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Originally published In Press as doi:10.1074/jbc.M312666200 on February 25, 2004

J. Biol. Chem., Vol. 279, Issue 20, 20576-20581, May 14, 2004
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Identification and Analysis of the Promoter Region of the Human Hyaluronan Synthase 2 Gene*

Jamie Monslow{ddagger}§, John D. Williams{ddagger}§, Carol A. Guy||, Iain K. Price{ddagger}, Kathrine J. Craig{ddagger}§, Hywel J. Williams||, Nigel M. Williams||, John Martin{ddagger}§, Sharon L. Coleman{ddagger}§, Nicholas Topley{ddagger}§, Andrew P. Spicer**, Paul R. Buckland||, Malcolm Davies{ddagger}, and Timothy Bowen{ddagger}§{ddagger}{ddagger}

From the {ddagger}Institute of Nephrology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom, §Cardiff Institute of Tissue Engineering and Repair, Cardiff Medicentre, Heath Park, Cardiff, CF14 4UJ, United Kingdom, ||Department of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom, and **Center for Extracellular Matrix Biology, Institute of Biosciences and Technology, Texas A&M University, Houston, Texas 77030

Received for publication, November 19, 2003 , and in revised form, February 24, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyaluronan (HA) is a linear glycosaminoglycan of the vertebrate extracellular matrix that is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2 and -3. The regulation of HA synthesis has been implicated in a variety of extracellular matrix-mediated and pathological processes, including renal fibrosis. We have recently described the genomic structures of each of the human HAS genes. In the present study, we analyzed the HAS2 promoter region. In 5'-rapid amplification of cDNA ends analysis of purified mRNA from human renal epithelial proximal tubular cells, we detected an extended sequence for HAS2 exon 1, relocating the transcription initiation site 130 nucleotides upstream of the reference HAS2 mRNA sequence, GenBankTM accession number NM_005328 [GenBank] . A luciferase reporter gene assay of nested fragments spanning the 5' terminus of NM_005328 [GenBank] demonstrated the constitutive promoter activity of sequences directly upstream of the repositioned transcription initiation site but not of the newly designated exonic nucleotides. Using reverse transcription-PCR, expression of this extended HAS2 mRNA was demonstrated in a variety of human cell types, and orthologous sequences were detected in mouse and rat kidney. Alignment of human, murine, and equine genomic DNA sequences upstream of the repositioned HAS2 exon 1 provided evidence for the evolutionary conservation of specific transcription factor binding sites. The location of the HAS2 promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts as well as in developmental models in which HAS2 null animals have an embryonic lethal phenotype.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The glycosaminoglycan hyaluronan (HA)1 is a key component of the vertebrate extracellular matrix and is involved in a wide range of cellular functions including migration, adhesion, and proliferation (1–3). HA is synthesized by the HA synthase (HAS) enzymes HAS1, HAS2, and HAS3 at the plasma membrane (4), with the corresponding HAS genes mapping to discrete autosomal loci (5–9).

Previous reports from this and other laboratories have demonstrated the importance of HA metabolism in clinical nephrology. End stage renal failure is most frequently characterized by progressive interstitial fibrosis and tubular atrophy (10), and increased expression of HA in the renal corticointerstitium is associated with this progression (11, 12). In high glucose concentrations, which mimic diabetic nephropathy, renal proximal tubular epithelial cells synthesize high levels of HA, which is coincident with specific up-regulation of transcription at the HAS2 locus (13). The mesothelial cells that line the peritoneal membrane synthesize HA as a normal constituent of human peritoneal effluent, and this synthesis is up-regulated during periods of peritonitis in patients undergoing peritoneal dialysis (14). In an in vitro model of peritoneal wound healing (15), the mechanical disruption of human peritoneal mesothelial cell monolayers led to increased levels of HAS2 mRNA transcription together with an increase in HA synthesis (16).

