SOX9, through Interaction with Microphthalmia-associated Transcription Factor (MITF) and OTX2, Regulates BEST1 Expression in the Retinal Pigment Epithelium

Abstract

BEST1 is highly and preferentially expressed in the retinal pigment epithelium (RPE) and causes Best macular dystrophy when mutated. We previously demonstrated that the human BEST1 upstream region ؊154 to ؉38 bp is sufficient to direct expression in the RPE of transgenic mice, and microphthalmiaassociated transcription factor (MITF) and OTX2 regulate this BEST1 promoter. However, a number of questions remained. Here, we show that yeast one-hybrid screen with bait corresponding to BEST1 ؊120 to ؊88 bp identified the SOX-E factors, SOX8, SOX9, and SOX10. A paired SOX site was found in this bait, and mutation of either of the paired sites significantly decreased BEST1 promoter activity in RPE primary cultures. Among the SOX-E genes, SOX9 is highly and preferentially expressed in the RPE, and chromatin immunoprecipitation with fresh RPE cells revealed binding of SOX9, but not SOX10, to the BEST1 region where the paired SOX site is located. BEST1 promoter activity was increased by SOX9 overexpression and decreased by siRNA-mediated SOX9 knockdown. Importantly, SOX9 physically interacted with MITF and OTX2 and orchestrated synergistic activation of the BEST1 promoter with the paired SOX site playing essential roles. A combination of the expression patterns of SOX9, MITF, and OTX2 yielded tissue distribution remarkably similar to that of BEST1. Lastly, the BEST1 promoter was also active in Sertoli cells of the testis in transgenic mice where SOX9 is highly expressed. These results define SOX9 as a key regulator of BEST1 expression and demonstrate for the first time its functional role in the RPE. The retinal pigment epithelium (RPE)2 has many specialized functions essential for vision and is indispensable for the survival and function of retinal photoreceptors (1, 2). RPE cells and melanocytes, two major pigmented cells in the body, share pigment-related genes such as tyrosinase (Tyr) and dopachrome tautomerase (Dct) and transcription factors regulating them such as microphthalmia-associated transcription factor (MITF); otherwise, these two cell types are distinct in many aspects. Although the molecular networks controlling gene expression in melanocytes have been extensively studied (3, 4), those in the RPE are still poorly understood. Among the key transcription factors required for RPE specification and development are MITF (3-9) and orthodenticle homeobox 2 (OTX2) (10 -14). MITF and OTX2 proteins physically interact with each other and cooperatively activate some pigment-related genes in the RPE, such as QNR71 and Tyr (15). Our previous studies found that human BEST1, a gene that is highly and preferentially expressed in the RPE but not related to pigment, is also regulated by . However, a number of questions remained to be answered. Recently, it has been found unintentionally that SRY (sex-determining region Y) box 9 (SOX9) is robustly expressed in mouse RPE throughout embryonic development and into postnatal stages (19).SOX9 is a member of the SOX family of transcription factors, which is characterized by the hig...

Transcription Factor SOX9 Plays a Key Role in the Regulation of Visual Cycle Gene Expression in the Retinal Pigment Epithelium

“…Plasmid Transfection-To analyze the effect of transcription factors on visual cycle gene promoters, cotransfection was performed using Dual-Luciferase assays as described previously (17,32). All plasmid transfections were carried out with D407 cells in 12-well plates using Lipofectamine Plus (Invitrogen).…”

“…To analyze the expression of transcription factors in human tissues and cells, we used total RNA prepared previously from human RPE, retina, and culture cells (D407 and ARPE19 RPE cell lines and M1 RPE primary cells) as well as total RNA from five human tissues (brain, liver, kidney, spleen, and testis; Clontech, Mountain View, CA) (17). The mRNA levels of SOX9, LHX2, and control GAPDH (supplemental Table S1) were analyzed by RT-qPCR as described above.…”

“…ChIP-ChIP with bovine RPE was performed as described previously (17,32). Antibodies used were anti-SOX9 (catalog no.…”

“…To construct an expression vector for human LHX2, cDNA was generated by RT-PCR using RNA from M1 human RPE primary cells (17) with the primers listed in supplemental Table S1 and inserted downstream of the CMV promoter in the pcDNA3.1/Myc-His(-) B vector (Invitrogen).…”

“…Its importance in maintaining adult RPE functions has also been reported recently (16). In our previous studies, we identified sex-determining region Y box 9 (SOX9) as a key regulator that activates the human BEST1 promoter in the RPE through interaction with MITF and OTX2 (17). SOX transcription factors are important regulators of organogenesis and promote the assembly of transcriptional complexes by facilitating interaction between distant binding sites (18).…”