Transcriptional regulation of the mouse αA-crystallin gene: Binding of USF to the -7/+5 region

Lens preferred-expression of the mouse αA-crystallin gene (αA-cry) is regulated at the transcriptional level by multiple elements located in the 5' flanking region of the gene. Here we present the first analysis of the functional role of the mouse αA-cry + 1 region and the protein(s) which bind to it. The -7/+5 region of this promoter exhibits sequence similarity with the consensus upstream stimulating factor (USF) transcription factor binding site. A wild type oligodeoxyribonucleotide (oligo) spanning the mouse αA-cry -15/+15 region specifically inhibited the activity of a mouse αA-cry promoter-cat gene fusion (pαA111(a)CAT) in competitive co-transfection studies in the mouse αTN4-1 lens cell line, as did an oligo containing the adenovirus 2 major late promoter strong USF binding site. In contrast, an αA-cry oligo mutated (-3/+3) within the USF-like binding site did not inhibit pαA111(a)CAT activity. Western blot analysis indicated that αTN4-1 cells express USF1. Co-transfection of pαA111(a)CAT and a USF1 cDNA expression vector in to αTN4-1 cells resulted in a repression of mouse αA-cry promoter activity. Electrophoretic mobility shift analyses (EMSA) demonstrated that proteins in an αTN4-1 nuclear extract form a single major complex on synthetic oligos spanning the mouse αA-cry -15/+15 region. The formation of this complex was inhibited by the presence of unlabeled -15/+15 oligos or an anti-USF1 antibody. In addition, purified USF1 bound to this region, producing a complex similar in size to that observed with αTN4-1 nuclear extracts. Taken together, our findings show that USF can bind to the mouse αA-cry + 1 site, and support the possibility that USF plays a role in promoter activity of this gene. Sequence similarities surrounding the +1 region of the αA-cry gene of the mouse, mole rat, hamster, and human, as well as the previously observed utilization of USF by different cry promoters suggest that USF contributes to the high expression of many crys in the ocular lens of diverse species.