The gene structure and promoter sequence of mouse ... - NCBI

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Biochem. J. (1998) 330, 1223–1227 (Printed in Great Britain)

The gene structure and promoter sequence of mouse hyaluronan synthase 1 (mHAS1) Yoichi YAMADA*†, Naoki ITANO*, Masahiro ZAKO*, Mamoru YOSHIDA*, Petros LENAS*, Atsushi NIIMI†, Minoru UEDA† and Koji KIMATA*1 *Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-11, Japan, and †Department of Oral Surgery, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan

The structure and organization of mouse hyaluronan synthase 1 gene, HAS1 were determined by direct sequencing of λ phage clones carrying the entire gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon–intron boundaries and all of the exons. This gene spans about 11 kb of genomic DNA and consists of 5 exons and 4 introns. A similarity in the exon–intron organization was found between the genes of mouse HAS1 and Xenopus laeŠis DG42 which was recently identified as Xenopus hyaluronan synthase.

The transcription initiation site was determined by rapid amplification of the cDNA ends (5«-RACE). Position ­1 is located 55 nucleotides upstream of the ATG initiation codon. The promoter region of the HAS1 gene has no typical TATA box, but contains a CCAAT box located 190 nucleotides upstream of the transcription initiation site. Further analysis of 1±4 kb of the 5« flanking region revealed several potential binding motifs for transcription factors. This information about the gene structure may be useful for further studies on the promoter activity.

INTRODUCTION

cells all led to hyaluronan synthesis, suggesting that they all play key roles in hyaluronan synthesis and, therefore, there are three putative mammalian hyaluronan synthases encoded by three distinct but related genes. The results have then raised many questions, such as how they are functionally different in their expressions. In order to have access to answering this question, we have, as the first step, investigated the genomic gene structure of mouse HAS1 and have also made a preliminary analysis of possible binding sites for transcription control factors on the promoter region. The results have suggested the involvement of various cis-elements such as a CCAAT box and binding sites for AP-2, CREB, p53, Sox-5, SRY, and MyoD which are known to be important for unique tissue differentiation and homeostasis.

Hyaluronan is a high-molecular-weight linear molecule, which is composed of β-1,4-linked repeating disaccharides of glucuronosyl β-1,3-N-acetylglucosamine. Although this macromolecule is a passive structural component in the matrix of a few connective tissues to support tissue architectures and in the capsule of certain strains of bacteria possibly to prevent host defence attack, hyaluronan is dynamically involved in many biological processes such as modulating cell migration, cell differentiation and cell–cell recognition as a major component of the extra- and pericellular matrix especially during the early stages of animal embryogenesis, and its synthesis is spatially and temporally regulated [1,2]. Hyaluronan also plays important roles in the complex processes of metastasis, wound healing, and inflammation [3–9]. In addition, hyaluronan is now recognized as a clinical medicine. A concentrated solution of hyaluronan has, through its tissue protective and rheological properties, become useful in ophthalmic surgery. Analysis of serum hyaluronan may also be useful for the diagnosis of liver disease and various inflammatory conditions, such as rheumatoid arthritis. In comparison with the findings related to biological and physiological significances of hyaluronan, the biosynthesis and its regulatory mechanisms have remained uncertain. Recently, we showed for the first time the gene, HAS1 for hyaluronan synthesis in mouse and human mammalian systems [10,11], which is related to the gene of the Streptococcus pyogenes hyaluronan synthase, HasA [12] and the one of the Xenopus laeŠis protein, DG42 [13]. DG42 was then shown to be a frog hyaluronan synthase [14,15]. More recently, other research groups identified two different mammalian genes, HAS2 and HAS3, encoding putative hyaluronan synthases which were isolated by the cloning of cDNA libraries using methods based on the molecular identity among these related proteins ([16–19], see also review [3]). Interestingly, expression of these genes by

