Cloning of the human phospholipase C-gamma1 promoter and identification of a DR6-type vitamin D-responsive element.
The 5'-flanking region of the human phospholipase C-gamma1 gene was isolated from a human P1 genomic DNA library. The S1-nuclease mapping and primer extension analysis revealed that there is a single transcriptional start site located at 135 bases upstream from the translation start codon in the human phospholipase C-gamma1 gene. DNA sequence analysis showed that the sequence around the transcriptional start site is very GC-rich and has no TATA box. The fragment +135 to -877 in the 5'-flanking region of the human phospholipase C-gamma1 gene was subcloned into a luciferase reporter vector. The chimeric gene produced a high level of luciferase activity and responded to 1,25-(OH)2D3 in transiently transfected human keratinocytes. Deletion and mutation studies of the fragment +135 to -877 demonstrated a vitamin D-responsive element that contains a motif arranged as two direct repeats separated by 6 bases (DR6), AGGTCAgaccacTGGACA, located between -786 and -803 base pairs. Incubation of the oligonucleotide containing the DR6 with keratinocyte nuclear extracts produced a specific protein-DNA complex that shifted to a higher molecular weight form upon the addition of an antibody specific to the 1,25-(OH)2D3 receptor. Therefore, the 5'-flanking region of the human phospholipase C-gamma1 gene confers promoter activity and contains a DR6-type vitamin D-responsive element that mediates, at least in part, the enhanced expression of this gene in human keratinocytes by 1, 25-(OH)2D3.
Xie
Z
Z
Endocrine Unit, Veterans Affairs Medical Center, University of California, San Francisco, California 94121, USA.
Bikle
D D
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D001483:Base Sequence; D001665:Binding Sites; D002117:Calcitriol / Q000502:physiology; D004268:DNA-Binding Proteins / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D015603:Keratinocytes / Q000166:cytology*; D008969:Molecular Sequence Data; D051966:Phospholipase C gamma; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D018167:Receptors, Calcitriol / Q000378:metabolism*; D015183:Restriction Mapping; D017384:Sequence Deletion; D014162:Transfection; D010738:Type C Phospholipases / Q000235:genetics*; D014807:Vitamin D / Q000502:physiology*
D004268:DNA-Binding Proteins; D007527:Isoenzymes; D018167:Receptors, Calcitriol; D014807:Vitamin D; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002117:Calcitriol