Identification of negative-acting and protein-binding elements in the mouse αA-crystallin -1556/-1165 region

The mouse αA-crystallin-encoding gene (αA-cry) is expressed in a highly lens-preferred manner. To date, it has been shown that this lens-preferred expression is controlled by four proximal positive-acting transcriptional regulatory elements: DEI (-111/-97), αA-CRYBPl (-66/-57), PE1/TATA (-35/-19) and PE2 (+24/+43). The present study extends our knowledge of mouse αA-cry transcriptional regulatory elements to the far upstream region of that gene by demonstrating that the - 1556 to - 1165 region contains negative-acting sequence elements which function in transfected lens cells derived from mouse, rabbit and chicken. This is the first negative-acting regulatory region identified in αA-cry. The - 1556 to - 1165 region contains sequences similar to repressor/silencer elements identified in other genes, including those highly expressed in the lens, such as the δl-crystallin (δ1-cry) and vimentin (vim) genes. The - 1480 to - 1401 region specifically interacts with nuclear proteins isolated from the αTN4-1 mouse lens cell line. Co within this protein-binding region and positioned at - 1453 to - 1444 is a sequence (RS1) similar to the chicker intron 3 repressor, and which competes for the formation of - 1480 to - 1401 DNA-protein complexes. Our findings suggest that lens nuclear proteins bind to the mouse αA-cry RS1 region. We demonstrate that the chicken δ1-cry intron represser binds similar nuclear proteins in chicken embryonic lens cells and mouse αTN4-1 lens cells. However, the mouse αA-cry RS1 region does not appear to bind the same protein(s) that bind to the chicken δ1-cry intron repressor, suggesting the existence of a family of silencer proteins that recognize a similar site in the diverse cry genes.