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Human Muscle Carbonic Anhydrase: Gene Structure and DNA Methylation Patterns in Fetal and Adult Tissues

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Human Muscle Carbonic Anhydrase: Gene Structure and DNA Methylation Patterns in Fetal and Adult Tissues Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Human muscle carbonic anhydrase: gene structure and DNA methylation patterns in fetal and adult tissues Julie Lloyd, Carol Brownson, ~ Susan Tweedie, Jillian Charlton, and Yvonne H. Edwards MRC, Human Biochemical Genetics Unit, The Galton Laboratory, University College London, London NW1 2HE, UK; ~Department of Biological Sciences, City of London Polytechnic, London E1 7NT, UK We report the isolation and analysis of genomic clones comprising the entire gene coding for the human muscle carbonic anhydrase, CAIII. The gene spans 10.3 kb and has a seven-exon/six-intron structure. A noncanonical TATA box, a CCAAT motif, and two CCGCCC elements are present in the sequences upstream of exon 1. Although the expression of CAIII shows strict tissue specificity, the gene exhibits a number of features normally associated with housekeeping enzymes. For example, there is 48% homology with a 25-bp consensus sequence between the TATA box and the cap site and there is a CpG-rich island spanning a 469-bp sequence near to the origin of transcription. Methylation studies suggest that some CCGG sites clustered in the CpG-rich island are undermethylated in DNA from fetal and adult muscle and in other tissues irrespective of CAIII expression. In contrast, several nonclustered CCGG sites show a methylation pattern that correlates with gene expression. However DNA from differentiated type II adult muscle fibers is undermethylated at these sites even though CAIII is not expressed. [Key Words: Carbonic anhydrase; tissue-specific gene; CpG-rich islands; methylation; muscle development] Received April 21, 1987; revised version accepted June 8, 1987. Carbonic anhydrase III (CAIII, carbonate dehydratase EC levels by 20 weeks and to about 50% by birth. CAIII ex- 4.2.1) is a monomeric zinc metalloenzyme catalyzing pression can also be manipulated experimentally by the reversible hydration of carbon dioxide. In man, CAIII muscle denervation (Wistrand et al. 1986)or electro- is an abundant muscle-specific protein (Carter et al. stimulation (Edwards et al., in prep.), procedures that 1979) involved in the maintenance of ionic balance and alter the contractile properties and fiber type distribu- acid-base homeostasis. CAIII is characteristic of slow- tion of the muscle. An unusual situation occurs in rat twitch, aerobic, type I muscle fibers. The structural gene tissues where CAIII is expressed in liver as well as skel- for CAIII is part of a multigene family, other members of etal muscle. In the liver the CAIII protein levels exhibit which show tissue- and organelle-specific expression. a sexual dimorphism which is apparently regulated by CAI activity is confined largely to erythrocytes. CMI is growth hormone (Jeffery et al. 1986). also a major protein in erythrocytes but is expressed in a To provide probes for the analysis of the regulation of wide variety of other tissues. Distinct mitochondrial and the CMII gene, we have recently isolated a cDNA clone, membrane isoforms of CA have also been described. pCA15, encoding the entire sequence of human muscle Protein and cDNA nucleotide sequence analyses of CAI, CAIII (Lloyd et al. 1986). In the present study this cDNA CAII, and CAIII have shown that these genes are evolu- clone was employed to isolate the CMII gene and to elu- tionarily related (for review of CA see Tashian and He- cidate its structure. Since methylation of cytosine res- wett-Emmett 1984). These three genes in man have been idues has been implicated in gene regulation (for review, assigned to chromosome 8 (Venta et al. 1983; Edwards et see Razin et al. 1984; Bird 1986), we have investigated al. 1986a, b), and more recently it has been shown that the pattern of methylation in DNA from CMII ex- CAI and CAIII are in the same region of this human pressing and nonexpressing tissues, using both cDNA chromosome at q22 (Davis et al. 1987). and genomic DNA probes. The CAIII gene appears to be differentially regulated both in adult tissues where high expression is confined Results and discussion to one cell type, and in fetal muscle at different stages of Isolation of the CAIII gene: mapping of exons and development (Jeffery et al. 1980; Lloyd et al. 1986). In introns human limb muscle, trace amounts of CMII protein and mRNA are detected at 10 weeks gestation. These levels The recombinant clones ~,CAI.1, 1.2, and 1.3 shown in increase to approximately 20% of mature adult muscle Figure 1 were isolated by screening a genomic