Transcription factor SOX9 plays a key role in the regulation of visual cycle gene expression in the retinal pigment epithelium.

The retinal pigment epithelium (RPE) performs specialized functions to support retinal photoreceptors, including regeneration of the visual chromophore. Enzymes and carrier proteins in the visual cycle function sequentially to regenerate and continuously supply 11-cis-retinal to retinal photoreceptor cells. However, it is unknown how the expression of the visual cycle genes is coordinated at the transcriptional level. Here, we show that the proximal upstream regions of six visual cycle genes contain chromatin-accessible sex-determining region Y box (SOX) binding sites, that SOX9 and LIM homeobox 2 (LHX2) are coexpressed in the nuclei of mature RPE cells, and that SOX9 acts synergistically with orthodenticle homeobox 2 (OTX2) to activate the RPE65 and retinaldehyde binding protein 1 (RLBP1) promoters and acts synergistically with LHX2 to activate the retinal G protein-coupled receptor (RGR) promoter. ChIP reveals that SOX9 and OTX2 bind to the promoter regions of RPE65, RLBP1, and RGR and that LHX2 binds to those of RPE65 and RGR in bovine RPE. ChIP with human fetal RPE cells shows that SOX9 and OTX2 also bind to the human RPE65, RLBP1, and RGR promoters. Conditional inactivation of Sox9 in mouse RPE results in reduced expression of several visual cycle genes, most dramatically Rpe65 and Rgr. Furthermore, bioinformatic analysis predicts that multiple common microRNAs (miRNAs) regulate visual cycle genes, and cotransfection of miRNA mimics with luciferase reporter constructs validated some of the predicted miRNAs. These results implicate SOX9 as a key regulator of visual cycle genes, reveal for the first time the functional role of LHX2 in the RPE, and suggest the possible regulation of visual cycle genes by common miRNAs.

RIT2, a neuron-specific small guanosine triphosphatase, is expressed in retinal neuronal cells and its promoter is modulated by the POU4 transcription factors.

PURPOSE: Ras-like without CAAX 2 (RIT2), a member of the Ras superfamily of small guanosine triphosphatases, is involved in regulating neuronal function. RIT2 is a unique member of the Ras family in that RIT2 is preferentially expressed in various neurons, including retinal neurons. The mechanisms that regulate RIT2 expression in neurons were studied. METHODS: Reverse transcription-quantitative PCR (RT-qPCR), immunohistochemistry, western blotting, bioinformatic prediction, electrophoretic mobility shift assay (EMSA), and cell transfection methods were used. RESULTS: With immunohistochemistry of the mouse retina, RIT2 protein was detected in the ganglion cell layer (GCL), inner plexiform layer, inner nuclear layer, and outer plexiform layer, with the strongest staining in the GCL and the inner plexiform layer. RT-qPCR combined with laser capture microdissection detected Rit2 messenger RNA in the GCL and the inner nuclear layer. Western blot analysis showed a large increase in the RIT2 protein in the retina during maturation from newborn to adult. Transient transfection identified the 1.3 kb upstream region of human RIT2 as capable of driving expression in neuronal cell lines. Based on the known expression pattern and biological activity, we hypothesized that POU4 family factors might modulate RIT2 expression in retinal ganglion cells (RGCs). Bioinformatic analyses predicted six POU4 factor-binding sites within the 1.3 kb human RIT2 promoter region. EMSA analyses showed binding of POU4 proteins to three of the six predicted sites. Cotransfection with expression vectors demonstrated that POU4 proteins can indeed modulate the human RIT2 promoter, and that ISL1, a LIM homeodomain factor, can further modulate the activity of the POU4 factors. CONCLUSIONS: These studies confirm the expression of RIT2 in retinal neuronal cells, including RGCs, begin to reveal the mechanisms responsible for neuronal expression of RIT2, and suggest a role for the POU4 family factors in modulating RIT2 expression in RGCs.

The many faces of SIRT6 in the retina and retinal pigment epithelium.