EXPERIMENTAL PROCEDURES Isolation and characterization of the mHAS1 gene A 129}sv mouse genomic DNA library in the λFixII vector (Stratagene, La Jolla, CA, U.S.A.) was used for screening. About 5¬10' plaques were screened with fluorescein-labelled cDNA probes corresponding to the mouse HAS1 ORF (open reading frame) sequence. The plaques were transferred onto membrane filters and hybridized with the cDNA probes for 12 h at 60 °C in 5¬SSC (sodium chloride}sodium citrate buffer), 0±5 % (w}v) blocking agent (Amersham International, Amersham, Bucks., U.K.), 0±1 % (w}v) SDS, 5 % (w}v) dextran sulphate and 100 mg}ml denatured salmon sperm DNA. The filters were first washed for 30 min at 60 °C in 1¬SSC and 0±1 % SDS, then for 30 min at 60 °C in 0±5¬SSC and 0±1 % SDS. Positive clones were detected with the ECL4 detection kit (Amersham). The genomic DNA inserts were excised from the phage DNA by digestion with NotI and then subcloned into pBluescript II KS vector (Stratagene) for further characterization. To map the exons, the results from restriction enzyme mapping and hybridization with the cDNA probes (Southern blots) were combined. Restriction

Abbreviations used : mHAS, mouse hyaluronan synthase ; PCR, polymerase chain reaction ; 5«-RACE, rapid amplification of cDNA ends ; SSC, sodium chloride/sodium citrate buffer ; ORF, open reading frame. 1 To whom correspondence should be addressed. The nucleotide sequences reported in this paper has been submitted to the GenBank/EBI Data Bank with accession numbers : AB005226 and AB005227.

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enzyme mapping and sequencing of the obtained clones showed that any of them coded for the 5« end of the mouse HAS1 gene. The same cDNA library was then rescreened with the 3« end of the cDNA probes which was obtained by the PCR with a pair of the primers corresponding to the dGGGGATGTGAGGATCCTTAACCCTC (position 856–880 of the mouse HAS1 ORF sequence) and the dAACCACTTTGATTCATGGTAA (the position 1525–1505). The additional clones thus obtained were further identified as described above. The size of each intron was determined by polymerase chain reaction (PCR) using primers located in the exons (see Figures 2a and 2b) and, in some cases, by restriction enzyme mapping (Figure 1). The PCR was performed using the Expand4 Long Template PCR system (Boehringer–Mannheim, GmbH, Germany). The PCR amplification was carried out by 35 cycles ; each cycle consisted of denaturation of 94 °C for 20 s, annealing at 58 °C for 30 s, and extension at 68 °C for 8 min. The PCR products were checked by electrophoresis on a 1 or 2 % agarose gel.

RESULTS AND DISCUSSION Isolation and characterization of the mHAS1 gene A 129}sv mouse genomic DNA library was initially screened with cDNA probes derived from the mouse HAS1 cDNA clones, and several positive clones were isolated. Restriction enzyme mapping and sequencing of these clones confirmed that they were overlapping but coded for the 5« end of the mouse HAS1 gene. The same cDNA library was, therefore, rescreened with the 3« end of the cDNA probes as described in the Experimental procedures. Several additional clones were further identified. As a result, clone λgc1 containing the longest 5« extension and clone λgc2 were selected for further analysis. These two clones gave the entire gene of mouse HAS1 (Figure 1). The gene spanned 11 kb from the transcription initiation site to the polyadenylation signal.

Structure and organization of the mHAS1 gene DNA sequence and analysis The nucleotide sequences of the isolated genomic DNAs were determined by repeated sequencing of both strands of alkalinedenatured plasmid DNA using the deazaGTP kit with Sequenase version 2.0 (U.S. Biochemical Corp., Cleveland, OH, U.S.A.). The sequencing was also performed in a Model 373A DNA Sequencer (Applied Biosystems Co., Tokyo, Japan). The oligonucleotide primers synthesized based on the sequences of cDNA and genomic DNA were used for the subsequent sequencing. The DNA sequences obtained were compiled and analysed using the - computer program (Software Development Co., Ltd., Tokyo, Japan) and  (Internet database).

