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Cloning of mouse uncoupling protein 3 cDNA and 5′-flanking region, and its genetic map1
Gene 215 (1998) 77–84
Cloning of mouse uncoupling protein 3 cDNA and 5∞-flanking region, and its genetic map1 Hideki Yoshitomi *, Kazuto Yamazaki, Isao Tanaka Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3, Tokodai, Tsukuba, Ibaraki 300-2635, Japan Received 9 April 1998; accepted 7 May 1998; Received by T. Sekiya
Abstract Brown adipose tissue and skeletal muscle are important sites of non-shivering thermogenesis. It has been known that UCP1 and UCP2 function as the main effector of the thermogenesis: the former is expressed exclusively in brown adipose tissue, whereas the latter is distributed widely. Recently, the third UCP homologue was discovered in humans, which was designated as UCP3. We now report molecular cloning of full-length mouse UCP3 cDNA and its 5∞-flanking genomic region. The mouse UCP3 cDNA sequence predicted a 308-amino acid protein, and the overall identity between the mouse and human UCP3 proteins was 85.6%. The mouse UCP3 amino acid sequence was 54.7% and 73.1% identical to the mouse UCP1 and UCP2, respectively. Expression of the mouse UCP3 was found to be abundant in skeletal muscle and somewhat less abundant in heart, but was minimally expressed in other critical organs. The sequences of 5∞-flanking regions of the mouse UCP1 and UCP3 were very different, resulting in different distributions of putative transcriptional factor binding sites. The differences could reflect tissue-specific expression of the UCPs. The mouse Ucp3 gene was mapped near Ucp2 on chromosome 7, suggesting that the Ucp2 and Ucp3 are clustered genes. This region is boundary of synteny between human chromosome 11q13 and 11p15. As Solanes et al. reported that both human UCP2 and UCP3 genes are assigned to chromosome 11q13, the region where the mouse Ucp2 and Ucp3 are localized is syntenic to human chromosome 11q13. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Mitochondrial energy-transfer-protein signature; MyoD; Clustered gene; Non-shivering thermogenesis
1. Introduction UCPs are inner mitochondrial membrane transporters that dissipate the proton gradient, releasing stored energy as heat, without coupling to other energyconsuming process. Therefore, UCPs are thought to be important determinants of the metabolic efficiency (Nicholls and Locke, 1984; Klingenberg, 1990). UCP1, the first uncoupling protein to be identified (Lin and Klingenberg, 1980; Jacobsson et al., 1985; Bouillaud * Corresponding author. Tel: +81 298 47 5920; Fax: +81 298 47 5367; e-mail: [email protected] 1 Sequence data from this article have been deposited with the GenBank/EMBL Data Libraries under the following Accession Nos: mouse UCP3 complete cDNA, AB010742; rat UCP2 complete cDNA, AB010743; mouse UCP3 5∞-flanking region, AB011070. Abbreviations: bp, base pair; cDNA, DNA complementary to mRNA; EST, expressed sequence tag; kb, kilobase pairs; MTN, multiple tissue Northern; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; UCP, uncoupling protein; UTR, untranslated region. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 27 9 - 0
et al., 1986), is expressed exclusively in brown adipose tissue, an important organ for energy expenditure in rodents (Rothwell and Stock, 1979; Brooks et al., 1980; Himms-Hagen, 1989). Human UCP1 gene, located in 4q31, is composed of six exons and encodes a 307-amino acid sequence. A second uncoupling protein, UCP2, which is expressed ubiquitously contrary to UCP1 ( Fleury et al., 1997), might play a role in energy balance. Moreover, a third uncoupling protein homologue was discovered in humans and designated as UCP3 (Boss et al., 1997; Vidal-Puig et al., 1997). The UCP3 is expressed abundantly and preferentially in skeletal muscle in humans, and in brown adipose tissue and skeletal muscle in rodents. Because skeletal muscle and brown adipose tissue are regarded as important sites for regulated energy expenditure in humans (Astrup et al., 1985; Zurlo et al., 1990) and rodents (Himms-Hagen, 1989), respectively, it is possible that the UCP3 is a critical mediator of adaptive thermogenesis. Recently, we succeeded in cloning full-length mouse UCP3 cDNA and its 5∞-flanking region and mapping its
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Table 1 Primers used for genome walking to subclone 5∞-flanking region of mouse UCP3 gene Gene-specific primer 1 for first PCR Gene-specific primer 2 for nested PCR (5∞3∞)
Position in cDNA
PCR product
Origin of library
mUcp3-3R: GAA AAG TGA GGA GGT CCG CAA A mUcp3-4R: AGG AAC TTC ACA ACC GTT GTG G
76 to 97 32 to 53
~1.2 kb ~1.5 kb
PvuII SspI
mUcp3-6R: TGC GTC TAG CCA AGG TTG GGT A mUcp3-7R: CCT AAG GCT CCA CTC CAT TAG
−118 to −97 −151 to −131
~600 bp
DraI
mUcp3-8R: ACA ATG GTT ACA GGT GTC ACT mUcp3-9R: TAG AGG CCT GAC ATT CTC AAG A
~700 bp
PvuII
mUcp3-12R: AGC CCT CAT GTG TCA ATG CTT AC mUcp3-13R: GAC TGT CAA GGA CAG GCA GCT A
~1.7 kb
DraI
The subcloning was done using the GenomeWalker kit for the mouse including EcoRV, ScaI, DraI, PvuII and SspI libraries. All primers listed are antisense. The nucleotide positions in the cDNA are numbered from the first residue of the initiation codon.
locus. We report their sequences and comparison among human, mouse and rat UCPs.
2. Materials and methods
Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and sequence-specific primers, as well as T7 and U19 primers, with ABI Prism 377 DNA Sequencer (Applied Biosystems). The PCR conditions for sequencing were as follows: 25 cycles of 10 s at 96°C, 5 s at 50°C and 4 min at 60°C.
