Identification of the Human α6 Integrin Gene Promoter

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Abstract
The α6 integrin subunit couples with either the β1 or the β4 subunit to form a laminin receptor. α6 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 α6 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 α6 promoter directs transcription initiation from a primary site 202 nucleotides from the translation initiation codon. Unlike most other integrin gene promoters, the α6 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 α6 mRNA levels in four tested cell lines. In the presence of the SV40 enhancer, α6 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 α6-expressing with non-α6-expressing cells suggested that DNA methylation is not involved in the silencing of the α6 gene in α6-negative cells. DNase I footprint analysis confirmed the binding of Sp1 and AP2 to their cognate sequences. A nuclear extract of high-α6-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 α6 promoter.