Sirtuin 6 (SIRT6) is a member of the mammalian sirtuin family of NAD+-dependent protein deacylases, homologues of the yeast silent information regulator 2 (Sir2). SIRT6 has remarkably diverse functions and plays a key role in a variety of biological processes for maintaining cellular and organismal homeostasis. In this review, our primary aim is to summarize recent progress in understanding SIRT6's functions in the retina and retinal pigment epithelium (RPE), with the hope of further drawing interests in SIRT6 to increase efforts in exploring the therapeutic potential of this unique protein in the vision field. Before describing SIRT6's role in the eye, we first discuss SIRT6's general functions in a wide range of biological contexts. SIRT6 plays an important role in gene silencing, metabolism, DNA repair, antioxidant defense, inflammation, aging and longevity, early development, and stress response. In addition, recent studies have revealed SIRT6's role in macrophage polarization and mitochondrial homeostasis. Despite being initially understudied in the context of the eye, recent efforts have begun to elucidate the critical functions of SIRT6 in the retina and RPE. In the retina, SIRT6 is essential for adult retinal function, regulates energy metabolism by suppressing glycolysis that affects photoreceptor cell survival, protects retinal ganglion cells from oxidative stress, and plays a role in Müller cells during early neurodegenerative events in diabetic retinopathy. In the RPE, SIRT6 activates autophagy in culture and protects against oxidative stress in mice. Taken together, this review demonstrates that better understanding of SIRT6's functions and their mechanisms, both in and out of the context of the eye, holds great promise for the development of SIRT6-targeted strategies for prevention and treatment of blinding eye diseases.

SIRT6 overexpression in the nucleus protects mouse retinal pigment epithelium from oxidative stress.

Retinal pigment epithelium (RPE) is essential for the survival of retinal photoreceptors. To study retinal degeneration, sodium iodate (NaIO3) has been used to cause oxidative stress-induced RPE death followed by photoreceptor degeneration. However, analyses of RPE damage itself are still limited. Here, we characterized NaIO3-induced RPE damage, which was divided into three regions: periphery with normal-shaped RPE, transitional zone with elongated cells, and center with severely damaged or lost RPE. Elongated cells in the transitional zone exhibited molecular characteristics of epithelial-mesenchymal transition. Central RPE was more susceptible to stresses than peripheral RPE. Under stresses, SIRT6, an NAD+-dependent protein deacylase, rapidly translocated from the nucleus to the cytoplasm and colocalized with stress granule factor G3BP1, leading to nuclear SIRT6 depletion. To overcome this SIRT6 depletion, SIRT6 overexpression was induced in the nucleus in transgenic mice, which protected RPE from NaIO3 and partially preserved catalase expression. These results demonstrate topological differences of mouse RPE and warrant further exploring SIRT6 as a potential target for protecting RPE from oxidative stress-induced damage.

IKKß Inhibition Attenuates Epithelial Mesenchymal Transition of Human Stem Cell-Derived Retinal Pigment Epithelium.

Epithelial-mesenchymal transition (EMT), which is well known for its role in embryonic development, malignant transformation, and tumor progression, has also been implicated in a variety of retinal diseases, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and diabetic retinopathy. EMT of the retinal pigment epithelium (RPE), although important in the pathogenesis of these retinal conditions, is not well understood at the molecular level. We and others have shown that a variety of molecules, including the co-treatment of human stem cell-derived RPE monolayer cultures with transforming growth factor beta (TGF-ß) and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), can induce RPE-EMT; however, small molecule inhibitors of RPE-EMT have been less well studied. Here, we demonstrate that BAY651942, a small molecule inhibitor of nuclear factor kapa-B kinase subunit beta (IKKß) that selectively targets NF-κB signaling, can modulate TGF-ß/TNF-α-induced RPE-EMT. Next, we performed RNA-seq studies on BAY651942 treated hRPE monolayers to dissect altered biological pathways and signaling events. Further, we validated the effect of IKKß inhibition on RPE-EMT-associated factors using a second IKKß inhibitor, BMS345541, with RPE monolayers derived from an independent stem cell line. Our data highlights the fact that pharmacological inhibition of RPE-EMT restores RPE identity and may provide a promising approach for treating retinal diseases that involve RPE dedifferentiation and EMT.

SOX9, through interaction with microphthalmia-associated transcription factor (MITF) and OTX2, regulates BEST1 expression in the retinal pigment epithelium.

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 microphthalmia-associated 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.

BEST1 expression in the retinal pigment epithelium is modulated by OTX family members.