We have recently described the genomic structures of the human HAS genes (17) and demonstrated that ~500 bp upstream of each HAS reference mRNA sequence showed constitutive promoter activity in a high-throughput luciferase analysis system (18, 19). The sequence upstream of HAS2 was the least active in this assay but still conformed to our criteria for promoter activity (18). In the present study, we describe further investigation of the transcriptional regulation of the HAS2 gene. Data from 5'-rapid amplification of cDNA ends (RACE) analysis revealed that the HAS2 transcription initiation site (TIS) was situated 130 bp upstream of the 5' terminus of reference mRNA sequence NM_005328 [GenBank] , thus extending HAS2 exon 1 (see Ref. 17). The results of luciferase reporter gene analysis of a nested set of constructs spanning the region directly upstream of NM_005328 [GenBank] concurred with these findings. Reverse transcription (RT)-PCR analysis confirmed the expression of this extended HAS2 exon 1 in selected renal and other human cell types, including primary human peritoneal mesothelial cells, together with murine kidney RNAs. Alignment of human, murine, and equine genomic DNA (gDNA) sequences upstream of the repositioned HAS2 TIS revealed the presence of putative Sp1, NF-Y/CCAAT, and NF-{kappa}B sites common to all HAS2 orthologues, suggesting evolutionary conservation of HAS2 transcriptional regulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—A variety of human cells was used in the present study. Culture conditions for renal proximal tubular HK-2 (20), lung fibroblast AG02222 (21), medulloblastoma TE-671, and renal embryonic HEK293t cell lines (18) together with those for primary peritoneal mesothelial cells (14) have been described previously.

5'-RACE—mRNA was isolated from total RNA extracts using magnetic Oligo(dT)25 Dynabeads (Dynal, Bromborough, Wirral, UK) prior to 5'-RACE analysis using the SMART RACE cDNA amplification kit (BD Biosciences) together with HAS2-specific oligonucleotide primers (all oligonucleotides from Invitrogen; see Fig. 1 for primer sequence data) according to the manufacturer's instructions. An aliquot of the reaction products generated with a primer binding at the RACE site was used as the template for a nested reaction primed from the RACE-N site, as recommended by the manufacturer. Reaction products were analyzed by 1.5% agarose gel electrophoresis, purified using the QIAquick gel extraction kit (Qiagen Ltd., Crawley, West Sussex, UK) according to the manufacturer's instructions, T-cloned into vector pCR-TOPO2.1 (Invitrogen), and sequenced using Big Dye terminator chemistry (Applied Biosystems, Warrington, Cheshire, UK) and M13 primers. The HAS2 proximal promoter sequence was analyzed using the MatInspector professional transcription factor binding site (TFBS) identification software (22) (see Fig. 1 for putative TFBSs).



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  		FIG. 1.
Primer binding sites for 5'-RACE, luciferase, and RT-PCR analyses of the human HAS2 gene. The first kbp (positions –1000 to –1) of gDNA upstream of the repositioned TIS of HAS2 is shown in lower case, with putative TFBSs highlighted in upper case and labeled: CEBP, CCAAT-enhancer binding protein; Nf{kappa}B; NF-Y, nuclear factor Y; CCAAT, CCAAT box; Sp1, GC-box factors Sp1/GC. The first 800 nucleotides (positions +1 to +800) of HAS2 mRNA are shown in upper case, with the extended 5'-UTR sequence identified in the present study, and which forms the 5' terminus of sequence AJ604570 [GenBank] shown in bold. This precedes reference HAS2 mRNA sequence NM_005328 [GenBank] , which begins at position +131. In light of in silico sequence data, nucleotide +131 has been revised from a cytosine (NM_005328 [GenBank] ) to an adenine residue (see Ref. 17). The boundary between HAS2 exons 1 and 2 (17) is denoted with a vertical bar, with the illustrated portion of exon 2 in italics. The sense-strand primer sequences used to amplify inserts for the initial set of luciferase reporter vectors are underlined and labeled F1–F10. Subsequent sense-strand promoter primers spanning the gDNA immediately upstream of AJ604570 [GenBank] are labeled as potential TFBSs and underlined, with the exception of primer Sp1–3', which is in italics. The common antisense primer binding site used in the generation of all luciferase constructs is underlined and labeled R1, with those for the primary and nested antisense primers used in 5'-RACE analysis shown as RACE and RACE-N, respectively. The sense Prom-RT-PCR-F oligonucleotide sequence is shown in bold italics, whereas the binding site of the antisense Prom-RT-PCR-R primer, in HAS2 exon 2, is both in italics and underlined.

