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S. Chen, G. Peng; Identification of Direct Targets for the Photoreceptor Transcription Factor Crx Using Chromatin Immunoprecipitation (ChIP) . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3538.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In vitro studies have implicated a number of transcription factors in the regulation of photoreceptor gene expression. Studies using gene knockouts or naturally occurring mutants confirmed that some of these transcription factors are required for regulating gene expression in the photoreceptor cells in vivo. Analyzing changes in gene expression that occur after the deletion or mutation of these transcription factors identified potential target genes. However, these studies do not distinguish between direct vs. indirect targets. We used chromatin immunoprecipitation (ChIP) to identify direct targets of Crx, a homeodomain transcription factor required for the development and maintenance of photoreceptor function in vivo. Methods: ChIP assays were performed using retina and liver from adult mice and cows. Tissues were treated with a fixative to cross-link chromatin-associated proteins and DNA was fragmented by sonication. Crx-associated chromatin fragments were immunoprecipitated using an antibody specific to Crx and analyzed by PCR for the presence of the promoter region of several potential Crx targets; the promoter regions of presumed non-target genes were included as controls. Results: Antibody to Crx immunoprecipitated the promoter regions of rhodopsin, M- and S-cone opsins, ß-PDE, rod arrestin (Sag), IRBP and Crx. The antibody did not precipitate the promoter of the liver-specific gene, albumin from retinal chromatin or the promoters of the photoreceptor genes from mouse liver chromatin. Irrelevant antibodies, such as anti-GST and a rabbit serum, did not precipitate the Crx targets from the retinal chromatin. Equivalent results were obtained from multiple independent assays using bovine or mouse retina. Conclusions: ChIP assays can identify the direct, in vivo targets of transcription factors in the retina. Seven of the photoreceptor genes analyzed, including Crx itself, appear to be direct targets of Crx. The role of Crx in regulating IRBP deserves further study, since IRBP was not identified as a target of Crx in Crx-/- mutant mice. ChIP assays are a useful tool for the identification of the direct targets of transcription factors in the retina.
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