Identification of the human alpha6 integrin gene promoter.
The alpha6 integrin subunit couples with either the beta1 or the beta4 subunit to form a laminin receptor. alpha6 expression is cell-type-specific and generally is present at high levels in epithelial and endothelial cells. To study its gene regulation, we isolated a genomic clone containing the human alpha6 integrin gene promoter. It includes 3 kb of the upstream flanking region, the first exon (385 bp), and 9 kb of the first intron. The alpha6 promoter directs transcription initiation from a primary site 202 nucleotides from the translation initiation codon. Unlike most other integrin gene promoters, the alpha6 promoter has a TATA box (GATAAA), which is located 22 nucleotides upstream from the primary transcription initiation site. A 190-bp region upstream from the TATA box is highly rich (78%) in C and G nucleotides and contains several Sp1 and AP2 binding sequences. However, full promoter activity (in the presence of the SV40 enhancer) requires only 78 bp of this C/G-rich sequence upstream from the TATA box. Slightly upstream from the C/G-rich region are a steroid receptor binding homolog and an epithelial-cell-specific E-pal sequence. Another possible epithelial cell-specific binding sequence (Ker1) is found immediately downstream from the TATA box. Cell-type-specific activities of the promoter paralleled the alpha6 mRNA levels in four tested cell lines. In the presence of the SV40 enhancer, alpha6 promoter activity increased approximately four-fold in primary keratinocytes and in HT1080 fibrosarcoma cells and 30-fold in T47D breast carcinoma cells, but remained undetectable in K562 leukemia cells. Genomic analysis that compared alpha6-expressing with non-alpha6-expressing cells suggested that DNA methylation is not involved in the silencing of the alpha6 gene in alpha6-negative cells. DNase I footprint analysis confirmed the binding of Sp1 and AP2 to their cognate sequences. A nuclear extract of high-alpha6-expressing HBL-100 cells also produced significant binding to these sites, suggesting that the two transcription factors are probably involved in the positive regulation of the alpha6 promoter.
Lin
C S
CS
Department of Stomatology, University of California, San Francisco 94143-0512, USA.
Chen
Y
Y
Huynh
T
T
Kramer
R
R
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000096:biosynthesis* / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D039503:Integrin alpha6; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012867:Skin / Q000166:cytology / Q000378:metabolism; D016385:TATA Box; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D004268:DNA-Binding Proteins; D039503:Integrin alpha6; D011993:Recombinant Fusion Proteins