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.

Fine Tuning of an Oxidative Stress Model with Sodium Iodate Revealed Protective Effect of NF-κB Inhibition and Sex-Specific Difference in Susceptibility of the Retinal Pigment Epithelium.

Oxidative stress of the retinal pigment epithelium (RPE) is a major risk factor for age-related macular degeneration (AMD). As a dry AMD model via oxidative stress, sodium iodate (NaIO3), which is primarily toxic to the RPE, has often been used at a high dose to cause RPE death for studying photoreceptor degeneration. Thus, characterization of RPE damage by a low dose of NaIO3 is still limited. To quantify RPE damage caused by NaIO3 in mice, we recently developed a morphometric method using RPE flat-mounts. Here, we report that NaIO3 has a narrow range of dose-effect correlation at 11-18 mg/kg body weight in male C57BL/6J mice. We evaluated the usefulness of our quantification method in two experimental settings. First, we tested the effect of NF-κB inhibition on NaIO3-induced RPE damage in male C57BL/6J mice. IKKß inhibitor BAY 651942 suppressed upregulation of NF-κB targets and protected the RPE from oxidative stress. Second, we tested sex-specific differences in NaIO3-induced RPE damage in C57BL/6J mice using a low dose near the threshold. NaIO3 caused more severe RPE damage in female mice than in male mice. These results demonstrate the usefulness of the quantification method and the importance of fine-tuning of the NaIO3 dose. The results also show the therapeutic potential of IKKß inhibition for oxidative stress-related RPE diseases, and reveal previously-unrecognized sex-specific differences in RPE susceptibility to oxidative stress.

Proteomic and phosphoproteomic analyses identify liver-related signaling in retinal pigment epithelial cells during EMT.

Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is associated with several blinding retinal diseases. Using proteomics and phosphoproteomics studies of human induced pluripotent stem cell-derived RPE monolayers with induced EMT, we capture kinase/phosphatase signaling cascades 1 h and 12 h after induction to better understand the pathways mediating RPE EMT. Induction by co-treatment with transforming growth factor ß and tumor necrosis factor alpha (TGNF) or enzymatic dissociation perturbs signaling in many of the same pathways, with striking similarity in the respective phosphoproteomes at 1 h. Liver hyperplasia and hepatocyte growth factor (HGF)-MET signaling exhibit the highest overall enrichment. We also observe that HGF and epidermal growth factor signaling, two cooperative pathways inhibited by EMT induction, regulate the RPE transcriptional profile.

Inducible expression of cre recombinase in the retinal pigmented epithelium.

PURPOSE: The retinal pigmented epithelium (RPE) expresses many genes that play important roles in the support and maintenance of photoreceptors. The present study was conducted to develop a system amenable to the dissection of the temporal function of these genes, specifically within RPE cells. Transgenic mice were generated and characterized in which the expression of Cre recombinase could be specifically induced within the RPE. METHODS: Transgenic mice carrying the human vitelliform macular dystrophy-2 (VMD2) promoter (P(VMD2))-directed reverse tetracycline-dependent transactivator (rtTA) and the tetracycline-responsive element (TRE)-directed cre were generated. Inducible Cre expression was achieved by feeding doxycycline to these mice and was characterized by using a Cre-activatable lacZ reporter mouse strain (R26R). RESULTS: A beta-galactosidase assay of rtTA/Cre-R26R mice demonstrated that the basal level of Cre expression without doxycycline induction was negligible. Addition of doxycycline led to induction of RPE-specific Cre expression/function at least from embryonic day 9 to postnatal day 60. The highest induction occurred at approximately postnatal day 4. As measured by ERG and histology, retinal function and morphology were normal in 10-month-old rtTA/Cre mice that were treated with doxycycline at weaning age. CONCLUSIONS: Transgenic mice were generated that express Cre recombinase in the RPE in an inducible fashion. These mice will be useful for studies of the RPE-specific role of genes that are expressed in the RPE as well as other cells, particularly for avoiding embryonic lethality and dissecting the function of genes that play dual roles in development and adulthood.

Genome-wide prediction and characterization of interactions between transcription factors in Saccharomyces cerevisiae.