2.1. Cloning of mouse UCP3 2.2. Northern blot analysis Identification of mouse UCP3 fragment was achieved by homology searches of nucleotide database using the BLAST service from the National Center for Biotechnology Information at http://www.ncbi.nlm. nih.gov. Thus, we found an expressed sequence tag ( EST ) sequence, GenBank Accession No. AA062091, and primers were constructed for 5∞- and 3∞-RACE based on this sequence. We subcloned full-length mouse UCP3 cDNA using Marathon-Ready cDNA from 17-day mouse embryo (Clontech, Palo Alto, CA) according to the manufacturer’s manual. The primers to amplify the 5∞-fragment were: an adaptor primer, AP1 (5∞-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3∞) and mUcp3RV (5∞-AGT TCC CAG GCG TAT CAT GGC TTG-3∞, antisense, nt 424–447 numbered from the first residue of the initiation codon of cDNA sequence) in the first PCR, and a nested adaptor primer 2, AP2 (5∞-ACT CAC TAT AGG GCT CGA GCG GC-3∞) and mUcp3RV2 (5∞-CTG TCG TGC AGC CTG CCA GAA TCC-3∞, antisense, nt 347–370) in the nested PCR, and those for the 3∞-fragment were: AP1 and mUcp3FW (5∞-CAA GCC ATG ATA CGC CTG GGA ACT-3∞, sense, nt 424–447) in the first PCR, and AP2 and mUcp3FW2 (5∞-TAT GGA TGC CTA CAG AAC CAT CG-3∞, sense, nt 474–496) in the nested PCR. The PCR conditions were 40 cycles of 30 s at 94°C and 4 min at 68°C with a final extension for 15 min at 72°C in GeneAmp PCR System 9600 (Perkin-Elmer, Palo Alto, CA). The nested PCR products were subcloned into pT7Blue(R)T-vector (Novagen, Madison, WI ). Each PCR product was sequenced with a Dye Terminator
Mouse MTN blot containing approximately 2 mg of poly(A)+ RNA per lane was purchased from Clontech Laboratories (#7762-1). The same blot filter was used to hybridize with the mouse UCP2 and UCP3 probes. The mouse UCP2 and UCP3 probes were 930- and 927-bp fragments, respectively, spanning the entire open reading frames. The probe labeling was conducted by random priming in the presence of [a-32P] dCTP. For the hybridization, ExpressHyb Hybridization solution (Clontech) was used, and its procedures were performed as described by the manufacturer. The blot was hybridized at the highest stringency: 68°C for 2 h, followed by washing for 15 min at room temperature with 2× SSC/0.05% SDS five times, and then for 30 min at 55°C with 0.1× SSC/0.1% SDS three times. The filter was exposed to a film at −80°C for 18–24 h. To control the mRNA sample processing quantitatively, the Northern blot was stripped and rehybridized with a b-actinspecific probe. 2.3. Subcloning of 5∞-flanking region and intron of mouse UCP3 We subcloned 5∞-flanking region of the mouse UCP3 using the GenomeWalker kit for the mouse (Clontech) according to the manufacturer’s manual. The primers used were: AP1 (5∞-GTA ATA CGA CTC ACT ATA GGG C-3∞) and a gene-specific primer 1 in the first PCR, and AP2 (5∞-ACT ATA GGG CAC GCG TGG T-3∞) and a gene-specific primer 2 in nested PCR. The
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gene-specific primers are summarized in Table 1. The PCR conditions were as follows: seven cycles of 25 s at 94°C and 4 min at 72°C, 32 cycles of 25 s at 94°C and 4 min at 67°C with a final extension for 4 min at 67°C in the first PCR; five cycles of 25 s at 94°C and 4 min at 72°C, 22 cycles of 25 s at 94°C and 4 min at 67°C with an additional 4 min at 67°C in the nested PCR. The PCR products were subcloned to pT7Blue(R)Tvector and subjected to sequencing using T7, U19 and gene-specific primers. The computer analysis for the potential transcription factor binding sites was carried out using GENETEX software (Software Development, Tokyo, Japan). Intron 1 of the mUCP3 was amplified using the following primers: mUcp3-16 (5∞-TTA GGT TTC AGG TCA GCT GGT G-3∞, sense) and mUcp3-3R. C57BL/6J genomic DNA purchased from the Jackson Laboratory (Bar Harbor, ME) was used as a template. PCR conditions were: seven cycles of 25 s at 95°C and 4 min at 72°C, 38 cycles of 25 s at 95°C and 4 min at 67°C with an additional 4 min at 67°C. We determined the 5∞- and 3∞-end partial sequences of the PCR product by direct sequencing with gene-specific primers to confirm its identity.
2.4. Genetic mapping of mouse Ucp3
Fig. 1. Nucleotide and deduced amino acid sequence of mouse uncoupling protein 3. The nucleotides are numbered from the first nucleotide of the translational initiation codon as +1. The initiation methionine codon and the translation stop codon are in bold. The amino acid sequence is presented below the DNA sequence in the one-letter code. The three mitochondrial energy-transfer-protein signature domains are boxed. Constructed primers to amplify 5∞-RACE and 3∞-RACE fragments are underlined. A polyadenylation signal (AATAAA) is doubleunderlined.
