Medical Science and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada
Received June 7, 2000
A novel human gene containing an ankyrin repeat and BTB/POZ domains (BPOZ) was isolated from a human leukocyte cDNA library. The cDNA sequence contains an open reading frame of 1434 bp that encodes 478 amino acid residues with a predicted molecular mass of 53.9 kDa. Sequence pattern analysis shows that BPOZ contains an N-terminal ankyrin repeat, a bipartite nuclear localization signal and two BTB/POZ domains. Using semiquantitative RT-PCR, the BPOZ transcript was found to be ubiquitously expressed in all fetal tissues examined (heart, brain, liver, and kidney) suggesting that BPOZ is involved in basic cellular function. Low expression of BPOZ in adult tissues (normal and hypertrophic heart) suggests that BPOZ mRNA is developmentally regulated and may play a role in developmental processes. Chromosomal localization by radiation hybrid mapping revealed that this gene is localized between D3S1269 and D3S3606 markers corresponding to the region of chromosome 3q21, a region frequently associated with leukemia. It is thus suggested that BPOZ may be functionally involved in protein-protein interaction, perhaps in forming protein complexes, and may have an important role in normal development and in the development of leukemia. © 2000 Academic Press Key Words: leukocyte; chromosomal mapping; BTB/ POZ domain; ankyrin repeat region; reverse transcription-polymerase chain reaction.
BTB/POZ domain, an evolutionarily conserved domain, was originally described for broad-complex, tramtrack, bric-a-brac (1, 2), and for poxvirus and zinc finger (3, 4). Subsequently, many genes having a variety of functions have been reported to contain this domain (5–9). The BTB/POZ domain has been reported to serve as a protein-protein interaction interface (2– 4) via homodimerization or multimerization (3, 5, 10), or
through heterophilic interactions (8, 11, 12). Furthermore, many studies have shown that additional conserved motifs were found in association with BTB/POZ domain. These include kelch repeats (13, 14), zinc finger domains (2– 4, 15), MATH domains (16), b-zip DNA binding domains, FYVE fingers and ankyrin repeats (17), histone fold domains, transmembrane domains, AT-hook and uncharacterized globular domains (18). The ankyrin repeat, a conserved domain, is one of these domains associated with BTB/POZ domains and has been found in a large number of functionally unrelated proteins (19, 20). It is thought that this domain also serves as a protein-protein interaction domain (20, 21). In this report, we identify and characterize BPOZ, a novel developmentally-regulated gene containing an N-terminal ankyrin repeat, a bipartite nuclear localization signal and two BTB/POZ domains. Chromosomal assignment suggests that BPOZ is a potential candidate gene involved in the development of leukemia. MATERIALS AND METHODS Analysis of a human leukocyte cDNA database. Expressed sequence tags (ESTs) generated from the large-scale PCR-based sequencing of the 5⬘-end of human leukocyte cDNA clones were compiled for sequence comparisons against the nonredundant nucleotide and protein databases at the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov) using the BLASTN and BLASTX programs (22–24), with a significance cutoff of P ⬍ 10 ⫺10 . Sequence pattern was characterized based on a search of the Swiss-Prot protein sequence database (http://www.expasy.ch/ sprot/prosite.html). Isolation of a cDNA clone. A cDNA clone exhibiting a nucleotide and amino acid sequence similar to the BTB/POZ domain was identified during the course of EST generation. The insert of this clone was subsequently excised in vivo from the ZAP Express vector using the ExAssist/XLOLR helper phage system (Stratagene). In brief, phagemid particles were excised by coinfecting XL1-BLUE MRF’ cells with ExAssist helper phage. The excised pBluescript phagemids were used to infect E. coli XLOLR cells, which lack the amber suppressor necessary for ExAssist phage replication. Infected XLOLR cells were selected using kanamycin resistance. Resultant
