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M312666200v1
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Papers In Press, published online ahead of print February 25, 2004
J. Biol. Chem, 10.1074/jbc.M312666200
Submitted on November 19, 2003
Revised on February 25, 2004
Accepted on February 25, 2004

Identification and analysis of the promoter region of the human hyaluronan synthase 2 gene

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

Institute of Nephrology, University of Wales, Cardiff, Cardiff CF14 4XN

Corresponding Author: bowent{at}cf.ac.uk

Hyaluronan (HA) is a linear glycosaminoglycan of the vertebrate extracellular matrix (ECM) that is synthesised 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 ECM-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 analysed 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 (TIS) 130 nucleotides upstream of the reference HAS2 mRNA sequence, NM_005328. A luciferase reporter gene assay of nested fragments spanning the 5’ terminus of NM_005328 demonstrated constitutive promoter activity of sequences directly upstream of the repositioned TIS, but not of the newly-designated exonic nucleotides. Using RT-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 developmental models, in which HAS2 null animals have an embryonic lethal phenotype.


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