We obtained 3∞-flanking genomic DNA of the Ucp3 gene using the GenomeWalker kit for the mouse. The primers used were: AP1 and mUcp3-1 (5∞-CAC CTT AGG GCA AGA ACG AGA A-3∞, sense, nt 1835–1857) in the first PCR, and AP2 and mUcp3-2 (5∞-TTC TCA TCT CAG GGT CGT ACC T-3∞, sense, nt 1889–1910) in the nested PCR. The PCR conditions were as follows: seven cycles of 25 s at 94°C and 4 min at 72°C, 33 cycles of 25 s at 94°C and 4 min at 67°C with a final extension for 4 min at 67°C in the first PCR; five cycles of 25 s at 94°C and 4 min at 72°C, 22 cycles of 25 s at 94°C and 4 min at 67°C with an additional 4 min at 67°C in the nested PCR. We determined the 3∞- end partial sequence of the nested PCR product of ~550 bp and set a primer to amplify the genomic DNA. A BSS intersubspecific backcross [(C57BL/6JEi X SPRET/Ei)F ×SPRET/Ei] 1 panel was obtained from the Jackson Laboratory, including one C57BL/6JEi and one SPRET/Ei, and 94 N individuals’ genomic DNA (Rowe et al., 1994). The 2 ~550-bp genomic DNA was amplified by the following primers: mUcp3-1 and mUcp3-5R (5∞-TCG GAT CTT TAG GCT CTC CAA G-3∞, antisense). The PCR conditions were 40 cycles of 30 s at 94°C, 30 s at 60°C and 1 min at 72°C with a final extension of 3 min 30 s at 72°C. The PCR products were digested with BspH I (New England BioLabs, MA): BspHI digestion identified an ~550 bp in C57BL/6J, and ~250 and 300 bp in SPRET/Ei. The digested PCR products were analyzed
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A
B
Fig. 2. (a) Comparison of human, rat and mouse UCP3 amino acid sequences. The amino acid sequences are denoted by a single-letter code. Identities among the sequences are indicated in asterisks. Gaps (—) are introduced to maximize the alignment among the sequences. The three mitochondrial energy-transfer-protein signature domains are double-underlined. The GenBank Accession Nos are as follows: human UCP3, AF001787; rat UCP3, RNU92069; mouse Ucp3, AB010742. (b) Phylogenic tree of human, rat and mouse UCPs. The amino acid sequences are compared to produce the tree, generated with DNASIS software program (Hitachi, Tokyo, Japan). The GenBank Accession Nos are as follows: human UCP1, U28480; human UCP2, U76367; human UCP3, AF001787; mouse UCP1, U63418; mouse UCP2, U69135; mouse UCP3, AB010742; rat UCP1, M11814; rat UCP2, AB010743; rat UCP3, RNU92069.
on 1.2% agarose (Gibco BRL, Gaithersburg, MD)/ ethidium bromide gel electrophoresis.
3. Results 3.1. Cloning of full-length mouse UCP3 The EST database was screened for sequences homologous to the mouse UCP1. One mouse EST was identified (GenBank Accession No. AA062091) that was similar, but not identical, to the mouse UCP1 and mouse UCP2 sequences. We constructed the primers based on the EST sequence, and 5∞- and 3∞-RACE were performed
to clone its fragments presumed to represent the mouse UCP3. In humans, long and short forms of the UCP3 were observed (Boss et al., 1997), but we only found a mouse UCP3 fragment corresponding to the long form in PCR from the mouse embryo cDNA library. The mouse UCP3 cDNA sequence contained a 5∞-UTR of 197 bp, an ORF of 927 bp and a 3∞-UTR of 1323 bp, with a polyadenylation signal (AATAAA) and polyA tail ( Fig. 1). The mouse UCP3 protein deduced from the ORF was composed of 308 amino acids and was estimated to have a molecular weight of ~34 kDa. At the amino acid level, the mouse UCP3 was 85.6% identical to the human UCP3 and 97.7% identical to the rat UCP3 ( Fig. 2a). The mitochondrial energy-
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a doublet ( Fig. 3). The sizes of the signals, as compared with the marker, were ~2.4 and ~2.6 kb. No UCP3 signals were detected in the other critical organs. Northern blots also showed the presence of a smaller mRNA transcript of ~1.6 kb, but rehybridization of the same blots with the mouse UCP2 probe revealed that this signal represented the mouse UCP2. This seemed to be because of the high identity of the ORF regions between the UCP2 and UCP3. 3.3. Cloning of 5∞-flanking region and intron of UCP3
Fig. 3. Tissue distribution analysis of mouse UCP2 and UCP3 transcripts by Northern blot. (A) Autoradiogram of a Northern blot of various mouse tissue poly(A)+ RNA hybridized with 32P-labeled mouse UCP3 probe. The molecular size markers are indicated in kb. (B) Autoradiogram of a Northern blot of various mouse tissue poly(A)+ RNA hybridized with 32P-labeled mouse UCP2 probe. The same blot was used in hybridizing with the UCP2 and UCP3 probes.
transfer-protein signature motifs were observed in the mouse UCP3 like in other UCPs (Palmier, 1994). Using a computer program analysis, we demonstrated a graphical representation showing relationships among the human, rat, and mouse UCPs ( Fig. 2b). The UCP3s have a higher homology of predicted amino acid sequences to the UCP2s than to the UCP1s. In addition, the UCP2 amino acid sequences were highly conserved among humans, rats and mice, in comparison with the UCP1 and UCP3 sequences. 3.2. Northern blot of mouse UCP3 The tissue distributions of mouse UCP3 mRNA were determined by Northern blot. The mouse UCP3 was abundantly expressed in skeletal muscle and somewhat less abundantly in heart. As Boss et al. reported that the human UCP3 mRNA signal exists in two sizes, it can also be seen that the mouse UCP3 mRNA signal is
PCR with antisense primers around the initiation codon revealed the existence of an intron 5∞-flanking to the exon including the initiation codon. The size of exon 1 was 104 bp (nt −197 to −94), and that of intron 1 was estimated to be ~6.5 kb. Repeating the PCR allowed us to obtain an ~2.7-kb 5∞-flanking region. The sequences of the regions were not similar between the mouse UCP1 and UCP3. In fact, the positions of the putative sites for transcriptional regulation, such as TATA-box, CCAAT-box, GC-box (Sp-1) and Ap-1, were different, as demonstrated by a computer search of the predicted promoter regions. Interestingly, two MyoD sites were seen in the putative promoter region of the mouse UCP3. One MyoD site was detected in 5∞-flanking region of the mouse Ucp1 (GenBank/EMBL Accession No. U63418). However, this position was ~−6.7 kb upstream from the transcription start point. The distributions of the representative transcriptional factor binding sites are illustrated in Fig. 4. 3.4. Genetic mapping of Ucp3 Genotyping of the BSS panel revealed that the Ucp3 locus was assigned to chromosome 7, cosegregating with Gucy2d, Art2a, and Art2b (Fig. 5). The gene order and recombination frequencies were as follows, expressed as genetic distances in centimorgans±SEM (number of recombinants/number of total mice scored ): centromere–Pcsk3–2.1±1.5 (2/94)–Tyr–2.1±1.5 (2/94)–Ucp3, Gucy2d, Art2a–1.1±1.1 (1/94)–Hbb, Cckbr–2.1±1.5 (2/94)–Adm–6.4±2.5 (6/94)–D7Mit98.