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0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 273, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS colonies contained the double stranded phagemid vector with the cloned cDNA insert. A single colony was grown overnight in LBkanamycin, and DNA was purified using a plasmid purification kit (Qiagen). Full length nucleotide sequencing and database comparisons. Phagemid DNA was sequenced using the Taq Dyedeoxy Terminator Cycle Sequencing Kit for Applied Biosystems 377 sequencing system (Perkin Elmer). Using the primer-walking approach, full-length sequence was determined. Nucleotide and protein searches were performed using BLAST against the NCBI nonredundant database. Assays of reverse transcriptase-polymerase chain reaction (RTPCR). To investigate the possible expression of BPOZ in cells other than leukocyte, RT-PCR assay was performed. Using TRIZOL reagents (Life Technology), total RNA was isolated from human tissues, e.g., pooled fetal heart, brain, liver and kidney (10 –12 weeks), adult normal heart (healthy 40-year-old male) and pooled adult hypertrophic heart samples (patients undergoing surgery). Tissues were powdered under liquid nitrogen before homogenization. RNA purity and integrity were assessed by absorbance at 260/280 nm and by agarose gel electrophoresis. The forward and reverse primers for BPOZ was 5⬘-AGGAGGCGGGATTACTATGACG-3⬘ and 5⬘-ACAGCTCAGTGTGGTCGCTGTA-3⬘, respectively. The expected size of this specific PCR fragment was 748 bp. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Accession No. M33197) was used as an internal control. The forward and reverse primers for GAPDH were: 5⬘-TGGGTGTGAACCATGAGAAG-3⬘ and 5⬘-GTGTCGCTGTTGAAGTCAGA-3⬘. The expected size of this PCR fragment was 472 bp. The RT-PCR experiment was performed according to the manufacturer’s instructions (Boehringer Mannheim). A 50 l reaction mixture containing 2 l total RNA (0.1 g/l), 1 l each primer (20 pM), 1 l each dNTP (10 mM), 2.5 l DTT solution (100 mM), 10 l 5⫻ RT-PCR buffer, 1 l enzyme mixture and 28.5 l sterile distilled water were subjected to reverse transcription at 60°C for 30 min followed by 35 cycles of denaturation at 94°C for 2 min, annealing at 60°C for 2 min, and extension at 68°C for 2 min. Five microliters (10%) of the amplified products mixed with 1 l of loading buffer were separated on a 1% horizontal agarose gel stained with ethidium bromide in 0.5⫻ TAE buffer. The gel was electrophoresed at 100 V for 45 min. The RT-PCR analysis was repeated twice to ensure reproducibility, for a total of three independent experiments. Human radiation hybrid (RH) mapping of BPOZ gene. To define the locus of the BPOZ gene, the RH mapping panel G3 (Stanford Human Genome Center), consisting of 83 hybrid cell lines, was used. PCR analysis of a radiation hybrid panel was performed with primers designed within the 3⬘-untranslated region of BPOZ. The forward primer (5⬘-GAGACAAGCATGTGTATGCGTT-3⬘) and the reverse primer (5⬘-GGAGAAGGAAAGTGCTAAGACC-3⬘) were designed to amplify a 257 bp fragment. PCR amplification conditions were 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 45 sec, and extension at 72°C for 30 sec. Raw scores were submitted to the RH server at the Stanford Human Genome center (http://wwwshgc.stanford.edu/RH/rhserverformnew.html).
RESULTS A novel clone, BPOZ, exhibiting amino acid sequence similarity to the ankyrin repeats and BTB/POZ domains of several known genes was isolated from a
FIG. 1. Full length sequence of BPOZ. Nucleotide and deduced amino acid residues are numbered on the right. The ankyrin repeat region is doubly underlined. A single underline represents the
bipartite nuclear localization signal. The double BTB/POZ domains are marked in bold. An asterisk denotes the termination codon. A polyadenylation signal is dot-underlined.
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FIG. 2. An alignment of the conserved domains. (a) The ankyrin repeat region of BPOZ is aligned with 15 known proteins. (b) Both BPOZ1 and BPOZ2 are aligned with 19 known proteins. A consensus sequence is presented at the bottom; the highly conserved amino acids (75–100% conservation) are indicated in bold. Gaps between conserved amino acids are indicated by dots.
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FIG. 3. Semiquantitative RT-PCR analysis of BPOZ in human fetal (FH), adult (AH), hypertrophic (Hy) hearts, fetal brain (FB), liver (FL), and kidney (FK). The products of RT-PCR, electrophoresed in 1% agarose gel, show the expected sizes of approximately 748 bp for BPOZ and 472 bp for GAPDH (internal control). Shown on the left (M) are 100 bp DNA ladder markers.
