Cloning and characterization of a Bovine Homeodomain-Zinc finger protein (BHZ) that interacts with the rhodopsin promoter and enhancer regions

Purpose. The goal of this work is to identify and clone the transcription factors that bind to cis-acting elements that, based on previous transgenic mouse, cell transfection and biochemical studies, have been implicated in the regulation of rhodopsin gene expression. Methods. Cell transfection, expression cloning, protein expression, and biochemical analyses were performed by published methods. Results. Using transfection of primary chick retinal cell cultures, we have identified several candidate regulatory regions within the bovine rhodopsin proximal promoter. One of them, from -162 to -123 bp, which contains several putative negative regulatory elements and is homologous to a sequence in the rhodopsin enhancer region, was used to screen a bovine retina cDNA expression library. Two independent partial length cDNAs, which encode the same homeodomain- and zinc finger-containing protein (BHZ), were isolated. BHZ is homologous to NIL-2A and to the delta-crystallin enhancer-binding protein delta EF1, which are repressors of E2 box-mediated gene activation. A histidine-tagged fusion protein was obtained by expressing one of the BHZ clones using the pTricHis B vector. Purified fusion protein was shown, by South-Western, gel mobility shift and DNase I footprint analysis, to bind specifically to rhodopsin promoter and enhancer regions containing the CACCT element. Binding did not occur when mutated or heterologous DNA fragments were used. Ongoing cell transfection experiments using retinal cultures are testing the ability of BHZ to act as a negative regulator of rhodopsin expression. Conclusion. BHZ may contribute to the rod photoreceptor cell-specificity of rhodopsin by repressing its expression in non-rod cells.