 
Luciferase Analysis—A nested set of HAS2 promoter fragments was created by PCR amplification using the oligonucleotide sequences shown in Fig. 1. In each case, the sense-strand primer was tailed with a KpnI restriction endonuclease recognition sequence, the single antisense-strand primer bearing a corresponding HindIII site. PCR amplification of promoter fragments was carried out from gDNA by standard means (23). PCR products were gel-purified as described above, digested (restriction endonucleases were from New England Biolabs, Hitchin, Hertfordshire, UK), cloned into a modified pGL-3 luciferase reporter vector (17), and sequenced to ensure fidelity of amplification using vector-specific primers RV3 (5'-CTAGCAAAATAGGCTGTCCC-3') and GL-2 (5'-CTTTATGTTTTTGGCGTCTTCC-3') according to the manufacturer's instructions. The ability of each HAS2 promoter fragment to drive transcription of the luciferase gene was tested transiently in human cell lines HEK293t and TE-671 as described in detail previously (17–19), with the following amendment to maximize luciferase output. The growth medium used was a 1:1 mixture of Dulbecco's modified Eagle's medium/Ham's F-12 nutrient mixture without L-glutamine (Invitrogen) supplemented with 20 mM HEPES and 2 mM L-glutamine (both from Invitrogen) together with 5 ng/ml sodium selenite, 5 µg/ml insulin, 5 µg/ml transferrin, and 0.4 µg/ml hydrocortisone (all from Sigma).

RT-PCR—RT-PCR was carried out from total RNA extracted from a range of human cells using TRI Reagent (Sigma) according to the manufacturer's instructions. cDNA was reverse-transcribed from 1 µg of total RNA using Superscript II RNase H– reverse transcriptase (Invitrogen) and random hexamers (100 µM) (Amersham Biosciences), as recommended by the manufacturer. RT-PCR primers were designed to amplify the extended 5'-UTR identified in the present study. As shown in Fig. 1, the Prom-RT-PCR-F binding site was positioned within 5'-RACE Band 2 and spanned positions 54–75 from the new TIS, amplifying 77 nucleotides of sequence upstream of the transcription start site described previously in NM_005328 [GenBank] . Prom-RT-PCR-R primed within HAS2 exon 2.

Gene-specific PCR reactions were performed using 2 µl of RT reaction product in a total volume of 50 µl comprising 1x PCR buffer containing 1.5 mM MgCl2 (Applied Biosystems), 0.2 mM dNTPs (Amersham Biosciences), 2.5 units of Amplitaq Gold Taq polymerase (Applied Biosystems), and 1 µM oligonucleotide PCR primers. A denaturation step of 94 °C for 2 min was followed by 38 cycles comprising 30 s at 94 °C, 30 s at 65 °C, and 90 s at 68 °C, after which cycling was concluded with a final extension step of 15 min at 68 °C. Reaction products were analyzed by electrophoresis on 1.5% agarose.

The sequences of the HAS2 RT-PCR primers used routinely in our laboratory (and not given above) were sense-strand H2-F primer, 5'-CATAAAGAAAGCTCGCAACACG-3' (positions 1025–1046 in NM_005328 [GenBank] ), and antisense-strand oligonucleotide H2-R, 5'-ACTGCTGAGGAATGAGATCCAG-3' (1287–1308). Extended 5'-UTRs were amplified from total RNA from mouse and rat kidneys (Ambion Europe Ltd., Cambridgeshire, UK) using primers specific to the corresponding region of the mouse (GenBankTM accession number NM_008216 [GenBank] ) and rat (GenBankTM accession number NM_013153 [GenBank] ) HAS2 mRNAs. Where appropriate, reaction products were processed for sequencing as described above.

DNA Sequence Alignment and Data Base Analysis—Alignments for the genomic sequences upstream of HAS2 from human (clone RP11–3G20, sequence AC104233 [GenBank] ), mouse (clone RP24–147N23, sequence AC140799 [GenBank] ), rat (clone CH230–228F12, sequence AC105559 [GenBank] ), and horse (AF508308 [GenBank] ) (24) were carried out using the ClustalW algorithm at the European Bioinformatics Institute (www.ebi.ac.uk/clustalw/index.html). BLAST (25) and BLAT (26) analyses were carried out comparing the above HAS2 sequences against expressed data from a range of organisms in the expressed sequence tag (EST) data base at www.ncbi.nlm.nih.gov/blast/Blast.cgi and the genome browser at genome.ucsc.edu/.

