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A complex enhancer of the chicken beta A3/A1-crystallin gene depends on an AP-1-CRE element for activity
J B McDermott1, A Cvekl, J Piatigorsky
1Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2730, USA.
Purpose:
To define transcriptional regulatory elements of the chicken beta A3/A1-crystallin gene.
Methods:
Reporter genes were made with fragments of the chicken beta A3/A1-crystallin gene fused to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). The reporter plasmids were transfected into primary cultures of chicken-patched lens epithelium or fibroblast cells, and the CAT activity of cellular extracts was measured. The binding of lens nuclear proteins to beta A3/A1 sequences was tested in electrophoretic mobility shift assays.
Results:
Sequences from -287 to -254 bp relative to the transcriptional start site function as an enhancer in transfected lens and nonlens cells. The length of a T-rich sequence downstream of the enhancer influences its activity. Minimal enhancer activity depends on sequences between -270 and -254 bp, and full activity requires additional upstream sequences. The minimal enhancer includes a consensus sequence (TGAGTCA) for basic region-leucine zipper (bZIP) proteins of the AP-1-CREB superfamily. Lens nuclear proteins bind the enhancer sequences to form several specific complexes, some of which are related antigenically to members of the AP-1 and CREB families of proteins.
Conclusions:
An enhancer of the chicken beta A3/A1-crystallin gene between -287 and -254 bp functions in both lens and nonlens cells and binds multiple nuclear proteins. Temporal and spatial regulation of beta A3/A1 expression in the lens may be regulated by the enhancer.