TY - JOUR
T1 - Protein-DNA interactions of the mouse αA-crystallin control regions
T2 - Differences between expressing and non-expressing cells
AU - Kantorow, Marc
AU - Cvekl, Ales
AU - Sax, Christina M.
AU - Piatigorsky, Joram
PY - 1993/3/20
Y1 - 1993/3/20
N2 - 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-I 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-I 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-I 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.
AB - 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-I 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-I 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-I 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.
KW - Gene expression
KW - In vivo footprinting
KW - αA-crystallin
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U2 - 10.1006/jmbi.1993.1160
DO - 10.1006/jmbi.1993.1160
M3 - Article
C2 - 8464058
AN - SCOPUS:0027208110
SN - 0022-2836
VL - 230
SP - 425
EP - 435
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -