title: Identification of a KRAB-zinc finger protein binding to the Rpe65 gene promoter.
abstract: OBJECTIVE
  We wish to identify transcriptional factors involved in regulation binding to the proximal promoter region of the RPE65 gene that confers RPE-specific expression.
  
  
  METHODS
  We incubated human D407 RPE cell nuclear extract with double-stranded (sense 5-prime biotinylated) oligonucleotides, based on the RPE65 proximal gene promoter, bound to streptavidin-Dynabeads. Bound nuclear proteins were eluted, separated on SDS-PAGE, and analyzed by mass spectrometry. Peptide sequence was used to identify cDNA clones that were subcloned into pCDNA3.1 for expression and co-transfection into D407 cells to assess transcriptional activation of mouse Rpe65 gene promoter/reporter constructs. SiRNA interference was used to suppress ZNF492 expression.
  
  
  RESULTS
  We identified a D407 nuclear protein binding to biotinylated-DNA/streptavidin beads as the product of clone KIAA1473 encoding a protein named ZNF492. ZNF492 has an open reading frame of 531 amino acids with a truncated N-terminus and lacks the usual Krüppel-associated box-A (KRAB-A) while KRAB-B remains intact and has 12 C2H2 zinc-fingers in tandem arrangement. Co-expression in D407 cells of ZNF492 protein did not activate TR1, a mouse Rpe65 gene promoter/reporter construct with 49-bp 5-prime flanking sequence, but did activate construct TR2, containing 188-bp 5-prime flanking sequence, by 2.5-fold, and the longer constructs TR4, containing 655-bp 5-prime flanking sequence, and TR5, containing 1240-bp 5-prime flanking sequence, by about 2-fold. SiRNA-mediated suppression of ZNF492 in D407 resulted in decreased Rpe65 promoter activity.
  
  
  CONCLUSIONS
  We have identified ZNF492, a KRAB-zinc finger protein, by its interaction with immobilized RPE65 promoter DNA sequence. This KRAB-zinc finger protein serves as a moderate transcriptional factor for Rpe65 gene upregulation. In ZNF492, absence of KRAB-A might reduce or prevent co-repressor binding to account for the modest upregulation of Rpe65 gene expression.
authors.affiliation: Laboratory of Retinal Cell and Molecular Biology, National Eye Institute/NIH, 7 Memorial Drive, Bethesda, MD 20892, USA.
authors.forename: Zhongjian
authors.initials: Z
authors.lastname: Lu
authors.forename: Eugenia
authors.initials: E
authors.lastname: Poliakov
authors.forename: T Michael
authors.initials: TM
authors.lastname: Redmond
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D015345:Oligonucleotide Probes; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics*; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C511417:ZNF492 protein, human