Statistical Analysis of Luciferase Assay Data—Data for each luciferase construct were calculated and plotted graphically as either the -fold increase of luciferase activity compared with the promoterless control vector (C) (18) or as a percentage of the luciferase activity of construct F3. Where appropriate, statistical analysis was performed using Friedman's two-way analysis of variance test from SPSS 11.5 for Windows (SPSS Inc., Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
5'-RACE Analysis of the HAS2 Gene Using Purified mRNA—Agarose gel electrophoresis resolved the 5'-RACE-nested reaction products into the two principal bands shown in Fig. 2, which were then cloned. Sequence analysis of three randomly selected Band 1 transformants revealed that each insert terminated at a different 5'-terminal nucleotide, as highlighted in Fig. 2. The longest of these inserts extended the HAS2 5'-UTR of NM_005328 [GenBank] by 49 bp. Corresponding analysis of transformants from the discrete Band 2 resulted in a single cloned product, which extended 130 bp upstream of NM_005328 [GenBank] (see Figs. 1 and 2). This has now been submitted to the GenBankTM/EBI Data Bank with the accession number AJ604570 [GenBank] . These results were replicated in analysis using mRNA from HK-2, TE-671, and HEK293t cells (data not shown). No bands corresponding to the predicted size of the product from sequence NM_005328 [GenBank] were detected (Fig. 2).



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  		FIG. 2.
5'-RACE analysis of the HAS2 gene. Agarose gel electrophoresis of size marker (M) and HAS2 5'-RACE products from purified mRNA using primers RACE (R) and RACE-N (R-N) (see Fig. 1 for primer details). The 5' termini of the sequences of Band 1 and Band 2 from the RACE-N primer reaction are illustrated. Nucleotides shared with reference sequence NM_005328 [GenBank] are shown in uppercase, the upstream sequence from the largest fragment cloned from Band 1 (see "Experimental Procedures" for details) is shown in lowercase, and an additional upstream sequence from Band 2 in lowercase italics. The 5'-terminal nucleotide from each Band 1 reaction product is in bold, the terminal adenine from the longest Band 1 product is shown in uppercase and underlined in both sequences. The sequences of 5'-RACE reaction products seen in bands a, b, and c were determined as described under "Experimental Procedures" and found to be the products of nonspecific amplification of messages originating from other autosomes or the mitochondrion.

 
Analysis of Luciferase Activity of Sequences Upstream of NM_005328 [GenBank] and AJ604570 [GenBank] —Luciferase assay data are shown in Fig. 3. From initial screening of the gDNA upstream of NM_005328 [GenBank] , Fig. 3A shows that both cell lines produced a bipartite distribution, the difference between low scoring constructs C, F1, and F2 and the high readings for F3–F10 being highly significant (p < 0.001). The significance of the variation of luciferase activity in promoter fragments F1 and F2 compared with C was equivocal. A marginal increase significant at the p < 0.05 level in TE-671 cells contrasted with a decrease significant at p < 0.01 in HEK293t cells.



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  		FIG. 3.
Luciferase activity of sequences upstream of NM_005328 [GenBank] and AJ604570 [GenBank] . Promoter fragments are identified by the sense primer designations given in Fig. 1. Data are displayed for TE-671 cells (shaded bars) and HEK293t cells (white bars). A, nested constructs upstream of NM_005328 [GenBank] . Luciferase activity is expressed as the magnitude of normalized luciferase activity relative to the pGL3-Basic promoterless negative control vector. Significance values are indicated by asterisks: *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, nested constructs upstream of AJ604570 [GenBank] . Luciferase activity is expressed as the magnitude of normalized luciferase activity relative to construct F3.

 
On the basis of the above findings, the sequence immediately upstream of AJ604570 [GenBank] and spanned by the insert in construct F3 was analyzed in detail. MatInspector analysis (22) highlighted one NF-Y/CCAAT motif at –80 and Sp1 sites at –60 (Sp1–5') and –40 (Sp1–3') (Fig. 1). Nested constructs spanning this region were cloned, along with a Minimal promoter fragment comprising 28 bp upstream of the revised HAS2 TIS (Fig. 1). The luciferase data from these constructs are plotted in Fig. 3B as a percentage of the activity of the F3 construct. In each cell line, there was an incremental rise in luciferase activity as the insert size increased, demonstrating that this sequence was functionally active as a promoter.