4. Discussion We subcloned the mouse UCP3 cDNA, and determined the tissue expression distributions by Northern blot using its ORF as a probe. The results showed that the tissue distributions of the mouse UCP3 were very different from those of the mouse UCP1 and UCP2. The mouse UCP2 was expressed ubiquitously, but the UCP1 and UCP3 were highly specific to brown adipose tissue (Jacobsson et al., 1985; Bouillaud et al., 1986) and skeletal muscle (Boss et al., 1997; Vidal-Puig et al., 1997), respectively. It is interesting that brown adipose
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Fig. 4. Schematic comparion of 5∞-flanking regions of mouse UCP1 and UCP3 genes. Boxes represent exons, in which the open and filled parts indicate untranslated and translated regions, respectively. Transcriptional start points are aligned vertically (dotted line). Potential binding sites for the transcriptional factors are shown as follows: TATA-box, small black box; CCAAT-box, black solid oval; C/EBP, black open triangle; Sp1, red open triangle; AP-1, purple open triangle; AP-2, green open triangle; AP-3, pale blue open triangle; CRE, black open oval; MyoD, black arrow head; NF-kB, black open diamond. The partial sequence of 5∞-flanking region of mouse UCP1 is derived from the sequence data with GenBank/EMBL Accession No. U63418.
Fig. 5. Genetic map of Ucp3 locus on chromosome 7. The numbers on the left show recombinant values±SEM (centimorgan). The locations for human homologues are given in parentheses next to the mouse genes. The information can be retrieved from the Jackson Laboratory homepage at http://www.informatics.jax.org. Complete haplotype data for all of the animals in this cross are also available from the Jackson Laboratory at http://www.jax.org/resources/documents/cmdata.
tissue and skeletal muscle have in common a rich blood supply and abundant mitochondria. A number of studies have demonstrated that brown adipose tissue plays an important role in regulating energy balance in rodents ( Rothwell and Stock, 1979; Brooks et al., 1980; HimmsHagen, 1989). In contrast to rodents, the amount of brown adipose tissue in humans is limited, suggesting that it is not a significant regulator of human energy expenditure. Skeletal muscle has been thought to be implicated as an important mediator of adaptive thermogenesis in humans (Astrup et al., 1985; Simonsen et al., 1992; Spaul et al., 1993). Recently, it was reported that the mice lacking in the UCP1 were cold-sensitive but not obese, and that the mRNA level of the UCP2 was up-regulated in brown adipose tissue ( Enerba¨ck et al., 1997). In these mice, UCP2 and UCP3 may help in the prevention of obesity by their augmented expressions. In other studies, it was shown that the UCP1 and UCP3 are up-regulated in brown adipose tissue by cold acclimation, but cold acclimation does not increase the UCP3 expression in skeletal muscle (Thurlby and Ellis, 1986; Himms-Hagen, 1990; Larkin et al., 1997; Boss et al., 1998). Futhermore, Boss et al. (1998) showed that the increase in UCP3 mRNA expression is modulated by fasting in skeletal muscle and is not modified by changes in environmental temperature. It has been known that the UCP expressions are controlled by various mechanisms. Especially, it should be noted that muscle UCP3 mRNA expression is dependent on food intake (Boss et al., 1998). Free fatty acids, plasma glucose and glucocorticoids are changed by fasting, and these factors may be messengers for modu-
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lating UCP3 mRNA expression in muscle. Further studies, such as the creation of transgenic and knockout mice for the UCP3, will be required to elucidate its physiological roles. Fleury et al. reported linkage of the Ucp2 with Arrb1 and Hbb on chromosome 7, suggesting that the Ucp2 and Ucp3 are clustered genes. Colocalization of the Ucp2 and Ucp3 within P1 and BAC mouse genomic clones (Solanes et al., 1997) document our findings. This region to which the Ucp3 is mapped is boundary of synteny between human chromosome 11q13 and 11p15 (Fig. 5). Radiation hybrid mapping of human UCP2 and UCP3 loci revealed that both are assigned to chromosome 11q13 (Solanes et al., 1997). Accordingly, the region in which the Ucp2 and Ucp3 are localized is syntenic to human chromosome 11q13. The phylogenic map of Fig. 2b shows that the amino acid sequences of the UCP3s are more similar to those of the UCP2s than those of the UCP1s. Therefore, it is likely that the Ucp2 and Ucp3 arose from the other via duplication. Two MyoD binding sites were observed in the 5∞-flanking proximal region of the mouse UCP3. It was proposed that the MyoD binding site works as a musclespecific enhancer of the murine neurofilament light chain ( Yaworsky et al., 1997). Thus, it is possible that MyoD binding sites are important for the mouse UCP3 to be expressed prominently in skeletal muscle. The UCP1 expression is unique in that it is localized in brown adipose tissue. Interestingly, no MyoD sites are observed in the ~2.8-kb 5∞-flanking region of the mouse UCP1, which includes a critical part for its brown fat-specific expression ( Kozak et al., 1994). To develop anti-obesity drugs, one approach is to search compounds to induce the uncoupling reaction by activation of the UCPs, for example, b -adrenergic 3 receptor agonists, which increase UCP1 expression and activity in brown adipose tissue, as well as the tissue volume. However, the amount of brown adipose tissue in humans is limited, and their effectiveness against human obesity is uncertain. Although the UCP2 is another potential target, its expression is widely in critical organs, with the resulting problem that unexpected side effects are caused by UCP2 activation. However, UCP3 expression is specific to skeletal muscle, which normally has a large capacity for adaptive energy expenditure. Thus, the UCP3 in muscle is a reasonable and potential target for anti-obesity drug development.
Acknowledgement We thank Drs Lucy B. Rowe and Lois Maltais, the Jackson Laboratory, for support in mapping the Ucp3 gene and naming it, respectively.
