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 first 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 oligodeoxyribonucleotide (oligo) spanning the mouse alpha A-cry -15/+15 region specifically inhibited the activity of a mouse alpha A-cry promoter-cat gene fusion (p alpha A 111aCAT) in competitive co-transfection studies 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 contrast, an alpha A-cry oligo mutated (-3/+3) within the USF-like binding site did not inhibit p alpha A111aCAT activity. Western blot analysis indicated that alpha TN4-1 cells express USF1. Co-transfection of p alpha A111aCAT and a USF1 cDNA expression vector into alpha TN4-1 cells resulted in a repression of mouse alpha A-cry promoter activity. Electrophoretic mobility shift analyses (EMSA) demonstrated that proteins in an alpha TN4-1 nuclear extract form a single major complex on synthetic oligos spanning the mouse alpha A-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 alpha TN4-1 nuclear extracts. Taken together, our findings show that USF can bind to the mouse alpha A-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 alpha A-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.
Sax
C M
CM
Laboratory of Molecular and Developmental Biology, National Eye Institute, NIH, Bethesda, MD 20892, USA. sax@helix.nih.gov
Cvekl
A
A
Piatigorsky
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002478:Cells, Cultured; D017124:Conserved Sequence; D006224:Cricetinae; D003459:Crystallins / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism; D051379:Mice; D009154:Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D051720:Upstream Stimulatory Factors
D003459:Crystallins; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C497181:USF1 protein, human; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; C497182:Usf1 protein, rat