RT-PCR—The results of RT-PCR analysis are shown in Fig. 4. Despite differences in intensity, evidence of transcription of an AJ604570 [GenBank] -specific extended HAS2 5'-UTR was detected in each of the cell types assayed. Primers Prom-RT-PCR-F and -R (Fig. 1) amplified the larger product spanning 686 bp in Fig. 1, specific to sequence AJ604570 [GenBank] and comprising the 5'-UTR and the N-terminal amino acid codons of HAS2. The smaller band of 282 bp amplified codons from the glycosyltransferase domain of HAS2 using primers H2-F and -R. To obviate the possibility of amplification from contaminating gDNA, both sets of RT-PCR primers amplified across one or more intron/exon boundaries spanning at least 11.5 kbp of intronic sequence (17). Orthologous products from the total RNA of mouse and rat kidneys were also amplified. In no case was the message for either 5'-RACE Band 1 mRNA or NM_005328 [GenBank] detected in the absence of AJ604570 [GenBank] (data not shown).



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  		FIG. 4.
RT-PCR analysis of AJ604570 [GenBank] -specific fragments. RT-PCR analysis using primers Prom-RT-PCR-F and -R (as detailed in Fig. 1) (upper band) and H2-F and H2-R (as detailed under "Experimental Procedures")(lower band) from a variety of human cells: lane 1, embryonic renal (HEK293t); lane 2, medulloblastoma (TE-671); lane 3, renal proximal tubular epithelial (HK-2); lane 4, lung fibroblast (AG02222); lane 5, primary peritoneal mesothelial; as well as total RNAs from lane 6, mouse kidney, and lane 7, rat kidney. M, double-stranded DNA size standard (kbp).

 
DNA Sequence Alignment and Data Base Analysis—Fig. 5 shows a nucleotide sequence alignment from 200 bp upstream of the 5' terminus of AJ604570 [GenBank] to 150 bp downstream and includes the TISs of the reference mRNA sequences for HAS2 orthologues from human, mouse, rat, and horse. A high level of sequence similarity was evident throughout the majority of the alignment, most clearly between positions –100 and +100. With respect to TFBSs, sequence similarity was seen at the NF-{kappa}B site at approximately –150; the NF-Y/CCAAT element at –80 and the Sp1–5' site at –60. Similarity was also evident at the core motif of the Sp1–3' site at –40 in both murine sequences, the remainder of this region being absent. In the equine orthologue, positions –8 to –51 surrounding Sp1–3' were absent. The longest available ESTs from human (CD654109 [GenBank] ), mouse (BY110777 [GenBank] ), and bovine HAS2 (AW429456 [GenBank] ) terminated upstream of their reference mRNA sequences.



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  		FIG. 5.
Sequence alignment of HAS2 orthologues and ESTs. The 5' terminus of sequence AJ604570 [GenBank] lies at nucleotide +1 of this ClustalW alignment of gDNA sequences upstream of reference mRNAs for human (h, NM_005328 [GenBank] ), mouse (m, NM_008216 [GenBank] ), rat (r, NM_013153 [GenBank] ), and equine (e, AF508308 [GenBank] ) HAS2 orthologues. The 5' termini of the above reference HAS2 mRNAs, together with that for Bos taurus bovine (b, GenBankTM accession number NM_174079 [GenBank] ) HAS2, are in bold and labeled. Sequence differences from hHAS2 are shown by a lowercase nucleotide, and gaps are shown by a dash. Putative TFBSs are underlined and labeled as in Fig. 1. The 5' termini of three HAS2 ESTs are labeled: hEST, CD654109 [GenBank] ; bEST, AW429456 [GenBank] ; and mEST, BY110777 [GenBank] .

