THE COOPERATIVE INTERACTION BETWEEN 2 MOTIFS OF AN ENHANCER ELEMENT OF THE CHICKEN ALPHA A-CRYSTALLIN GENE, ALPHA-CE1 AND ALPHA-CE2, CONFERS LENS-SPECIFIC EXPRESSION

An 84 bp element located between nucleotides - 162 and - 79 of the chicken alpha-A-crystallin gene exhibits lens-specific enhancer activity. Transient transfection experiments using 5' deletion and linker scanner mutants has indicated that the 84 bp enhancer element is composed of three motifs, alpha-CE1 (- 162 to - 134), alpha-CE3 (- 135 to - 121) and alpha-CE2 (- 119 to - 99). Neither alpha-CE1 or alpha-CE3 motif alone can exhibit enhancer activity even when trimerized, whereas together they can direct some degree of lens-specific expression. Alpha-CE2 alone shows low transcriptional activity when trimerized. A combination of alpha-CE1 with alpha-CE2 exerts full lens-specific enhancer activity comparable with that of the 84 bp enhancer element, indicating that alpha-CE1 and alpha-CE2 motifs are sufficient to confer lens-specific expression. Transcriptional activation by these two motifs from a distance required the additional presence of either or both motifs adjacent to the beta-actin basal promoter. Gel shift experiments indicated that the alpha-CE1, alpha-CE2 and alpha-CE3 motifs specifically bind nuclear proteins. Alpha-CE1 binds a protein predominantly present in lens cells, whereas alpha-CE2- and alpha-CE3-binding proteins differ between lens and lung cells. Mutations within the alpha-CE1 and alpha-CE2 motifs that failed to bind nuclear factors in vitro resulted in loss of transcriptional activation, indicating that these nuclear factors play a key role in controlling lens-specific expression.