human leukocyte cDNA library for full-length sequencing. The nucleotide and predicted amino acid sequences of BPOZ are shown in Fig. 1. The 1953 bp clone contains a 1434 bp open reading frame extending from positions 61 to 1494, which corresponds to an encoded protein of 478 amino acid residues with a predicted molecular mass of 53.9 kDa. The sequence around the initiation ATG codon at nucleotide 61– 63 was matched with the Kozak consensus (A/GCCATGG) (25, 26). In the 3⬘-untranslated region, a potential polyadenylation signal AATAAA (27) was detected 18 bases upstream of a poly(A) tail. A search of GenBank indicated that the full length sequence of BPOZ was novel. Sequence analysis indicated that BPOZ contains an ankyrin repeat region (aa 1–76), a bipartite nuclear localization signal (aa 82–98) and two BTB/POZ domains, named BPOZ1 (aa 99 – 213) and BPOZ2 (aa 251–373). A comparison of the amino acid sequence of the ankyrin repeat of BPOZ and 15 known ankyrin repeat-containing proteins is shown in Figure 2a. The alignment reveals that a total of 31 amino acids are conserved among these sequences, as shown in the consensus sequence at the bottom of this figure (i.e., TPLH.AC..G....V..LL....A..NA..K....TPLH.A...GH...V..LL..GA.P......G.T.L..A). Of these 31 amino acids, 19 are highly conserved (75– 100% conservation; shown in bold) and 12 are partially conserved (50 –75% conservation). A comparison of the amino acid sequence of the BTB/POZ domains of BPOZ (BPOZ1 and BPOZ2) and 19 known BTB/POZ domaincontaining proteins revealed striking conservation of 40 amino acid residues among these sequences, as shown in the consensus sequence at the bottom of Fig. 2b (i.e., .H.....L..LN.QR.....LCDV...V.G..F.......AHRAVLAACS..YF...F..........................P..F..LL.F.YT...L..........L.A....A..L.............C...L). Of these 40 amino acids, 19 are highly conserved (75–100% conservation; shown in bold) and 21 are partially conserved (50 –75% conservation). To determine whether BPOZ transcript could be detected in cells other than leukocyte, human fetal, adult, and hypertrophic hearts, fetal brain, liver, and kidney
tissues were used for RT-PCR analysis (Fig. 3). Semiquantitative analysis of BPOZ expression patterns obtained by coamplifying BPOZ and an internal control gene (GAPDH) in a single tube showed that BPOZ was expressed at similar levels in all fetal tissues examined. In cardiac tissues, BPOZ expression was higher in the fetal tissues than in the adult tissues (normal and hypertrophic heart). Chromosomal localization of this gene was performed using a PCR-based radiation hybrid panel. The data vector for BPOZ was 0000000000 0000100000 0000000000 0000100000 0001000000 1000000000 0000000010 0000001000 000. Statistical analysis of the data vector processed using the RH server at Stanford Human Genome Center (http://www-shgc.stanford.edu/ RH/rhserverform new.html) mapped BPOZ 17cR from SHGC-30693 (LOD ⬎ 6.0), which is located between AFM markers D3S1269 and D3S3606 on chromosome 3. Searches of GeneMap’99 (http://www.ncbi.nlm.nih. gov/genemap/), UniGene (http://www.ncbi.nlm.nih.gov/ UniGene/), and OMIM (http://www.ncbi.nlm.nih.gov/ Omim/) showed that both markers lie on chromosome 3q21 (Fig. 4). DISCUSSION A clone, Bood POZ containing gene (BPOZ), with an amino acid sequence similarity to many ankyrin repeat-containing genes and BTB/POZ domain-containing genes, was isolated for full-length sequencing. Sequence analysis showed that BPOZ contained an N-terminal ankyrin repeat region, a nuclear localization signal (NLS) and two BTB/POZ domains. The presence of NLS suggests that BPOZ is potentially a nuclear protein (28). It is interesting to note that BTB/ POZ domain is usually found in a single copy per protein (18) with only a few exceptions such as YIL001w in yeast (GenBank S48434) and LZTR-1 in human (29). The presence of two BTB/POZ domains associated with the ankyrin repeat region suggests that BPOZ may function as a interface to form protein complexes via interacting with other proteins, since
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Radiation hybrid mapping localized BPOZ to a position between D3S1269 and D3S3606 markers corresponding to the region of chromosome 3q21, a leukemia breakpoint region frequently associated with leukemia (33, 34). This chromosomal assignment suggests that BPOZ may be a potential candidate gene involved in normal tissue development and perhaps in aberrant development in leukemia. In conclusion, we have identified a novel developmentally regulated gene, BPOZ, containing a nuclear localization signal and 3 protein-protein interacting domains (i.e., 1 ankyrin repeat and 2 BTB/POZ domains). Chromosomal analysis suggests that BPOZ is essential to the development of normal tissues and perhaps involved in diseases such as leukemia. Further elucidation of its functional roles may contribute to our understanding of the BPOZ regulation in the development and pathogenesis of the leukocyte. ACKNOWLEDGMENTS This work was supported by The Medical Research Council of Canada, The Heart and Stroke Foundation of Ontario, and The Canadian Genome Analysis and Technology Program. K.-S. Dai is a recipient of a Heart and Stroke Foundation traineeship.
