Zeb1 represses Mitf and regulates pigment synthesis, cell proliferation, and epithelial morphology.

PURPOSE Epithelial-mesenchymal transition (EMT) is important in fibrotic responses, formation of cancer stem cells, and acquisition of a metastatic phenotype. Zeb1 represses epithelial specification genes to enforce epithelial-mesenchymal phenotypic boundaries during development, and it is one of several E-box-binding repressors whose overexpression triggers EMT. The purpose of this study was to investigate the potential role for Zeb1 in EMT leading to the dedifferentiation of retinal pigment epithelial (RPE) cells. METHODS Real-time PCR was used to examine mRNA expression during RPE dedifferentiation in primary cultures of RPE cells from Zeb1(+/-) mice and after knockdown of Zeb1 by lentivirus shRNA. Chromatin immunoprecipitation was used to detect Zeb1 at gene promoters in vivo. RESULTS Zeb1 is overexpressed during RPE dedifferentiation. Heterozygous mutation or shRNA knockdown to prevent this overexpression eliminates the onset of proliferation, loss of epithelial morphology, and pigment, which characterizes RPE dedifferentiation. Zeb1 binds to the Mitf A promoter in vivo, and Zeb1 mutation or shRNA knockdown derepresses the gene. The authors link Zeb1 expression to cell-cell contact and demonstrate that forcing dedifferentiated RPE cells to adopt cell-cell only contacts via sphere formation reverses the overexpression of Zeb1 and reprograms RPE cells back to a pigmented phenotype. CONCLUSIONS Overexpression of the EMT transcription factor Zeb1 has an important role in RPE dedifferentiation via its regulation of Mitf. Expression of Zeb1 and, in turn, RPE dedifferentiation, is linked to cell-cell contact, and these contacts can be used to diminish Zeb1 expression and reprogram dedifferentiated RPE cells.