Identification and analysis of the promoter region of the human hyaluronan synthase 2 gene.
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, GenBank accession number NM_005328. A luciferase reporter gene assay of nested fragments spanning the 5' terminus of NM_005328 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.
Monslow
Jamie
J
Institute of Nephrology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, Wales, UK.
Williams
John D
JD
Guy
Carol A
CA
Price
Iain K
IK
Craig
Kathrine J
KJ
Williams
Hywel J
HJ
Williams
Nigel M
NM
Martin
John
J
Coleman
Sharon L
SL
Topley
Nicholas
N
Spicer
Andrew P
AP
Buckland
Paul R
PR
Davies
Malcolm
M
Bowen
Timothy
T
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D020224:Expressed Sequence Tags; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D000076002:Hyaluronan Synthases; D007668:Kidney; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D014453:Glucuronosyltransferase; C101754:HAS2 protein, human; C491099:Has2 protein, mouse; C491097:Has2 protein, rat; D000076002:Hyaluronan Synthases