Determination of 5«-RNA ends The rapid amplification of cDNA ends (RACE) method was used to identify transcription initiation sites [20,21], using a 5« Ampli Finder RACE kit (Clontech, Palo Alto, CA, U.S.A.). Briefly, 2 µg of poly(A)+ RNA derived from FM3A HA1 cells [10] was annealed with an antisense primer corresponding to the cDNA sequence derived from the second exon (primer a, 5«-dCACCACCATGAGCACGCGTAACCTCGTGTG-3«). To prevent an artificial termination due to the secondary structure of mRNA, the reverse transcription was performed using Molony Murine Leukaemia virus reverse transcriptase at 52 °C for 30 min. The 3« end of the resulting first-strand cDNA was ligated to a single-stranded oligonucleotide anchor provided in the kit by T4 RNA ligase. An aliquot of the reaction product was then used as a template for PCR amplification using a primer complementary to the anchor sequence (primer c) and a nested genespecific primer corresponding to the second exon (primer b, 5«-dCGCGGAGGTCAAGCACTGGCGCAGGTAAGC-3«). PCR amplification was carried out by 35 cycles ; each cycle consisted of denaturation at 94 °C for 45 s, annealing at 60 °C for 45 s, and extension at 72 °C for 2 min. The PCR product was analysed by 2 % agarose gel electrophoresis. Amplified products (see Figure 4) were purified from the gels by Jetsorb (Genomed GmbH, Oeynhausen, Germany) and cloned into BamHI and EcoRI sites of pBluescript II KS vector. The inserted cDNA sequences were determined by repeated sequencing of both strands of alkaline-denatured plasmid DNA using the deazaGTP kit with Sequenase version 2±0 (U.S. Biochemical Corp.). The experiment was repeated five times in order to examine the reproducibility of the above reaction processes.

The organization of the mouse HAS1 gene was shown by boundary architectures between exons and introns determined by restriction enzyme mapping and partial sequencing of PCR products (Figures 1 and 2). The mouse HAS1 gene consisted of 5 exons. All exon sequences including part of the exon-flanking regions were identified bidirectionally. Sequences for the exon– intron splicing junctions almost matched with consensus sequences for donor and acceptor sites [22] (Figure 3). Exon I was the shortest with 64 bp, but it contained a 5« untranslated sequence, the ATG starting codon which is in the 9 bp upstream from Exon I}Intron I boundary, and the transcription initiation site which was located 55 bp upstream from the ATG codon as described below. Hence, only three amino acid residues from Exon I participated in the ORF. Several amino acid residues near and on the Exon III}Exon IV boundary which was located at 998 bp of HAS1 cDNA were found to be conserved in the HAS protein family (HAS1, 2 and 3, HasA and DG42) and therefore might correspond to some specific residues necessary to create an active HAS. For example, the cysteine residue in the Exon III which was numbered 309 residues from the N-terminal end of mouse HAS1 protein was conserved in all other HAS family proteins [3]. It is likely that part of this exon

Figure 1 clones

Organization of mHAS1 and alignment of isolated genomic DNA

Two phage clones, λgc1 and λgc2 are shown as horizontal lines. Exons are indicated by closed boxes and numbered from I to V. The name and characteristics of each domain are described in the Results and Discussion. Introns as well as 5« and 3« flanking regions are indicated by lines. The initiation (ATG) and termination (TGA) codons are indicated by arrowheads. The upper thin line depicts the positions of restriction sites for B, Bam HI ; E, Eco RI ; and H, Hin dIII, respectively. The scale for 1 kb is indicated.