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The importance of HA metabolism in a multiplicity of biological and pathological contexts is becoming increasingly evident and an understanding of the mechanisms controlling its synthesis ever more desirable. HA has been implicated in chondrogenesis and cartilage homeostasis, embryonic development, immunomodulation, wound healing, and a variety of disease processes, including arthritis, malignancies, and pulmonary and vascular disease, together with other immune and inflammatory disorders (27–35). In addition, the transcriptional regulation of HAS2 has potential significance in renal fibrosis (10–12), diabetic nephropathy (13), and peritoneal inflammation (16). We have recently described the genomic structures of the human HAS genes (17) and, in the present study, set out to investigate further the HAS2 promoter region.

Previously, we carried out 5'-RACE analysis on HAS2 from total RNA (17). In the present study, mRNA was purified from HK-2, HEK293t, and TE-671 cells and used as the template for 5'-RACE reactions. Because no products of the predicted size for NM_005328 [GenBank] were recovered, it appears that this data base entry is 5'-truncated. A significant number of similarly truncated sequences are deposited in the public data bases (18), because imperfect methodologies for creating libraries have generated incomplete cDNAs (36). Each of the 5'-RACE products from Band 1 terminated at a different nucleotide ~50 bp upstream of NM_005328 [GenBank] and 80 bp downstream of the single 5' terminus of Band 2. This suggests that Band 1 products were prematurely terminated, possibly due to a strong secondary structure in the HAS2 5'-UTR, as discussed below.

We have shown a high throughput luciferase system to be sensitive, accurate, and reproducible in analyses of a large number of human promoters, including naturally occurring polymorphic promoter variants (18, 19). This system was used to establish our quantitative definition of promoter activity at 10 times that of the promoterless vector control value (i.e. 10 x C) (18) and demonstrated that 500 bp of gDNA immediately upstream of the reference mRNA sequence of each HAS isoform had basal promoter activity (17). In the present study, we have used this system to analyze the ability of a number of HAS2 promoter constructs to drive luciferase transcription in a human renal (HEK293t) and human medulloblastoma (TE-671) cell line (17–19). In addition, we have used Friedman's two-way analysis of variance test to compare the significance of the variation in promoter activity with our previously established criterion of luciferase activity.

Luciferase activity data (Fig. 3A) provided highly significant evidence that promoter function was restricted to constructs F3–F10 clustered ~250 bp upstream of the 5' terminus of HAS2 reference mRNA sequence NM_005328 [GenBank] . In TE-671 cells, these constructs also exceeded our previously stated threshold for promoter activity (18). There was suggestive evidence for a significant increase in luciferase activity of constructs F1 and F2 in TE-671 cells, but this was contradicted by a significant decrease in activity for these constructs in HEK293t cells (Fig. 3A) and fell below our previous threshold in both cell lines. It thus appeared that the sequence directly upstream of NM_005328 [GenBank] was not active in our promoter assay. It was also seen that, in the absence of stimulation, basal promoter activity was approximately consistent for F3–F10. An overall decrease in activity in larger vectors is seen in Fig. 3A, but because this system was optimized for 500-bp inserts, we cannot discount the possibility that insert size might affect luciferase activity and are currently investigating this possibility. The luciferase activity of constructs immediately upstream of AJ604570 [GenBank] showed an incremental increase in luciferase activity, reaching a maximum at construct F3 as predicted from our previous data (see Fig. 3A). Once again, higher relative activities were obtained in TE-671 cells, but in each case the ability of constructs containing larger inserts to drive luciferase transcription was demonstrated.

Considering the 5'-RACE results in the light of the luciferase assay data, constructs F1 and F2 contained inserts spanning 74 and 130 nucleotides, respectively, upstream of NM_005328 [GenBank] , with F2 also spanning ~80 bp upstream from 5'-RACE Band 1. If transcription were initiated at either of these positions, the inserts in the respective vectors would contain sufficient proximal upstream sequence to contain core promoter elements and the binding site for RNA polymerase II (37). They would thus be expected to drive luciferase transcription in the manner in which we demonstrated for sequences upstream of AJ604570 [GenBank] (Fig. 3B). Comparison of each putative TIS with the consensus sequence YYANWYY (where Y = pyrimidine; W = A or T) described for transcription initiator elements between positions –2 and +5 (38) showed that only AJ604570 [GenBank] (Fig. 1) concurred fully with this or the key nucleotide consensus –2Y, +1A, and +3 W (38). The combined data thus suggest very strongly that the 5' terminus of 5'-RACE Band 2 (sequence AJ604570 [GenBank] ) represents the TIS of the HAS2 gene, and the promoter lies upstream of this site.

