Identification of a KRAB Zinc–Finger Protein Binding to the Rpe65 Gene Promoter

Abstract

Abstract: : Purpose: RPE65 protein is necessary for regeneration of the visual chromophore 11–cis retinal from all–trans retinyl ester. It is critical to understand the regulation of this gene indispensable for vision. We wished to use biochemical and molecular biological approaches to identify transcriptional factors involved in regulation of this gene. Methods: Sense (5' biotinylated) and antisense oligonucleotides (30–40 base pairs) based on RPE65 proximal gene promoter were synthesized and bound to streptavidin–Dynabeads. D407 cell nuclear extract was incubated with the tethered DNA in the presence of excess poly(dI–dC). Nuclear proteins bound to the DNA–beads were washed, eluted and separated on SDS–PAGE. Protein bands were excised for MALDI–TOF analysis. Peptide sequence was used to identify cDNA clones. Identified cDNA was subcloned into pCDNA3.1 for expression. The luciferase reporter constructs containing nested fragments of mouse Rpe65 gene promoter were co–transfected into D407 cells to assess transcriptional activity. Results: Nuclear proteins binding to biotinylated–DNA/streptavidin beads were analyzed by MALDI–TOF. One band was identified as protein KIAA1473, listed in GenBank as an unidentified human gene. Comparison of the cDNA sequence with the human genomic sequence identified the translation start side and the open reading frame of 531 amino acids for this protein, named ZNF492. It has a truncated N–terminus and consequently lacks the usual Kruppel–associated box–A (KRAB–A), while KRAB–B remains intact. Twelve C2H2 zinc–fingers are also found in tandem arrangement from amino acid 143 to 531. The transcriptional activity of ZNF492 was assessed using a series of mouse Rpe65 gene promoter/luciferase reporter constructs. For the shortest promoter fragment (TR1, 49 bp 5’ flanking), co–expression of ZNF492 protein had no effect on luciferase activity in D407 cells. Interestingly, ZNF492 expression activates construct TR2, containing 188 bp 5’ flanking sequence, by 2.5 fold. For longer promoter constructs, TR4 (655 bp) and TR5 (1240 bp), ZNF492 also confers an activation of about 2 fold. Conclusions: A KRAB–zinc finger protein–ZNF492 is identified by its interaction with immobilized Rpe65 promoter DNA sequence. This KRAB protein, devoid of Kruppel–associated box–A, serves as a moderate transcriptional factor for Rpe65 gene up–regulation. In this zinc–finger protein, absence of KRAB–A might reduce or prevent co–repressor binding to account for the modest up–regulation of Rpe65 gene expression.

Keywords: gene/expression • retinal pigment epithelium • transcription factors