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Vidal-Puig, A., Solanes, G., Grujic, D., Flier, J.S., Lowell, B.B., 1997. UCP3: an uncoupling protein homologue expressed preferentially and abundantly in skeletal muscle and brown adipose tissue. Biochem. Biophys. Res. Commun. 235, 79–82. Yaworsky, P.J., Gardner, D.P., Kappen, C., 1997. Transgenic analyses reveal developmentally regulated neuron- and muscle-specific elements in the murine neurofilament light chain gene promoter. J. Biol. Chem. 272, 25112–25120. Zurlo, F., Larson, K., Bogardus, C., Ravussin, E., 1990. Skeletal muscle metabolism is a major determinant of resting energy expenditure. J. Clin. Invest. 86, 1423–1427.
Cloning of mouse uncoupling protein 3 cDNA and 5∞-flanking region, and its genetic map1 Hideki Yoshitomi *, Kazuto Yamazaki, Isao Tanaka Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3, Tokodai, Tsukuba, Ibaraki 300-2635, Japan Received 9 April 1998; accepted 7 May 1998; Received by T. Sekiya
Abstract Brown adipose tissue and skeletal muscle are important sites of non-shivering thermogenesis. It has been known that UCP1 and UCP2 function as the main effector of the thermogenesis: the former is expressed exclusively in brown adipose tissue, whereas the latter is distributed widely. Recently, the third UCP homologue was discovered in humans, which was designated as UCP3. We now report molecular cloning of full-length mouse UCP3 cDNA and its 5∞-flanking genomic region. The mouse UCP3 cDNA sequence predicted a 308-amino acid protein, and the overall identity between the mouse and human UCP3 proteins was 85.6%. The mouse UCP3 amino acid sequence was 54.7% and 73.1% identical to the mouse UCP1 and UCP2, respectively. Expression of the mouse UCP3 was found to be abundant in skeletal muscle and somewhat less abundant in heart, but was minimally expressed in other critical organs. The sequences of 5∞-flanking regions of the mouse UCP1 and UCP3 were very different, resulting in different distributions of putative transcriptional factor binding sites. The differences could reflect tissue-specific expression of the UCPs. The mouse Ucp3 gene was mapped near Ucp2 on chromosome 7, suggesting that the Ucp2 and Ucp3 are clustered genes. This region is boundary of synteny between human chromosome 11q13 and 11p15. As Solanes et al. reported that both human UCP2 and UCP3 genes are assigned to chromosome 11q13, the region where the mouse Ucp2 and Ucp3 are localized is syntenic to human chromosome 11q13. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Mitochondrial energy-transfer-protein signature; MyoD; Clustered gene; Non-shivering thermogenesis
1. Introduction UCPs are inner mitochondrial membrane transporters that dissipate the proton gradient, releasing stored energy as heat, without coupling to other energyconsuming process. Therefore, UCPs are thought to be important determinants of the metabolic efficiency (Nicholls and Locke, 1984; Klingenberg, 1990). UCP1, the first uncoupling protein to be identified (Lin and Klingenberg, 1980; Jacobsson et al., 1985; Bouillaud * Corresponding author. Tel: +81 298 47 5920; Fax: +81 298 47 5367; e-mail: [email protected] 1 Sequence data from this article have been deposited with the GenBank/EMBL Data Libraries under the following Accession Nos: mouse UCP3 complete cDNA, AB010742; rat UCP2 complete cDNA, AB010743; mouse UCP3 5∞-flanking region, AB011070. Abbreviations: bp, base pair; cDNA, DNA complementary to mRNA; EST, expressed sequence tag; kb, kilobase pairs; MTN, multiple tissue Northern; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; UCP, uncoupling protein; UTR, untranslated region. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 27 9 - 0
et al., 1986), is expressed exclusively in brown adipose tissue, an important organ for energy expenditure in rodents (Rothwell and Stock, 1979; Brooks et al., 1980; Himms-Hagen, 1989). Human UCP1 gene, located in 4q31, is composed of six exons and encodes a 307-amino acid sequence. A second uncoupling protein, UCP2, which is expressed ubiquitously contrary to UCP1 ( Fleury et al., 1997), might play a role in energy balance. Moreover, a third uncoupling protein homologue was discovered in humans and designated as UCP3 (Boss et al., 1997; Vidal-Puig et al., 1997). The UCP3 is expressed abundantly and preferentially in skeletal muscle in humans, and in brown adipose tissue and skeletal muscle in rodents. Because skeletal muscle and brown adipose tissue are regarded as important sites for regulated energy expenditure in humans (Astrup et al., 1985; Zurlo et al., 1990) and rodents (Himms-Hagen, 1989), respectively, it is possible that the UCP3 is a critical mediator of adaptive thermogenesis. Recently, we succeeded in cloning full-length mouse UCP3 cDNA and its 5∞-flanking region and mapping its
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Table 1 Primers used for genome walking to subclone 5∞-flanking region of mouse UCP3 gene Gene-specific primer 1 for first PCR Gene-specific primer 2 for nested PCR (5∞3∞)
Position in cDNA
PCR product
Origin of library
mUcp3-3R: GAA AAG TGA GGA GGT CCG CAA A mUcp3-4R: AGG AAC TTC ACA ACC GTT GTG G
76 to 97 32 to 53
~1.2 kb ~1.5 kb
PvuII SspI
mUcp3-6R: TGC GTC TAG CCA AGG TTG GGT A mUcp3-7R: CCT AAG GCT CCA CTC CAT TAG
−118 to −97 −151 to −131
~600 bp
DraI
mUcp3-8R: ACA ATG GTT ACA GGT GTC ACT mUcp3-9R: TAG AGG CCT GAC ATT CTC AAG A
~700 bp
PvuII
mUcp3-12R: AGC CCT CAT GTG TCA ATG CTT AC mUcp3-13R: GAC TGT CAA GGA CAG GCA GCT A
~1.7 kb
DraI
The subcloning was done using the GenomeWalker kit for the mouse including EcoRV, ScaI, DraI, PvuII and SspI libraries. All primers listed are antisense. The nucleotide positions in the cDNA are numbered from the first residue of the initiation codon.
locus. We report their sequences and comparison among human, mouse and rat UCPs.
2. Materials and methods
Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and sequence-specific primers, as well as T7 and U19 primers, with ABI Prism 377 DNA Sequencer (Applied Biosystems). The PCR conditions for sequencing were as follows: 25 cycles of 10 s at 96°C, 5 s at 50°C and 4 min at 60°C.