Mouse HAS1 gene Table 1

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Comparative analysis of the mHAS1 and DG42 genes

The sizes of all domains are given in base pairs. The size of exon 1 was based on the major transcription start site as shown in Figure 5. The size of exon V was evaluated from the cDNA sequence. Data for the DG42 gene is from ref. [13]. Size (bp) Exon

HAS1

DG42

I II III IV V

64 711 223 134 ! 945

76 691 225 132 ! 1351

Figure 2 Length between exons of mHAS1 determined by PCR and subsequent gel electrophoresis (a) Primers used for PCR amplifications. a Letters in parentheses indicate sense (S) or antisense (A) primers. b Sequences are written from the 5« to 3« direction. c The primer positions [10]. (b) The primer positions used for PCR amplification to detect various intron length of mHAS1 are schematically shown (see a). (c) PCR products were analysed by agarose gel electrophoresis. DNA size markers are shown in lanes 1 and 6. Lanes 2 and 3, PCR products for the distance of exon IV/exon V with the primer pair d and i or j ; lanes 4 and 5, those for the distance of exon II/exon III with the primer pair a and e or f ; lanes 7 and 8, those for the distance of exon III/exon IV with the primer pair b and h and with the primer pair c and g, respectively.

Figure 4 method

Determination of mHAS1 gene transcription site by 5«-RACE

(a) The positions of three different primers used for the reverse transcription reaction and PCR amplification are indicated by arrowheads a–c. Exon boundaries are indicated by vertical arrows. An Eco RI site is indicated by open inverted triangles and a Bam HI site by close inverted triangles. The mRNA and first-strand cDNA are indicated by thin lines. The anchor is indicated by the thick line. (b) The products from the first PCR amplification were analysed by agarose gel electrogenesis. The amplified DNA fragments are indicated on the right lane. DNA size markers are shown on the left lane and their base pairs are shown by arrowheads.

Figure 3 Alignments of exon–intron boundaries and comparison with the consensus sequences for splicing Exon sequences are indicated by upper-case letters and intron sequences by lower-case letters. Consensus sequences for splice sites are shown at the top and are adapted from the work of Shapiro and Senapathy [22].

signal. Table 1 summarizes the actual exon sizes of the mouse HAS1 gene in comparison with those of the DG42 gene [13]. DG42 was almost identical in the genomic gene structure to mouse HAS1. It is likely, therefore, that DG42 may correspond to frog HAS1.

Possible transcription initiation site boundary may be responsible for the active site common to all proteins in the HAS family. Exon V coded the termination codon and 3« untranslated region including a potential polyadenylation

The transcription starting site on the mouse HAS1 gene was determined by the modified 5«-RACE method (Figure 4a). The electrophoresis of the amplified DNA fragments on 2 % agarose gel gave only one major band (Figure 4b). Sequence analysis of

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Figure 5 Nucleotide sequence of the 5« flanking promoter region of mHAS1 and potential binding motifs for transcription factors Nucleotide­1 denotes the A (shown in bold) residue of the transcription initiation sites, which was determined by the 5«-RACE method, and residues preceding it are indicated by negative numbers. The sequences of potential regulatory cis-elements are underlined. The CCAAT boxes are indicated by bold lines and the nucleotide sequences for TATA boxes are indicated by dashed lines.

this fragment suggested that the transcription initiation site was located at 55 bp upstream from the ATG translation initiation site and 7 bp upstream from the 5« end of the cDNA which we had described previously for the molecular cloning [10]. Five independent PCR products gave the identical sequence, which suggests the reliability of the above conclusion although further analysis is necessary to confirm the actual transcription site. The site started at an A that was numbered as position­1 (Figure 5).

Potential binding motifs for transcription factors The 5« flanking region (®1435 bp) of the mouse HAS1 gene was examined to determine whether there are sites with any structural or functional features of mammalian promoters. The region contained a number of potential transcription factor binding sites. There were two CCAAT-box sequence located 190 and 680 bp upstream from the transcription initiation site, and also three overlapping TATA box sequences located more than 850 bp upstream from the transcription initiation site. The CCAAT box at the 680 bp upstream and three TATA boxes appear to be too far from the transcription initiation site to have any promoter function. Following potential regulatory cis-elements that have been shown to be associated with functional activity in the promoters of other eukaryotic genes were detected throughout the 5« flanking region using - and Internet  programs