Combinatorial regulation by transcription factor complexes is an important feature of eukaryotic gene regulation. Here, we propose a new method for identification of interactions between transcription factors (TFs) that relies on the relationship of their binding sites, and we test it using Saccharomyces cerevisiae as a model system. The algorithm predicts interacting TF pairs based on the co-occurrence of their binding motifs and the distance between the motifs in promoter sequences. This allows investigation of interactions between TFs without known binding motifs or expression data. With this approach, 300 significant interactions involving 77 TFs were identified. These included more than 70% of the known protein-protein interactions. Approximately half of the detected interacting motif pairs showed strong preferences for particular distances and orientations in the promoter sequences. These one dimensional features may reflect constraints on allowable spatial arrangements for protein-protein interactions. Evidence for biological relevance of the observed characteristic distances is provided by the finding that target genes with the same characteristic distances show significantly higher co-expression than those without preferred distances. Furthermore, the observed interactions were dynamic: most of the TF pairs were not constitutively active, but rather showed variable activity depending on the physiological condition of the cells. Interestingly, some TF pairs active in multiple conditions showed preferences for different distances and orientations depending on the condition. Our prediction and characterization of TF interactions may help to understand the transcriptional regulatory networks in eukaryotic systems.

Cadherins in the retinal pigment epithelium (RPE) revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo.

The retinal pigment epithelium (RPE) supports the health and function of retinal photoreceptors and is essential for normal vision. RPE cells are post-mitotic, terminally differentiated, and polarized epithelial cells. In pathological conditions, however, they lose their epithelial integrity, become dysfunctional, even dedifferentiate, and ultimately die. The integrity of epithelial cells is maintained, in part, by adherens junctions, which are composed of cadherin homodimers and p120-, ß-, and α-catenins linking to actin filaments. While E-cadherin is the major cadherin for forming the epithelial phenotype in most epithelial cell types, it has been reported that cadherin expression in RPE cells is different from other epithelial cells based on results with cultured RPE cells. In this study, we revisited the expression of cadherins in the RPE to clarify their relative contribution by measuring the absolute quantity of cDNAs produced from mRNAs of three classical cadherins (E-, N-, and P-cadherins) in the RPE in vivo. We found that P-cadherin (CDH3) is highly dominant in both mouse and human RPE in situ. The degree of dominance of P-cadherin is surprisingly large, with mouse Cdh3 and human CDH3 accounting for 82-85% and 92-93% of the total of the three cadherin mRNAs, respectively. We confirmed the expression of P-cadherin protein at the cell-cell border of mouse RPE in situ by immunofluorescence. Furthermore, we found that oxidative stress induces dissociation of P-cadherin and ß-catenin from the cell membrane and subsequent translocation of ß-catenin into the nucleus, resulting in activation of the canonical Wnt/ß-catenin pathway. This is the first report of absolute comparison of the expression of three cadherins in the RPE, and the results suggest that the physiological role of P-cadherin in the RPE needs to be reevaluated.

Identification of regulatory targets of tissue-specific transcription factors: application to retina-specific gene regulation.

Identification of tissue-specific gene regulatory networks can yield insights into the molecular basis of a tissue's development, function and pathology. Here, we present a computational approach designed to identify potential regulatory target genes of photoreceptor cell-specific transcription factors (TFs). The approach is based on the hypothesis that genes related to the retina in terms of expression, disease and/or function are more likely to be the targets of retina-specific TFs than other genes. A list of genes that are preferentially expressed in retina was obtained by integrating expressed sequence tag, SAGE and microarray datasets. The regulatory targets of retina-specific TFs are enriched in this set of retina-related genes. A Bayesian approach was employed to integrate information about binding site location relative to a gene's transcription start site. Our method was applied to three retina-specific TFs, CRX, NRL and NR2E3, and a number of potential targets were predicted. To experimentally assess the validity of the bioinformatic predictions, mobility shift, transient transfection and chromatin immunoprecipitation assays were performed with five predicted CRX targets, and the results were suggestive of CRX regulation in 5/5, 3/5 and 4/5 cases, respectively. Together, these experiments strongly suggest that RP1, GUCY2D, ABCA4 are novel targets of CRX.

Increased expression of brain-derived neurotrophic factor preserves retinal function and slows cell death from rhodopsin mutation or oxidative damage.