2.1. Cloning of mouse UCP3 2.2. Northern blot analysis Identification of mouse UCP3 fragment was achieved by homology searches of nucleotide database using the BLAST service from the National Center for Biotechnology Information at http://www.ncbi.nlm. nih.gov. Thus, we found an expressed sequence tag ( EST ) sequence, GenBank Accession No. AA062091, and primers were constructed for 5∞- and 3∞-RACE based on this sequence. We subcloned full-length mouse UCP3 cDNA using Marathon-Ready cDNA from 17-day mouse embryo (Clontech, Palo Alto, CA) according to the manufacturer’s manual. The primers to amplify the 5∞-fragment were: an adaptor primer, AP1 (5∞-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3∞) and mUcp3RV (5∞-AGT TCC CAG GCG TAT CAT GGC TTG-3∞, antisense, nt 424–447 numbered from the first residue of the initiation codon of cDNA sequence) in the first PCR, and a nested adaptor primer 2, AP2 (5∞-ACT CAC TAT AGG GCT CGA GCG GC-3∞) and mUcp3RV2 (5∞-CTG TCG TGC AGC CTG CCA GAA TCC-3∞, antisense, nt 347–370) in the nested PCR, and those for the 3∞-fragment were: AP1 and mUcp3FW (5∞-CAA GCC ATG ATA CGC CTG GGA ACT-3∞, sense, nt 424–447) in the first PCR, and AP2 and mUcp3FW2 (5∞-TAT GGA TGC CTA CAG AAC CAT CG-3∞, sense, nt 474–496) in the nested PCR. The PCR conditions were 40 cycles of 30 s at 94°C and 4 min at 68°C with a final extension for 15 min at 72°C in GeneAmp PCR System 9600 (Perkin-Elmer, Palo Alto, CA). The nested PCR products were subcloned into pT7Blue(R)T-vector (Novagen, Madison, WI ). Each PCR product was sequenced with a Dye Terminator
Mouse MTN blot containing approximately 2 mg of poly(A)+ RNA per lane was purchased from Clontech Laboratories (#7762-1). The same blot filter was used to hybridize with the mouse UCP2 and UCP3 probes. The mouse UCP2 and UCP3 probes were 930- and 927-bp fragments, respectively, spanning the entire open reading frames. The probe labeling was conducted by random priming in the presence of [a-32P] dCTP. For the hybridization, ExpressHyb Hybridization solution (Clontech) was used, and its procedures were performed as described by the manufacturer. The blot was hybridized at the highest stringency: 68°C for 2 h, followed by washing for 15 min at room temperature with 2× SSC/0.05% SDS five times, and then for 30 min at 55°C with 0.1× SSC/0.1% SDS three times. The filter was exposed to a film at −80°C for 18–24 h. To control the mRNA sample processing quantitatively, the Northern blot was stripped and rehybridized with a b-actinspecific probe. 2.3. Subcloning of 5∞-flanking region and intron of mouse UCP3 We subcloned 5∞-flanking region of the mouse UCP3 using the GenomeWalker kit for the mouse (Clontech) according to the manufacturer’s manual. The primers used were: AP1 (5∞-GTA ATA CGA CTC ACT ATA GGG C-3∞) and a gene-specific primer 1 in the first PCR, and AP2 (5∞-ACT ATA GGG CAC GCG TGG T-3∞) and a gene-specific primer 2 in nested PCR. The
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gene-specific primers are summarized in Table 1. The PCR conditions were as follows: seven cycles of 25 s at 94°C and 4 min at 72°C, 32 cycles of 25 s at 94°C and 4 min at 67°C with a final extension for 4 min at 67°C in the first PCR; five cycles of 25 s at 94°C and 4 min at 72°C, 22 cycles of 25 s at 94°C and 4 min at 67°C with an additional 4 min at 67°C in the nested PCR. The PCR products were subcloned to pT7Blue(R)Tvector and subjected to sequencing using T7, U19 and gene-specific primers. The computer analysis for the potential transcription factor binding sites was carried out using GENETEX software (Software Development, Tokyo, Japan). Intron 1 of the mUCP3 was amplified using the following primers: mUcp3-16 (5∞-TTA GGT TTC AGG TCA GCT GGT G-3∞, sense) and mUcp3-3R. C57BL/6J genomic DNA purchased from the Jackson Laboratory (Bar Harbor, ME) was used as a template. PCR conditions were: seven cycles of 25 s at 95°C and 4 min at 72°C, 38 cycles of 25 s at 95°C and 4 min at 67°C with an additional 4 min at 67°C. We determined the 5∞- and 3∞-end partial sequences of the PCR product by direct sequencing with gene-specific primers to confirm its identity.
2.4. Genetic mapping of mouse Ucp3
Fig. 1. Nucleotide and deduced amino acid sequence of mouse uncoupling protein 3. The nucleotides are numbered from the first nucleotide of the translational initiation codon as +1. The initiation methionine codon and the translation stop codon are in bold. The amino acid sequence is presented below the DNA sequence in the one-letter code. The three mitochondrial energy-transfer-protein signature domains are boxed. Constructed primers to amplify 5∞-RACE and 3∞-RACE fragments are underlined. A polyadenylation signal (AATAAA) is doubleunderlined.
