ZBED4, a BED-type zinc-finger protein in the cones of the human retina.
OBJECTIVE
To characterize the ZBED4 cDNA identified by subtractive hybridization and microarray of retinal cone degeneration (cd) adult dog mRNA from mRNA of normal dog retina.


METHODS
The cDNA library obtained from subtractive hybridization was arrayed and screened with labeled amplicons from normal and cd dog retinas. Northern blot analysis was used to verify ZBED4 mRNA expression in human retina. Flow cytometry sorted peanut agglutinin (PNA)-labeled cones from dissociated mouse retinas, and quantitative RT-PCR (QPCR) was used to measure ZBED4 mRNA levels in these cone cells. Immunohistochemistry localized ZBED4 in human retinas. Expression of ZBED4 mRNA transiently transfected into HEK293 cells was analyzed by immunofluorescence. ZBED4 subcellular localization was determined with Western blot analysis.


RESULTS
One of 80 cDNAs differentially expressed in normal and cd dog retinas corresponded to a novel gene, ZBED4, which is also expressed in human and mouse retinas. ZBED4 mRNA was found to be present in cone photoreceptors. When ZBED4 cDNA was transfected into HEK293 cells, the expressed protein showed nuclear localization. However, in human retinas, ZBED4 was localized to cone nuclei, inner segments, and pedicles, as well as to Müller cell endfeet. Confirming these immunohistochemical results, the 135-kDa ZBED4 was found in both the nuclear and cytosolic extracts of human retinas. ZBED4 has four predicted DNA-binding domains, a dimerization domain, and two LXXLL motifs characteristic of coactivators/corepressors of nuclear hormone receptors.


CONCLUSIONS
ZBED4 cellular/subcellular localization and domains suggest a regulatory role for this protein, which may exert its effects in cones and Müller cells through multiple ways of action.
Saghizadeh
Mehrnoosh
M
Jules Stein Eye Institute, David Geffen School of Medicine, University of California Los Angeles (UCLA), Los Angeles, California, USA.
Akhmedov
Novrouz B
NB
Yamashita
Clyde K
CK
Gribanova
Yekaterina
Y
Theendakara
Veena
V
Mendoza
Emmanuel
E
Nelson
Stanley F
SF
Ljubimov
Alexander V
AV
Farber
Debora B
DB
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics*; D004283:Dog Diseases / Q000235:genetics* / Q000473:pathology; D004285:Dogs; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D017403:In Situ Hybridization; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D046228:Microarray Analysis; D009457:Neuroglia / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism* / Q000473:pathology; D012162:Retinal Degeneration / Q000235:genetics* / Q000473:pathology / Q000662:veterinary*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection; D016335:Zinc Fingers
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; C542680:ZBED4 protein, human; C575736:Zbed4 protein, mouse