![]() ![]() In mice, homozygous Mitf mutations ( Mitf Mi/Mitf Mi) cause pigmentation defects and microphthalmia with thickening of the RPE that forms a multilayered structure resembling the early stage of neural retina ( 6, 8, 10, 17–19). In humans, heterozygous MITF mutations result in Waardenburg syndrome type IIA and Tietz syndrome that are characterized by hearing loss and pigmentation defects ( 15, 16). The MITF-TFE (MiT) subfamily of proteins, which includes MITF, TFE3, TFEB and TFEC, forms homodimers or heterodimers within the subfamily and binds to E-box sequences in vitro ( 6, 12). ![]() ![]() MITF is a member of the basic helix-loop-helix leucine zipper (bHLH-ZIP) family of transcription factors and is expressed in several cell lineages, including melanocytes, RPE, osteoclasts and mast cells ( 6, 10, 12–14). MITF and OTX2 co-localize in the nuclei of the RPE, interact with each other, and can cooperatively activate some genes, such as QNR71 and tyrosinase ( Tyr) ( 11). Two of the key transcription factors required for RPE specification and development are microphthalmia-associated transcription factor (MITF) and orthodenticle homeobox 2 (OTX2) ( 3–10). RPE shares its origin with the neural retina, as both tissues are derived from the neuroepithelium of the forebrain ( 3, 4). The retinal pigment epithelium (RPE), a monolayer of cuboidal cells with melanin pigment located between the photoreceptors and choroid of the eye, has many specialized functions that nourish and support retinal photoreceptors ( 1, 2). 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. 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. 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. 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. 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). 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. 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. One of these, BEST1, encodes bestrophin-1, a protein that when mutated causes Best macular dystrophy. A number of genes preferentially expressed in the retinal pigment epithelium (RPE) are associated with retinal degenerative disease. ![]()
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