REFERENCES
FIG. 4. Chromosomal localization of BPOZ gene. The idiogram of chromosome 3 is shown and the position of BPOZ relative to known markers on 3q21 are depicted in the adjacent diagram which was constructed using the framework provided by the GeneMap’99 G3 integrated map.
both BTB/POZ domain and ankyrin repeat have been shown to be involved in protein-protein interactions (3, 20). Previous studies have shown that many BTB/POZ domain-containing proteins (e.g., BCL6, PLZF, ZID, GAGA, and SalF17R) interact with common cellular target proteins, such as N-CoR (nuclear receptor corepressor)/SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), mediated by the BTB/ POZ domain (30 –32). BPOZ may therefore play a role in this regulatory network. A semiquantitative RT-PCR expression pattern showed that BPOZ transcript is widely distributed in all fetal tissues examined with no apparent differences in their mRNA levels, suggesting that BPOZ plays a role in basic cellular function. The low expression level of BPOZ mRNA in adult hearts (normal and hypertrophic) suggests that this gene is developmentally regulated and may play a role in the developmental processes. 995
1. Godt, D., Couderc, J. L., Cramton, S. E., and Laski, F. A. (1993) Pattern formation in the limbs of Drosophila: Bric a brac is expressed in both a gradient and a wave-like pattern and is required for specification and proper segmentation of the tarsus. Development 119, 799 – 812. 2. Zollman, S., Godt, D., Prive, G. G., Couderc, J. L., and Laski, F. A. (1994) The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc. Natl. Acad. Sci. USA 91, 10717–10721. 3. Bardwell, V. J., and Treisman, R. (1994) The POZ domain: A conserved protein-protein interaction motif. Genes Dev. 8, 1664 – 1677. 4. Albagli, O., Dhordain, P., Deweindt, C., Lecocq, G., and Leprince, D. (1995) The BTB/POZ domain: A new protein-protein interaction motif common to DNA- and actin-binding proteins. Cell Growth Differ. 6, 1193–1198. 5. Dhordain, P., Albagli, O., Ansieau, S., Koken, M. H., Deweindt, C., Quief, S., Lantoine, D., Leutz, A., Kerckaert, J. P., and Leprince, D. (1995) The BTB/POZ domain targets the LAZ3/ BCL6 oncoprotein to nuclear dots and mediates homomerisation in vivo. Oncogene 11, 2689 –2697. 6. Robinson, D. N., and Cooley, L. (1997) Drosophila kelch is an oligomeric ring canal actin organizer. J. Cell. Biol. 138, 799 – 810. 7. Kim, T. A., Lim, J., Ota, S., Raja, S., Rogers, R., Rivnay, B., Avraham, H., and Avraham, S. (1998) NRP/B, a novel nuclear matrix protein, associates with p110(RB) and is involved in neuronal differentiation. J. Cell. Biol. 141, 553–566. 8. Lin, R. J., Nagy, L., Inoue, S., Shao, W., Miller, W. H., Jr., and Evans, R. M. (1998) Role of the histone deacetylase complex in acute promyelocytic leukaemia. Nature 391, 811– 814. 9. Soltysik-Espanola, M., Rogers, R. A., Jiang, S., Kim, T. A.,
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Gaedigk, R., White, R. A., Avraham, H., and Avraham, S. (1999) Characterization of Mayven, a novel actin-binding protein predominantly expressed in brain. Mol. Biol. Cell. 10, 2361–2375. Li, X., Lopez-Guisa, J. M., Ninan, N., Weiner, E. J., Rauscher, F. J., 3rd, and Marmorstein, R. (1997) Overexpression, purification, characterization, and crystallization of the BTB/POZ domain from the PLZF oncoprotein. J. Biol. Chem. 272, 27324 – 27329. David, G., Alland, L., Hong, S. H., Wong, C. W., DePinho, R. A., and Dejean, A. (1998) Histone deacetylase associated with mSin3A mediates repression by the acute promyelocytic leukemia-associated PLZF protein. Oncogene 16, 2549 –2556. Grignani, F., De Matteis, S., Nervi, C., Tomassoni, L., Gelmetti, V., Cioce, M., Fanelli, M., Ruthardt, M., Ferrara, F. F., Zamir, I., Seiser, C., Grignani, F., Lazar, M. A., Minucci, S., and Pelicci, P. G. (1998) Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukaemia. Nature 391, 815– 818. Koonin, E. V., Senkevich, T. G., and Chernos, V. I. (1992) A family of DNA virus genes that consists of fused portions of unrelated cellular genes. Trends Biochem. Sci. 17, 213–214. Bork, P., and Doolittle, R. F. (1994) Drosophila kelch motif is derived from a common enzyme fold. J. Mol. Biol. 236, 1277–1282. Ahmad, K. F., Engel, C. K., and Prive, G. G. (1998) Crystal structure of the BTB domain from PLZF. Proc. Natl. Acad. Sci. USA 95, 12123–12128. Uren, A. G., and Vaux, D. L. (1996) TRAF proteins and meprins share a conserved domain. Trends Biochem. Sci. 21, 244 –245. Stenmark, H., Aasland, R., Toh, B. H., and D’Arrigo, A. (1996) Endosomal localization of the autoantigen EEA1 is mediated by a zinc-binding FYVE finger. J. Biol. Chem. 271, 24048 –24054. Aravind, L., and Koonin, E. V. (1999) Fold prediction and evolutionary analysis of the POZ domain: structural and evolutionary relationship with the potassium channel tetramerization domain. J. Mol. Biol. 285, 1353–1361. Bennett, V. (1992) Ankyrins. Adaptors between diverse plasma membrane proteins and the cytoplasm. J. Biol. Chem. 267, 8703– 8706. Bork, P. (1993) Hundreds of ankyrin-like repeats in functionally diverse proteins: Mobile modules that cross phyla horizontally? Proteins 17, 363–374. Breeden, L., and Nasmyth, K. (1987) Similarity between cellcycle genes of budding yeast and fission yeast and the Notch gene of Drosophila. Nature 329, 651– 654.
22. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403– 410. 23. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25, 3389 –3402. 24. Gish, W., and States, D. J. (1993) Identification of protein coding regions by database similarity search. Nature Genetics 3, 266 – 272. 25. Kozak, M. (1987) An analysis of 5⬘-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125– 8148. 26. Kozak, M. (1991) An analysis of vertebrate mRNA sequences: Intimations of translational control. J. Cell. Biol. 115, 887–903. 27. Proudfoot, N. J., and Brownlee, G. G. (1976) 3⬘ Noncoding region sequences in eukaryotic messenger RNA. Nature 263, 211–214. 28. Dingwall, C., and Laskey, R. A. (1991) Nuclear targeting sequences—a consensus? Trends Biochem. Sci. 16, 478 – 481. 29. Kurahashi, H., Akagi, K., Inazawa, J., Ohta, T., Niikawa, N., Kayatani, F., Sano, T., Okada, S., and Nishisho, I. (1995) Isolation and characterization of a novel gene deleted in DiGeorge syndrome. Hum. Mol. Genet. 4, 541–549. 30. Dhordain, P., Albagli, O., Lin, R. J., Ansieau, S., Quief, S., Leutz, A., Kerckaert, J. P., Evans, R. M., and Leprince, D. (1997) Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein. Proc. Natl. Acad. Sci. USA 94, 10762– 10767. 31. Hong, S. H., David, G., Wong, C. W., Dejean, A., and Privalsky, M. L. (1997) SMRT corepressor interacts with PLZF and with the PML-retinoic acid receptor alpha (RARalpha) and PLZFRARalpha oncoproteins associated with acute promyelocytic leukemia. Proc. Natl. Acad. Sci. USA 94, 9028 –9033. 32. Huynh, K. D., and Bardwell, V. J. (1998) The BCL-6 POZ domain and other POZ domains interact with the co-repressors N-CoR and SMRT. Oncogene 17, 2473–2484. 33. Rynditch, A., Pekarsky, Y., Schnittger, S., and Gardiner, K. (1997) Leukemia breakpoint region in 3q21 is gene rich. Gene 193, 49 –57. 34. Lindquist, R., Forsblom, A. M., Ost, A., and Gahrton, G. (2000) Mutagen exposures and chromosome 3 aberrations in acute myelocytic leukemia. Leukemia 14, 112–118.
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Molecular Cloning and Characterization of a Novel Human Gene Containing Ankyrin Repeat and Double BTB/POZ Domain Ken-Shwo Dai, Wei Wei, and Choong-Chin Liew Institute of Medical Science and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada
Received June 7, 2000
A novel human gene containing an ankyrin repeat and BTB/POZ domains (BPOZ) was isolated from a human leukocyte cDNA library. The cDNA sequence contains an open reading frame of 1434 bp that encodes 478 amino acid residues with a predicted molecular mass of 53.9 kDa. Sequence pattern analysis shows that BPOZ contains an N-terminal ankyrin repeat, a bipartite nuclear localization signal and two BTB/POZ domains. Using semiquantitative RT-PCR, the BPOZ transcript was found to be ubiquitously expressed in all fetal tissues examined (heart, brain, liver, and kidney) suggesting that BPOZ is involved in basic cellular function. Low expression of BPOZ in adult tissues (normal and hypertrophic heart) suggests that BPOZ mRNA is developmentally regulated and may play a role in developmental processes. Chromosomal localization by radiation hybrid mapping revealed that this gene is localized between D3S1269 and D3S3606 markers corresponding to the region of chromosome 3q21, a region frequently associated with leukemia. It is thus suggested that BPOZ may be functionally involved in protein-protein interaction, perhaps in forming protein complexes, and may have an important role in normal development and in the development of leukemia. © 2000 Academic Press Key Words: leukocyte; chromosomal mapping; BTB/ POZ domain; ankyrin repeat region; reverse transcription-polymerase chain reaction.