There are no effective treatments for inherited retinal degenerations, which are prevalent causes of visual disability. Several proteins promote the survival of various types of neurons, and increasing expression of one or more of these survival factors is a promising strategy for a new treatment. Studies examining the effects of intravitreous injections of brain-derived neurotrophic factor (BDNF) in models of inherited retinal degenerations have suggested that BDNF has little survival-promoting activity for photoreceptors. In this study, we generated double transgenic mice with doxycycline-inducible expression of BDNF in the retina. In a model of primary rod photoreceptor degeneration, expression of BDNF resulted in significant delay in photoreceptor cell death and maintenance of retinal function assessed by electroretinogram recordings. Expression of BDNF also caused strong protection of photoreceptors from oxidative damage-induced cell death. These data suggest that continuous expression of BDNF, unlike intravitreous injections, results in morphologic and functional benefit in animal models of inherited retinal degeneration. Double transgenic mice with inducible expression of survival factors provide valuable tools for selection of survival factor candidates for gene therapy.

Studies on retinal and retinal pigment epithelial gene expression.

The 'completion' of the murine and human genomes and creation of high-density expressed sequence tag (EST) databases from multiple tissues and multiple species, coupled with the development of high-throughput expression profiling approaches such as microarrays and Serial Analysis of Gene Expression (SAGE), is making possible the in-depth analysis of gene expression patterns in health and disease to an extent that was not previously possible. Such new information is providing insight into normal function, and into how normal function is altered in disease. Efforts have begun, and are accelerating, in the application of expression profiling to the study of the retina and retinal pigment epithelium (RPE). In this chapter we will review progress in this area. We will also discuss technical issues that make expression studies of the RPE particularly challenging, and share our experience in methodological approaches to overcome these challenges.

Protein Phosphatase 1 binds strongly to the retinoblastoma protein but not to p107 or p130 in vitro and in vivo.

PURPOSE: To identify and characterize retinoblastoma protein (pRb) binding proteins that may influence retinoblast proliferation and retinal pigment epithelial cell survival. METHODS: The yeast two-hybrid system was used to screen a bovine retinal cDNA library and to characterize positive clones. DNA sequencing and site-directed mutagenesis were used for further analysis. Co-immunoprecipitation experiments were used to confirm the results of the two-hybrid system in vivo. RESULTS: In the two-hybrid system, Protein Phosphatase 1alpha1 (PP1alpha1) binds the retinoblastoma protein. Unlike several other pRb binding proteins, PP1alpha1 binds only weakly to the Rb family member p107, and does not demonstrate detectable binding to p130. Confirming the two-hybrid results, endogenous PP1 in a human retinal pigment epithelial (RPE) cell line co-immunoprecipitates with endogenous pRb but not p107 or p130. Site directed mutagenesis of two pRb binding motifs in PP1alpha1 from LXSXE to LXCXE leads to slight increases in its two-hybrid interaction with pRb but does not alter its binding preference for pRb over the other family members. The complete sequence of bovine PP1alpha1 is reported. CONCLUSIONS: The strong two-hybrid interaction between PP1alpha1 and pRb, but not p107 or p130, suggests that the phosphorylation status of members of the pRb family may be regulated by different phosphatases, contributing to fine control of cell cycle progression. Conversely, PP1 activity may be specifically regulated by pRb and not p107 or p130. Mutagenesis studies suggest that PP1alpha1's LXSXE motif is not responsible for its binding preference for pRb over p107 and p130. Disruption of the PP1-pRb interaction may influence retinoblastoma tumorigenesis as well as RPE cell proliferation and survival.

Identification of a therapeutic dose of continuously delivered erythropoietin in the eye using an inducible promoter system.