(Figure 5). Sites for AP-2 which mediates transcriptional activation in response to signal transduction pathways activated by cAMP and phorbol esters were detected at nucleotides ®306 to ®299 [23,24], and also for CREB which stimulates transcription of some cAMP-responsive genes at nucleotides ®70 to ®63 [25]. These sites may be related to the observation that an increase of cAMP enhances hyaluronan synthesis [26]. A site for H4TF-2 which is known to regulate gene expression during the cell cycle [27] was located at nucleotides ®166 to ®162 and may be involved in the cell-growth phase-dependent hyaluronan synthesis [28]. There were also sites for GAGA at nucleotides ®1043 to ®994 and ®347 to ®338 which has been shown to cause nucleosome disruption [29]. Possible binding sites for MyoD were detected at nucleotides ®1278 to ®1274, ®242 to ®237, and ®229 to ®224 which is a well-known master regulatory gene for skeletal myogenesis and activate the muscle differentiation programme by the direct binding to those regions [30,31], suggesting that HAS may also be related to muscle differentiation during its morphogenesis. There were many binding sites for GATA at nucleotides ®257 to ®252, ®832 to ®827, ®1147 to ®1142, and ®1192 to ®1186, and also the ones for LBP-1 at nucleotides ®496 to ®492, ®750 to ®746, ®814 to ®810, ®822 to ®818 and ®1349 to ®1345. However, it is difficult so far to evaluate these sites in relation to known hyaluronan functions. Partial sequences of possible binding sites for SRY and Sox-5 were detected at nucleotides ®1421 to ®1417 and ®501 to ®497, and at nucleotides ®501 to ®497, respectively. The SRY and Sox-5 genes are expressed during embryogenesis [32]. It has been shown that SRY contains a HMG box and functions in embryos as a transcription factor to regulate some effector genes that directly or indirectly determine the testis development pathway [33]. Sox-5 is also a DNA binding protein with a sequence specificity overlapping that of SRY [34,35]. These sites might also be related to the possible involvement of HAS1 in embryo morphogenesis. An important aspect of roles of hyaluronan may be in tumour cell growth and metastasis. Tumours are often rich in hyaluronan and in most cases elevated levels of hyaluronan synthesis in tumours are correlated with such malignant phenotypes [4], although many extracellular matrix receptors and ligands also participate in regulating these phenotypes in tumour cells [2,36]. Sites for transcriptional factors correlated with these malignant potentials were detected in the promoter region ; ones for the tumour suppresser p53 at nucleotides ®81 to ®71, for IRF-1 at nucleotides ®904 to ®894, and for IRF-2 at nucleotides ®1171 to ®1160. IRF-2 has oncogenic potentials by suppressing a set of genes whose products are required for the negative regulation of cell growth [37]. IRF-1 and p53, on the other hand, activate those same genes [38,39]. Their mutations and abnormal expressions induce abnormal cell growth and invasive abilities of a variety of cancer cells [40–42]. The detection of these sites in the promoter regions suggests that HAS1 may play some roles in cell cycle progression and malignant phenotype expression of tumour cells. In summary, a variety of potential binding sites for transcription factors, including CCAAT box, AP-2, H4TF-2, CREB, MyoD, SRY, Sox-5, IRF-1, IRF-2, p53, LBP-1, GATA, and GAGA were found in the promoter region of the HAS1 gene. Their possible functions could be tested by generating sitedirected mutations in the gene and then expressing them in transgenic mice, which may confirm the usefulness of this approach and would also be helpful for considering the application of the HAS1 gene in clinical treatment such as gene therapy.

Mouse HAS1 gene We are grateful to Drs. Masahiko Yoneda and Hidekazu Takagi for their discussions, and Mrs Atsuko Iida for technical assistance. This work was supported by special coordination funds of the Science and Technology Agency of the Japanese Government, and a special research fund from the Seikagaku Corp.

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Received 1 August 1997/28 October 1997 ; accepted 28 November 1997

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