BTB/POZ domain, an evolutionarily conserved domain, was originally described for broad-complex, tramtrack, bric-a-brac (1, 2), and for poxvirus and zinc finger (3, 4). Subsequently, many genes having a variety of functions have been reported to contain this domain (5–9). The BTB/POZ domain has been reported to serve as a protein-protein interaction interface (2– 4) via homodimerization or multimerization (3, 5, 10), or
through heterophilic interactions (8, 11, 12). Furthermore, many studies have shown that additional conserved motifs were found in association with BTB/POZ domain. These include kelch repeats (13, 14), zinc finger domains (2– 4, 15), MATH domains (16), b-zip DNA binding domains, FYVE fingers and ankyrin repeats (17), histone fold domains, transmembrane domains, AT-hook and uncharacterized globular domains (18). The ankyrin repeat, a conserved domain, is one of these domains associated with BTB/POZ domains and has been found in a large number of functionally unrelated proteins (19, 20). It is thought that this domain also serves as a protein-protein interaction domain (20, 21). In this report, we identify and characterize BPOZ, a novel developmentally-regulated gene containing an N-terminal ankyrin repeat, a bipartite nuclear localization signal and two BTB/POZ domains. Chromosomal assignment suggests that BPOZ is a potential candidate gene involved in the development of leukemia. MATERIALS AND METHODS Analysis of a human leukocyte cDNA database. Expressed sequence tags (ESTs) generated from the large-scale PCR-based sequencing of the 5⬘-end of human leukocyte cDNA clones were compiled for sequence comparisons against the nonredundant nucleotide and protein databases at the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov) using the BLASTN and BLASTX programs (22–24), with a significance cutoff of P ⬍ 10 ⫺10 . Sequence pattern was characterized based on a search of the Swiss-Prot protein sequence database (http://www.expasy.ch/ sprot/prosite.html). Isolation of a cDNA clone. A cDNA clone exhibiting a nucleotide and amino acid sequence similar to the BTB/POZ domain was identified during the course of EST generation. The insert of this clone was subsequently excised in vivo from the ZAP Express vector using the ExAssist/XLOLR helper phage system (Stratagene). In brief, phagemid particles were excised by coinfecting XL1-BLUE MRF’ cells with ExAssist helper phage. The excised pBluescript phagemids were used to infect E. coli XLOLR cells, which lack the amber suppressor necessary for ExAssist phage replication. Infected XLOLR cells were selected using kanamycin resistance. Resultant
991
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 273, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS colonies contained the double stranded phagemid vector with the cloned cDNA insert. A single colony was grown overnight in LBkanamycin, and DNA was purified using a plasmid purification kit (Qiagen). Full length nucleotide sequencing and database comparisons. Phagemid DNA was sequenced using the Taq Dyedeoxy Terminator Cycle Sequencing Kit for Applied Biosystems 377 sequencing system (Perkin Elmer). Using the primer-walking approach, full-length sequence was determined. Nucleotide and protein searches were performed using BLAST against the NCBI nonredundant database. Assays of reverse transcriptase-polymerase chain reaction (RTPCR). To investigate the possible expression of BPOZ in cells other than leukocyte, RT-PCR assay was performed. Using TRIZOL reagents (Life Technology), total RNA was isolated from human tissues, e.g., pooled fetal heart, brain, liver and kidney (10 –12 weeks), adult normal heart (healthy 40-year-old male) and pooled adult hypertrophic heart samples (patients undergoing surgery). Tissues were powdered under liquid nitrogen before homogenization. RNA purity and integrity were assessed by absorbance at 260/280 nm and by agarose gel electrophoresis. The forward and reverse primers for BPOZ was 5⬘-AGGAGGCGGGATTACTATGACG-3⬘ and 5⬘-ACAGCTCAGTGTGGTCGCTGTA-3⬘, respectively. The expected size of this specific PCR fragment was 748 bp. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Accession No. M33197) was used as an internal control. The forward and reverse primers for GAPDH were: 5⬘-TGGGTGTGAACCATGAGAAG-3⬘ and 5⬘-GTGTCGCTGTTGAAGTCAGA-3⬘. The expected size of this PCR fragment was 472 bp. The RT-PCR experiment was performed according to the manufacturer’s instructions (Boehringer Mannheim). A 50 l reaction mixture containing 2 l total RNA (0.1 g/l), 1 l each primer (20 pM), 1 l each dNTP (10 mM), 2.5 l DTT solution (100 mM), 10 l 5⫻ RT-PCR buffer, 1 l enzyme mixture and 28.5 l sterile distilled water were subjected to reverse transcription at 60°C for 30 min followed by 35 cycles of denaturation at 94°C for 2 min, annealing at 60°C for 2 min, and extension at 68°C for 2 min. Five microliters (10%) of the amplified products mixed with 1 l of loading buffer were separated on a 1% horizontal agarose gel stained with ethidium bromide in 0.5⫻ TAE buffer. The gel was electrophoresed at 100 V for 45 min. The RT-PCR analysis was repeated twice to ensure reproducibility, for a total of three independent experiments. Human radiation hybrid (RH) mapping of BPOZ gene. To define the locus of the BPOZ gene, the RH mapping panel G3 (Stanford Human Genome Center), consisting of 83 hybrid cell lines, was used. PCR analysis of a radiation hybrid panel was performed with primers designed within the 3⬘-untranslated region of BPOZ. The forward primer (5⬘-GAGACAAGCATGTGTATGCGTT-3⬘) and the reverse primer (5⬘-GGAGAAGGAAAGTGCTAAGACC-3⬘) were designed to amplify a 257 bp fragment. PCR amplification conditions were 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 45 sec, and extension at 72°C for 30 sec. Raw scores were submitted to the RH server at the Stanford Human Genome center (http://wwwshgc.stanford.edu/RH/rhserverformnew.html).
RESULTS A novel clone, BPOZ, exhibiting amino acid sequence similarity to the ankyrin repeats and BTB/POZ domains of several known genes was isolated from a
FIG. 1. Full length sequence of BPOZ. Nucleotide and deduced amino acid residues are numbered on the right. The ankyrin repeat region is doubly underlined. A single underline represents the
bipartite nuclear localization signal. The double BTB/POZ domains are marked in bold. An asterisk denotes the termination codon. A polyadenylation signal is dot-underlined.
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FIG. 2. An alignment of the conserved domains. (a) The ankyrin repeat region of BPOZ is aligned with 15 known proteins. (b) Both BPOZ1 and BPOZ2 are aligned with 19 known proteins. A consensus sequence is presented at the bottom; the highly conserved amino acids (75–100% conservation) are indicated in bold. Gaps between conserved amino acids are indicated by dots.
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FIG. 3. Semiquantitative RT-PCR analysis of BPOZ in human fetal (FH), adult (AH), hypertrophic (Hy) hearts, fetal brain (FB), liver (FL), and kidney (FK). The products of RT-PCR, electrophoresed in 1% agarose gel, show the expected sizes of approximately 748 bp for BPOZ and 472 bp for GAPDH (internal control). Shown on the left (M) are 100 bp DNA ladder markers.
human leukocyte cDNA library for full-length sequencing. The nucleotide and predicted amino acid sequences of BPOZ are shown in Fig. 1. The 1953 bp clone contains a 1434 bp open reading frame extending from positions 61 to 1494, which corresponds to an encoded protein of 478 amino acid residues with a predicted molecular mass of 53.9 kDa. The sequence around the initiation ATG codon at nucleotide 61– 63 was matched with the Kozak consensus (A/GCCATGG) (25, 26). In the 3⬘-untranslated region, a potential polyadenylation signal AATAAA (27) was detected 18 bases upstream of a poly(A) tail. A search of GenBank indicated that the full length sequence of BPOZ was novel. Sequence analysis indicated that BPOZ contains an ankyrin repeat region (aa 1–76), a bipartite nuclear localization signal (aa 82–98) and two BTB/POZ domains, named BPOZ1 (aa 99 – 213) and BPOZ2 (aa 251–373). A comparison of the amino acid sequence of the ankyrin repeat of BPOZ and 15 known ankyrin repeat-containing proteins is shown in Figure 2a. The alignment reveals that a total of 31 amino acids are conserved among these sequences, as shown in the consensus sequence at the bottom of this figure (i.e., TPLH.AC..G....V..LL....A..NA..K....TPLH.A...GH...V..LL..GA.P......G.T.L..A). Of these 31 amino acids, 19 are highly conserved (75– 100% conservation; shown in bold) and 12 are partially conserved (50 –75% conservation). A comparison of the amino acid sequence of the BTB/POZ domains of BPOZ (BPOZ1 and BPOZ2) and 19 known BTB/POZ domaincontaining proteins revealed striking conservation of 40 amino acid residues among these sequences, as shown in the consensus sequence at the bottom of Fig. 2b (i.e., .H.....L..LN.QR.....LCDV...V.G..F.......AHRAVLAACS..YF...F..........................P..F..LL.F.YT...L..........L.A....A..L.............C...L). Of these 40 amino acids, 19 are highly conserved (75–100% conservation; shown in bold) and 21 are partially conserved (50 –75% conservation). To determine whether BPOZ transcript could be detected in cells other than leukocyte, human fetal, adult, and hypertrophic hearts, fetal brain, liver, and kidney
