TRANSCRIPTIONAL REGULATION OF THE MOUSE ALPHA-A-CRYSTALLIN GENE - BINDING OF USF TO THE -7 +5 REGION/

Lens preferred-expression of the mouse alpha A-crystallin gene (alpha A-cry) is regulated at the transcriptional level by multiple elements located in the 5' flanking region of the gene. Here we present the fir st analysis of the functional role of the mouse alpha A-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 oligodeoxy ribonucleotide (oligo) spanning the mouse alpha A-cry -15/+15 region s pecifically inhibited the activity of a mouse alpha A-cry promoter-cat gene fusion (p alpha A111(a)CAT) in competitive co-transfection studi es in the mouse alpha TN4-1 lens cell line, as did an oligo containing the adenovirus 2 major late promoter strong USF binding site. In cont rast, an alpha A-cry oligo mutated (-3/+3) within the USE-like binding site did not inhibit p alpha A111(a)CAT activity. Western blot analys is indicated that alpha TN4-1 cells express USF1. Co-transfection of p alpha A111(a)CAT and a USF1 cDNA expression vector into alpha TN4-1 c ells resulted in a repression of mouse alpha A-cry promoter activity. Electrophoretic mobility shift analyses (EMSA) demonstrated that prote ins in an alpha TN4-1 nuclear extract form a single major complex on s ynthetic oligos spanning the mouse alpha A-cry - 15/+15 region. The fo rmation 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 alpha TN4-1 nuclear extracts. Taken together, our findings show t hat USF can bind to the mouse alpha A-cry +1 site, and support the pos sibility that USF plays a role in promoter activity of this gene. Sequ ence similarities surrounding the +1 region of the alpha A-cry gene of the mouse, mole rat, hamster, and human, as well as the previously ob served 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.