library in 594 GENES & DEVELOPMENT 1:594-602 © 1987 by Cold Spring Harbor Laboratory ISSN 0890-9369/87 $1.00 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Human muscle carbonic anhydrase P T L -'I~T T I kb A A H T E H E E Hh T T :-..._: E e- E E ° E L////J'///////////~ f/ZJ~///////////7] E Hh Hh T R T _ =_ =_ - 8 1 1,1 , 11 I ll , , !" I I1___ ~l ~1~1 1-6 7 8 10 1 ),CA2.1 (15kb) ~.CA2.2 ( 17 kt)) C ,~CAI-1 (l'Tkb) ~.A2-z XcA1-3 pc.A 3.4 Figure 1. (A) The sequencing strategy employed to determine the exon-intron boundaries in the human CAIII gene. Subcloned fragments are indicated: (E)EcoRI; (T) TaqI; (P) PstI; (Hh) HhaI; (R) RsaI. Their size and position relate exactly to the physical map given below in B. The hatched boxes indicate subcloned fragments that were sequenced in full after the generation of multiple, small, random M13mp8 subclones. (B) Physical map of the CAIII gene, with coding sequences indicated by filled boxes and nontranslated mRNA sequences by open boxes. (C) Various recombinant phage clones are shown, and the relative positions of two subcloned genomic fragments (pCA2.2 and pCA3.4)referred to in the text are indicated. Charon 4A (Lawn et al. 1978)using the CAIII cDNA quence. The most 3' exon contains 147 bp of coding se- pCA15 (Lloyd et al. 1986) as a probe. The most 5' 2.2-kb quence and 877 bp of 3' noncoding sequence. The other EcoRI fragment from ~,CAI.1 was subcloned into pUC8 exons vary in size between 63 and 198 bp. (pCA 2.2) and used to screen the Charon 4A library and The overall architecture of the muscle carbonic anhy- another human genomic library in ?~2001 (LeFranc et al. drase gene CAIII appears to be very similar to that de- 1986) to derive the CAIII recombinants kCA2.1 and scribed for the reticulocyte gene CAII isolated from the kCA2.2. An EcoRI, HindIII, and TaqI restriction enzyme mouse (Venta et al. 1985). The CAII gene is also inter- map extending across a 22-kb region of genomic DNA rupted by six introns. The sizes of IVS 5 and 6 are similar and encompassing the whole of the CAIII gene was de- in the two genes but the sizes of the other introns show rived by a combination of single and double digests of considerable variation; in particular, IVS 2, which is 2.4 DNA from the various ~,CA genomic clones. All of the kb in the CAIII gene, is 7.2 kb in the CAII gene. The EcoRI and TaqI fragments from kCAI.1 and ~,CA2.2, positions of five of the introns are identical to those shown in Figure 1B, were subcloned into pUC8. Exon found in CAII. However, IVS 4 which in the CAIII se- sequences were localized by hybridization of restriction quence occurs between codons 147 (Lys)and 148 (Ile)is enzyme digests (HhaI, RsaI, A vaII, PstI) of these sub- found 14 bp upstream in CAII where it interrupts the clones to the 32P-labeled CAIII cDNA clone and to DdeI codon for Gly-143. This difference between CAII and fragments derived from the 5' end (278 bp), the center CAIII is unexpected and may represent a peculiarity of (576 bp), and the 3' end (419 bp) of the cDNA clone (see the mouse CAII sequence since there is some recent evi- Lloyd et al. 1986 for cDNA sequence). dence that human CAI and CAII (Venta et al. 1987; R. In some cases further subclones were prepared which Tashian, pers. comm.) and chicken CAII (Yoshihara et contained smaller DNA inserts cross-hybridizing to the al. 1987)have IVS 4 in the same position as described cDNA. The DNA inserts from the various subclones here for CAIII. In general, intron positions within multi- (Fig. 1A)were sequenced from both ends to obtain the gene families are conserved between homologous mam- precise location of each of the exon-intron boundaries. malian and avian genes. Although long evolutionary dis- In addition, the 2.2-kb 5'-terminal EcoRI fragment of tances may lead to insertion or loss of introns, it is diffi- kCAI.1, containing exon 2, and the 2.8-kb 5'-terminal cult to envisage a mechanism by which a shift in the EcoRI fragment of kCA2.1 were sequenced in full using position of an intron might occur. the dideoxy method and 57 independent, randomly gen- erated M13 subclones (hatched boxes in Fig. 1A). 3' End of the CAIII gene These analyses showed that the gene for human CAIII is about 10.3 kb long and comprises seven exons. The The nucleotide sequence extending 270 bp downstream sequences at the 5' and 3' junction of each intron are of the poly(AI addition site was determined. The se- shown in Figure 2. All the introns, which vary in size quence TTGTGTTTT is found 25 bp downstream of the from 750 bp (WS 4) to 2360 bp (IVS 2), conform to the AATAAA polyadenylation motif and this fits the con- consensus eukaryotic structure beginning with the din- sensus sequence YGTGTTYY discussed by McLauchlan ucleotide GT and ending with AG.
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