tissues were used for RT-PCR analysis (Fig. 3). Semiquantitative analysis of BPOZ expression patterns obtained by coamplifying BPOZ and an internal control gene (GAPDH) in a single tube showed that BPOZ was expressed at similar levels in all fetal tissues examined. In cardiac tissues, BPOZ expression was higher in the fetal tissues than in the adult tissues (normal and hypertrophic heart). Chromosomal localization of this gene was performed using a PCR-based radiation hybrid panel. The data vector for BPOZ was 0000000000 0000100000 0000000000 0000100000 0001000000 1000000000 0000000010 0000001000 000. Statistical analysis of the data vector processed using the RH server at Stanford Human Genome Center (http://www-shgc.stanford.edu/ RH/rhserverform new.html) mapped BPOZ 17cR from SHGC-30693 (LOD ⬎ 6.0), which is located between AFM markers D3S1269 and D3S3606 on chromosome 3. Searches of GeneMap’99 (http://www.ncbi.nlm.nih. gov/genemap/), UniGene (http://www.ncbi.nlm.nih.gov/ UniGene/), and OMIM (http://www.ncbi.nlm.nih.gov/ Omim/) showed that both markers lie on chromosome 3q21 (Fig. 4). DISCUSSION A clone, Bood POZ containing gene (BPOZ), with an amino acid sequence similarity to many ankyrin repeat-containing genes and BTB/POZ domain-containing genes, was isolated for full-length sequencing. Sequence analysis showed that BPOZ contained an N-terminal ankyrin repeat region, a nuclear localization signal (NLS) and two BTB/POZ domains. The presence of NLS suggests that BPOZ is potentially a nuclear protein (28). It is interesting to note that BTB/ POZ domain is usually found in a single copy per protein (18) with only a few exceptions such as YIL001w in yeast (GenBank S48434) and LZTR-1 in human (29). The presence of two BTB/POZ domains associated with the ankyrin repeat region suggests that BPOZ may function as a interface to form protein complexes via interacting with other proteins, since
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Radiation hybrid mapping localized BPOZ to a position between D3S1269 and D3S3606 markers corresponding to the region of chromosome 3q21, a leukemia breakpoint region frequently associated with leukemia (33, 34). This chromosomal assignment suggests that BPOZ may be a potential candidate gene involved in normal tissue development and perhaps in aberrant development in leukemia. In conclusion, we have identified a novel developmentally regulated gene, BPOZ, containing a nuclear localization signal and 3 protein-protein interacting domains (i.e., 1 ankyrin repeat and 2 BTB/POZ domains). Chromosomal analysis suggests that BPOZ is essential to the development of normal tissues and perhaps involved in diseases such as leukemia. Further elucidation of its functional roles may contribute to our understanding of the BPOZ regulation in the development and pathogenesis of the leukocyte. ACKNOWLEDGMENTS This work was supported by The Medical Research Council of Canada, The Heart and Stroke Foundation of Ontario, and The Canadian Genome Analysis and Technology Program. K.-S. Dai is a recipient of a Heart and Stroke Foundation traineeship.
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FIG. 4. Chromosomal localization of BPOZ gene. The idiogram of chromosome 3 is shown and the position of BPOZ relative to known markers on 3q21 are depicted in the adjacent diagram which was constructed using the framework provided by the GeneMap’99 G3 integrated map.
both BTB/POZ domain and ankyrin repeat have been shown to be involved in protein-protein interactions (3, 20). Previous studies have shown that many BTB/POZ domain-containing proteins (e.g., BCL6, PLZF, ZID, GAGA, and SalF17R) interact with common cellular target proteins, such as N-CoR (nuclear receptor corepressor)/SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), mediated by the BTB/ POZ domain (30 –32). BPOZ may therefore play a role in this regulatory network. A semiquantitative RT-PCR expression pattern showed that BPOZ transcript is widely distributed in all fetal tissues examined with no apparent differences in their mRNA levels, suggesting that BPOZ plays a role in basic cellular function. The low expression level of BPOZ mRNA in adult hearts (normal and hypertrophic) suggests that this gene is developmentally regulated and may play a role in the developmental processes. 995
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