We obtained 3∞-flanking genomic DNA of the Ucp3 gene using the GenomeWalker kit for the mouse. The primers used were: AP1 and mUcp3-1 (5∞-CAC CTT AGG GCA AGA ACG AGA A-3∞, sense, nt 1835–1857) in the first PCR, and AP2 and mUcp3-2 (5∞-TTC TCA TCT CAG GGT CGT ACC T-3∞, sense, nt 1889–1910) in the nested PCR. The PCR conditions were as follows: seven cycles of 25 s at 94°C and 4 min at 72°C, 33 cycles of 25 s at 94°C and 4 min at 67°C with a final extension for 4 min at 67°C in the first PCR; five cycles of 25 s at 94°C and 4 min at 72°C, 22 cycles of 25 s at 94°C and 4 min at 67°C with an additional 4 min at 67°C in the nested PCR. We determined the 3∞- end partial sequence of the nested PCR product of ~550 bp and set a primer to amplify the genomic DNA. A BSS intersubspecific backcross [(C57BL/6JEi X SPRET/Ei)F ×SPRET/Ei] 1 panel was obtained from the Jackson Laboratory, including one C57BL/6JEi and one SPRET/Ei, and 94 N individuals’ genomic DNA (Rowe et al., 1994). The 2 ~550-bp genomic DNA was amplified by the following primers: mUcp3-1 and mUcp3-5R (5∞-TCG GAT CTT TAG GCT CTC CAA G-3∞, antisense). The PCR conditions were 40 cycles of 30 s at 94°C, 30 s at 60°C and 1 min at 72°C with a final extension of 3 min 30 s at 72°C. The PCR products were digested with BspH I (New England BioLabs, MA): BspHI digestion identified an ~550 bp in C57BL/6J, and ~250 and 300 bp in SPRET/Ei. The digested PCR products were analyzed
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A
B
Fig. 2. (a) Comparison of human, rat and mouse UCP3 amino acid sequences. The amino acid sequences are denoted by a single-letter code. Identities among the sequences are indicated in asterisks. Gaps (—) are introduced to maximize the alignment among the sequences. The three mitochondrial energy-transfer-protein signature domains are double-underlined. The GenBank Accession Nos are as follows: human UCP3, AF001787; rat UCP3, RNU92069; mouse Ucp3, AB010742. (b) Phylogenic tree of human, rat and mouse UCPs. The amino acid sequences are compared to produce the tree, generated with DNASIS software program (Hitachi, Tokyo, Japan). The GenBank Accession Nos are as follows: human UCP1, U28480; human UCP2, U76367; human UCP3, AF001787; mouse UCP1, U63418; mouse UCP2, U69135; mouse UCP3, AB010742; rat UCP1, M11814; rat UCP2, AB010743; rat UCP3, RNU92069.
on 1.2% agarose (Gibco BRL, Gaithersburg, MD)/ ethidium bromide gel electrophoresis.
3. Results 3.1. Cloning of full-length mouse UCP3 The EST database was screened for sequences homologous to the mouse UCP1. One mouse EST was identified (GenBank Accession No. AA062091) that was similar, but not identical, to the mouse UCP1 and mouse UCP2 sequences. We constructed the primers based on the EST sequence, and 5∞- and 3∞-RACE were performed
to clone its fragments presumed to represent the mouse UCP3. In humans, long and short forms of the UCP3 were observed (Boss et al., 1997), but we only found a mouse UCP3 fragment corresponding to the long form in PCR from the mouse embryo cDNA library. The mouse UCP3 cDNA sequence contained a 5∞-UTR of 197 bp, an ORF of 927 bp and a 3∞-UTR of 1323 bp, with a polyadenylation signal (AATAAA) and polyA tail ( Fig. 1). The mouse UCP3 protein deduced from the ORF was composed of 308 amino acids and was estimated to have a molecular weight of ~34 kDa. At the amino acid level, the mouse UCP3 was 85.6% identical to the human UCP3 and 97.7% identical to the rat UCP3 ( Fig. 2a). The mitochondrial energy-
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a doublet ( Fig. 3). The sizes of the signals, as compared with the marker, were ~2.4 and ~2.6 kb. No UCP3 signals were detected in the other critical organs. Northern blots also showed the presence of a smaller mRNA transcript of ~1.6 kb, but rehybridization of the same blots with the mouse UCP2 probe revealed that this signal represented the mouse UCP2. This seemed to be because of the high identity of the ORF regions between the UCP2 and UCP3. 3.3. Cloning of 5∞-flanking region and intron of UCP3
Fig. 3. Tissue distribution analysis of mouse UCP2 and UCP3 transcripts by Northern blot. (A) Autoradiogram of a Northern blot of various mouse tissue poly(A)+ RNA hybridized with 32P-labeled mouse UCP3 probe. The molecular size markers are indicated in kb. (B) Autoradiogram of a Northern blot of various mouse tissue poly(A)+ RNA hybridized with 32P-labeled mouse UCP2 probe. The same blot was used in hybridizing with the UCP2 and UCP3 probes.
transfer-protein signature motifs were observed in the mouse UCP3 like in other UCPs (Palmier, 1994). Using a computer program analysis, we demonstrated a graphical representation showing relationships among the human, rat, and mouse UCPs ( Fig. 2b). The UCP3s have a higher homology of predicted amino acid sequences to the UCP2s than to the UCP1s. In addition, the UCP2 amino acid sequences were highly conserved among humans, rats and mice, in comparison with the UCP1 and UCP3 sequences. 3.2. Northern blot of mouse UCP3 The tissue distributions of mouse UCP3 mRNA were determined by Northern blot. The mouse UCP3 was abundantly expressed in skeletal muscle and somewhat less abundantly in heart. As Boss et al. reported that the human UCP3 mRNA signal exists in two sizes, it can also be seen that the mouse UCP3 mRNA signal is
PCR with antisense primers around the initiation codon revealed the existence of an intron 5∞-flanking to the exon including the initiation codon. The size of exon 1 was 104 bp (nt −197 to −94), and that of intron 1 was estimated to be ~6.5 kb. Repeating the PCR allowed us to obtain an ~2.7-kb 5∞-flanking region. The sequences of the regions were not similar between the mouse UCP1 and UCP3. In fact, the positions of the putative sites for transcriptional regulation, such as TATA-box, CCAAT-box, GC-box (Sp-1) and Ap-1, were different, as demonstrated by a computer search of the predicted promoter regions. Interestingly, two MyoD sites were seen in the putative promoter region of the mouse UCP3. One MyoD site was detected in 5∞-flanking region of the mouse Ucp1 (GenBank/EMBL Accession No. U63418). However, this position was ~−6.7 kb upstream from the transcription start point. The distributions of the representative transcriptional factor binding sites are illustrated in Fig. 4. 3.4. Genetic mapping of Ucp3 Genotyping of the BSS panel revealed that the Ucp3 locus was assigned to chromosome 7, cosegregating with Gucy2d, Art2a, and Art2b (Fig. 5). The gene order and recombination frequencies were as follows, expressed as genetic distances in centimorgans±SEM (number of recombinants/number of total mice scored ): centromere–Pcsk3–2.1±1.5 (2/94)–Tyr–2.1±1.5 (2/94)–Ucp3, Gucy2d, Art2a–1.1±1.1 (1/94)–Hbb, Cckbr–2.1±1.5 (2/94)–Adm–6.4±2.5 (6/94)–D7Mit98.
