Protein-DNA Interactions of the Mouse αA-Crystallin Control Regions: Differences Between Expressing and Non-expressing Cells

Abstract

Genomic footprinting, in vitro footprinting and mobility shift assays were used to investigate the molecular basis for expression of mouse αA-crystallin, a major structural protein of the transparent lens of vertebrates. The putative control region of the mouse αA-crystallin gene was footprinted by DNase I digestion in nuclear extracts, by dimethylsulfate treatment in cultured cells, and by micrococcal nuclease digestion in isolated nuclei. The resulting digestion patterns were compared between αTN4-1 lens cells, which express αA crystallin, and L929 fibroblasts, which do not express αA-crystallin. Four regions of DNA were found occupied in both cell types. These included positions -111 to -97 (DE-1 region), positions -75 to -55 (αA-CRYBP1 region), positions -35 to -12 (TATA box and PE-1 region), and positions +23 to +43 (an AP-1 consensus sequence). The DNase I footprints of the DE-1 and αA-CRYBP1 regions, previously implicated as functional control elements, were substantially more pronounced using nuclear extract from the αTN4-1 cells than from the L929 fibroblasts, suggesting more stable protein binding with the former than with the latter. Numerous in vivo binding variations were noted between the two cell types in all four of the footprinted regions examined. Finally, two complexes (A and B) were formed specifically with nuclear extracts from the αTN4-1 cells and a synthetic deoxyoligonucleotide comprising the αA-CRYBP1 region. These data indicate that specific differences in protein-DNA interactions with putative control regions are associated with tissue-preferred expression of the mouse αA-crystallin gene.