Exon 1 of the human HAS2 gene forms a discrete 5'-UTR, with the methionine codon at the 5' terminus of exon 2 initiating translation (17); sequence AJ604570 [GenBank] extends this first exon to 669 bp (Fig. 1). Approximately 10% of mRNAs contain such complex 5'-UTRs and frequently encode regulatory proteins that may be subject to post-transcriptional regulation (39, 40). Interestingly, this level of control is evident in certain key genes during embryonic development (40), and HAS2 knockout animals have an embryonic lethal phenotype (30). Untranslated AUG (uAUG) motifs and/or secondary structure in excess of –50 kcal/mol in a complex 5'-UTR inhibit significantly the process of cap-dependent ribosomal scanning prior to the initiation of translation (39, 40). Our extended HAS2 exon 1 contains one uAUG motif at +650, 20 bp upstream of the predicted translation initiation site (Fig. 1) and not within a Kozak consensus signal (41); thus its functional effect may be limited (39). Analysis of our extended HAS2 exon 1 sequence using software at the MFOLD web server (www.bioinfo.rpi.edu/applications/mfold) and default parameters (42) gave a range of free energies of folding between –129.4 and –134.9 kcal/mol. We thus found evidence for post-transcriptional control of HAS2 expression and a possible mechanism for premature termination of 5'-RACE products downstream of the HAS2 TIS.

RT-PCR showed that the extended HAS2 exon 1 was expressed in two human renal cell lines and total RNAs from murine kidney, as well as outside the kidney in a human lung fibroblast cell line and in primary human peritoneal mesothelial cells. RT-PCR products specific for either AJ604570 [GenBank] (5'-RACE Band 1) or for NM_005328 [GenBank] were detected throughout, and thus no evidence for transcript 5' length variation was found. There is evidence for a natural antisense mRNA synthesized from the opposite gDNA strand to HAS2, and that includes the complementary sequence to the HAS2 TIS (HASNT).2 Because this is a potential confounder of molecular biological analysis of this locus, we designed primers to amplify trans-intronic HAS2-specific RT-PCR products.

Alignment of gDNA sequences surrounding the 5' terminus of AJ604570 [GenBank] for human, murine, and equine HAS2 loci illustrated the high similarity suggested previously (24). Fig. 5 provides evidence for the evolutionary conservation of the TFBSs Sp1–5', NF-Y/CCAAT, and NF-{kappa}B but not Sp1–3'. However, the above luciferase data did not show a large increase in activity in response to the addition of any one of these motifs, and further analysis will be required to confirm the role of these or other sites within this region. The EST data suggested that 5'-truncated sequences for human, mouse, and bovine HAS2 orthologues were deposited in the NCBI data base.

In summary, we have identified the TIS of the human HAS2 gene 130 bp upstream of the 5' terminus of the reference HAS2 mRNA sequence deposited in the public data bases. Expression of our extended HAS2 5'-UTR sequence in human and murine kidney and in other human cells was detected. We suggest that, because of the high secondary structure of complex 5'-UTRs, the reference sequences for several HAS2 orthologues may also have 5' truncations and that HAS2 may be subject to post-transcriptional regulation. Furthermore, we provide evidence for the evolutionary conservation of transcriptional regulatory elements upstream of our repositioned HAS2 TIS in human, murine, and equine genomic DNAs.


   FOOTNOTES
 
* This work was funded by Project Grant J1146W25K from the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ604570 [GenBank] . Back

Recipient of a Ph.D. studentship from the Kidney Research Unit for Wales Foundation. Back

{ddagger}{ddagger} To whom correspondence should be addressed. Tel.: 44-29-2074-8389; Fax: 44-29-2074-8470; E-mail: bowent{at}cf.ac.uk.

1 The abbreviations used are: HA, hyaluronan (hyaluronic acid); RACE, rapid amplification of cDNA ends; UTR, untranslated region; EST, expressed sequence tag; gDNA, genomic DNA; HAS, hyaluronan synthase; RT, reverse transcription; TFBS, transcription factor binding site; TIS, transcription initiation site. Back

2 H. Chao and A. P. Spicer, unpublished data. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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