4. Discussion We subcloned the mouse UCP3 cDNA, and determined the tissue expression distributions by Northern blot using its ORF as a probe. The results showed that the tissue distributions of the mouse UCP3 were very different from those of the mouse UCP1 and UCP2. The mouse UCP2 was expressed ubiquitously, but the UCP1 and UCP3 were highly specific to brown adipose tissue (Jacobsson et al., 1985; Bouillaud et al., 1986) and skeletal muscle (Boss et al., 1997; Vidal-Puig et al., 1997), respectively. It is interesting that brown adipose
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Fig. 4. Schematic comparion of 5∞-flanking regions of mouse UCP1 and UCP3 genes. Boxes represent exons, in which the open and filled parts indicate untranslated and translated regions, respectively. Transcriptional start points are aligned vertically (dotted line). Potential binding sites for the transcriptional factors are shown as follows: TATA-box, small black box; CCAAT-box, black solid oval; C/EBP, black open triangle; Sp1, red open triangle; AP-1, purple open triangle; AP-2, green open triangle; AP-3, pale blue open triangle; CRE, black open oval; MyoD, black arrow head; NF-kB, black open diamond. The partial sequence of 5∞-flanking region of mouse UCP1 is derived from the sequence data with GenBank/EMBL Accession No. U63418.
Fig. 5. Genetic map of Ucp3 locus on chromosome 7. The numbers on the left show recombinant values±SEM (centimorgan). The locations for human homologues are given in parentheses next to the mouse genes. The information can be retrieved from the Jackson Laboratory homepage at http://www.informatics.jax.org. Complete haplotype data for all of the animals in this cross are also available from the Jackson Laboratory at http://www.jax.org/resources/documents/cmdata.
tissue and skeletal muscle have in common a rich blood supply and abundant mitochondria. A number of studies have demonstrated that brown adipose tissue plays an important role in regulating energy balance in rodents ( Rothwell and Stock, 1979; Brooks et al., 1980; HimmsHagen, 1989). In contrast to rodents, the amount of brown adipose tissue in humans is limited, suggesting that it is not a significant regulator of human energy expenditure. Skeletal muscle has been thought to be implicated as an important mediator of adaptive thermogenesis in humans (Astrup et al., 1985; Simonsen et al., 1992; Spaul et al., 1993). Recently, it was reported that the mice lacking in the UCP1 were cold-sensitive but not obese, and that the mRNA level of the UCP2 was up-regulated in brown adipose tissue ( Enerba¨ck et al., 1997). In these mice, UCP2 and UCP3 may help in the prevention of obesity by their augmented expressions. In other studies, it was shown that the UCP1 and UCP3 are up-regulated in brown adipose tissue by cold acclimation, but cold acclimation does not increase the UCP3 expression in skeletal muscle (Thurlby and Ellis, 1986; Himms-Hagen, 1990; Larkin et al., 1997; Boss et al., 1998). Futhermore, Boss et al. (1998) showed that the increase in UCP3 mRNA expression is modulated by fasting in skeletal muscle and is not modified by changes in environmental temperature. It has been known that the UCP expressions are controlled by various mechanisms. Especially, it should be noted that muscle UCP3 mRNA expression is dependent on food intake (Boss et al., 1998). Free fatty acids, plasma glucose and glucocorticoids are changed by fasting, and these factors may be messengers for modu-
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lating UCP3 mRNA expression in muscle. Further studies, such as the creation of transgenic and knockout mice for the UCP3, will be required to elucidate its physiological roles. Fleury et al. reported linkage of the Ucp2 with Arrb1 and Hbb on chromosome 7, suggesting that the Ucp2 and Ucp3 are clustered genes. Colocalization of the Ucp2 and Ucp3 within P1 and BAC mouse genomic clones (Solanes et al., 1997) document our findings. This region to which the Ucp3 is mapped is boundary of synteny between human chromosome 11q13 and 11p15 (Fig. 5). Radiation hybrid mapping of human UCP2 and UCP3 loci revealed that both are assigned to chromosome 11q13 (Solanes et al., 1997). Accordingly, the region in which the Ucp2 and Ucp3 are localized is syntenic to human chromosome 11q13. The phylogenic map of Fig. 2b shows that the amino acid sequences of the UCP3s are more similar to those of the UCP2s than those of the UCP1s. Therefore, it is likely that the Ucp2 and Ucp3 arose from the other via duplication. Two MyoD binding sites were observed in the 5∞-flanking proximal region of the mouse UCP3. It was proposed that the MyoD binding site works as a musclespecific enhancer of the murine neurofilament light chain ( Yaworsky et al., 1997). Thus, it is possible that MyoD binding sites are important for the mouse UCP3 to be expressed prominently in skeletal muscle. The UCP1 expression is unique in that it is localized in brown adipose tissue. Interestingly, no MyoD sites are observed in the ~2.8-kb 5∞-flanking region of the mouse UCP1, which includes a critical part for its brown fat-specific expression ( Kozak et al., 1994). To develop anti-obesity drugs, one approach is to search compounds to induce the uncoupling reaction by activation of the UCPs, for example, b -adrenergic 3 receptor agonists, which increase UCP1 expression and activity in brown adipose tissue, as well as the tissue volume. However, the amount of brown adipose tissue in humans is limited, and their effectiveness against human obesity is uncertain. Although the UCP2 is another potential target, its expression is widely in critical organs, with the resulting problem that unexpected side effects are caused by UCP2 activation. However, UCP3 expression is specific to skeletal muscle, which normally has a large capacity for adaptive energy expenditure. Thus, the UCP3 in muscle is a reasonable and potential target for anti-obesity drug development.
Acknowledgement We thank Drs Lucy B. Rowe and Lois Maltais, the Jackson Laboratory, for support in mapping the Ucp3 gene and naming it, respectively.
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