TY - JOUR
T1 - BEST1 expression in the retinal pigment epithelium is modulated by OTX family members
AU - Esumi, Noriko
AU - Kachi, Shu
AU - Hackler, Laszlo
AU - Masuda, Tomohiro
AU - Yang, Zhiyong
AU - Campochiaro, Peter A.
AU - Zack, Donald J.
N1 - Funding Information:
We thank Shiming Chen for providing the anti-CRX antibody p261. This study was supported in part by unrestricted funds from Research to Prevent Blindness, Inc. and generous gifts from Robert and Clarice Smith and the Guerrieri Family Foundation. P.A.C. is the George S. and Dolores Dore Eccles Professor of Ophthalmology and Neuroscience; D.J.Z. is the Guerrieri Professor of Genetic Engineering and Molecular Ophthalmology at the Wilmer Eye Institute and is a recipient of a Research to Prevent Blindness Senior Investigator Award.
PY - 2009
Y1 - 2009
N2 - A number of genes preferentially expressed in the retinal pigment epithelium (RPE) are associated with retinal degenerative disease. One of these, BEST1, encodes bestrophin-1, a protein that when mutated causes Best macular dystrophy. As a model for RPE gene regulation, we have been studying the mechanisms that control BEST1 expression, and recently demonstrated that members of the MITF-TFE family modulate BEST1 transcription. The human BEST1 upstream region from -154 to +38 bp is sufficient to direct expression in the RPE, and positive-regulatory elements exist between -154 and -104 bp. Here, we show that the -154 to -104 bp region is necessary for RPE expression in transgenic mice and contains a predicted OTX-binding site (Site 1). Since another non-canonical OTX site (Site 2) is located nearby, we tested the function of these sites using BEST1 promoter/luciferase constructs by in vivo electroporation and found that mutation of both sites reduces promoter activity. Three OTX family proteins - OTX1, OTX2 and CRX - bound to both Sites 1 and 2 in vitro, and all of them increased BEST1 promoter activity. Surprisingly, we found that human and bovine RPE expressed not only OTX2 but also CRX, the CRX genomic region in bovine RPE was hypersensitive to DNase I, consistent with active transcription, and that both OTX2 and CRX bound to the BEST1 proximal promoter in vivo. These results demonstrate for the first time CRX expression in the RPE, and suggest that OTX2 and CRX may act as positive modulators of the BEST1 promoter in the RPE.
AB - A number of genes preferentially expressed in the retinal pigment epithelium (RPE) are associated with retinal degenerative disease. One of these, BEST1, encodes bestrophin-1, a protein that when mutated causes Best macular dystrophy. As a model for RPE gene regulation, we have been studying the mechanisms that control BEST1 expression, and recently demonstrated that members of the MITF-TFE family modulate BEST1 transcription. The human BEST1 upstream region from -154 to +38 bp is sufficient to direct expression in the RPE, and positive-regulatory elements exist between -154 and -104 bp. Here, we show that the -154 to -104 bp region is necessary for RPE expression in transgenic mice and contains a predicted OTX-binding site (Site 1). Since another non-canonical OTX site (Site 2) is located nearby, we tested the function of these sites using BEST1 promoter/luciferase constructs by in vivo electroporation and found that mutation of both sites reduces promoter activity. Three OTX family proteins - OTX1, OTX2 and CRX - bound to both Sites 1 and 2 in vitro, and all of them increased BEST1 promoter activity. Surprisingly, we found that human and bovine RPE expressed not only OTX2 but also CRX, the CRX genomic region in bovine RPE was hypersensitive to DNase I, consistent with active transcription, and that both OTX2 and CRX bound to the BEST1 proximal promoter in vivo. These results demonstrate for the first time CRX expression in the RPE, and suggest that OTX2 and CRX may act as positive modulators of the BEST1 promoter in the RPE.
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U2 - 10.1093/hmg/ddn323
DO - 10.1093/hmg/ddn323
M3 - Article
C2 - 18849347
AN - SCOPUS:57649225477
SN - 0964-6906
VL - 18
SP - 128
EP - 141
JO - Human molecular genetics
JF - Human molecular genetics
IS - 1
ER -