Erythropoietin (EPO) can protect the retina from acute damage, but long-term systemic treatment induces polycythemia. Intraocular gene delivery of EPO is not protective despite producing high levels of EPO likely due to its bellshaped dose curve. The goal of this study was to identify a therapeutic dose of continuously produced EPO in the eye. We packaged a mutated form of EPO (EPOR76E) that has equivalent neuroprotective activity as wild-type EPO and attenuated erythropoietic activity into a recombinant adeno-associated viral vector under the control of the tetracycline inducible promoter. This vector was injected into the subretinal space of homozygous postnatal 5-7 day retinal degeneration slow mice, that express the tetracycline transactivators from a retinal pigment epithelium specific promoter. At weaning, mice received a single intraperitoneal injection of doxycycline and were then maintained on water with or without doxycycline until postnatal day 60. Intraocular EPO levels and outer nuclear layer thickness were quantified and correlated. Control eyes contained 6.1 ± 0.1 (SEM) mU/ml EPO. The eyes of mice that received an intraperitoneal injection of doxycycline contained 11.8 ± 2.0 (SEM) mU/ml EPO-R76E. Treatment with doxycycline water induced production of 35.9 ± 2.4 (SEM) mU/ml EPO-R76E in the eye. The outer nuclear layer was approximately 8 µm thicker in eyes of mice that received doxycycline water as compared to the control groups. Our data indicates that drug delivery systems should be optimized to deliver at least 36 mU/ml EPO into the eye since this dose was effective for the treatment of a progressive retinal degeneration.

Generation of Cre transgenic mice with postnatal RPE-specific ocular expression.

PURPOSE: To generate and characterize a constitutively active, RPE-specific, cre-expressing transgenic mouse line. This line can be used to create RPE-specific knockouts by crossing with mice harboring loxP-flanked (floxed) genes. METHODS: A transgene construct was assembled with the BEST1 promoter driving cre expression. Transgenic mice were generated on a C57BL/6 background. Cre expression was assessed by immunofluorescence and Western blot analysis. Cre enzymatic activity was tested by crossing to three lines with floxed DNA regions and detecting deletion of the intervening sequences or through histochemical detection of lacZ activity. Potential cre-mediated toxicity was assessed by retinal histology up to 24 months of age and by electroretinography. RESULTS: The BEST1-cre line with expression in the highest percentage of RPE cells displayed a patchy mosaic expression pattern, with 50% to 90% of RPE cells expressing cre. In mice outcrossed to a mixed B6/129 background, expression was consistently found in 90% of RPE cells. Within the eye, only the RPE cells were immunoreactive with an anti-cre antibody. Maximum cre expression quantified by Western blot analysis occurred at P28. Crosses with three lines containing floxed sequences revealed RPE-specific cre activity in the eye and extraocular expression limited to the testes. Histology and electroretinography showed no cre-mediated RPE toxicity. CONCLUSIONS: This BEST1-cre transgenic line enables generation of RPE-specific knockout mice. The mosaic expression pattern provides an internal control; the non-cre-expressing RPE cells continue to express the floxed genes. These mice should facilitate study of the multifunctional RPE and the generation of mouse models of human retinal disease.

VMD2 promoter requires two proximal E-box sites for its activity in vivo and is regulated by the MITF-TFE family.

The retinal pigment epithelium (RPE) is crucial for the function and survival of retinal photoreceptors. VMD2 encodes bestrophin, an oligomeric chloride channel that is preferentially expressed in the RPE and, when mutated, causes Best macular dystrophy. Previously, we defined the VMD2 upstream region from -253 to +38 bp as being sufficient to direct RPE-specific expression in the eye, and we suggested microphthalmia-associated transcription factor (MITF) as a possible positive regulator. Here we show that in transgenic mice the -154 to +38 bp region is sufficient for RPE expression, and mutation of two E-boxes, 1 and 2, within this region leads to loss of promoter activity. A yeast one-hybrid screen using bait containing E-box 1 identified clones encoding MITF, TFE3, and TFEB, and chromatin immunoprecipitation with antibodies against these proteins enriched the VMD2 proximal promoter. Analysis using in vivo electroporation with constructs containing mutation of each E-box indicated that expression in native RPE requires both E-boxes, yet in vitro DNA binding studies suggested that MITF binds well to E-box 1 but only minimally to E-box 2. MITF knockdown by small interfering RNA (siRNA) in cell culture revealed a strong correlation between MITF and VMD2 mRNA levels. Sequential transfection of a luciferase construct with expression vectors following MITF siRNA revealed that TFE3 and TFEB can also transactivate the VMD2 promoter. Taken together, we suggest that VMD2 is regulated by the MITF-TFE family through two E-boxes, with E-box 1 required for a direct interaction of MITF-TFE factors and E-box 2 for binding of the as yet unidentified factor(s).