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G.-H. Peng, S. Chen; Crx Interacts With Two Coactivators Cbp and p300 Promoting Chromatin Remodeling. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2923.
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The homeodomain transcription factor Crx is required for expression of many photoreceptor genes in the mammalian retina. How Crx regulates transcription remains to be determined. A previous report suggested that Crx interacts with Cbp and p300, two closely related proteins that act as coactivators for many transcription factors. Both proteins harbor histone acetyl-transferase activity for remodeling chromatin during transcriptional activation. The purpose of this study was to determine if Crx directly binds Cbp/p300, and whether this interaction plays a role in target gene transcription.
Direct interactions between Crx and Cbp/p300 were examined using co-immunoprecipitation assays (co-ip) with in vitro translated proteins. The in vivo interactions were studied using 1) co-ip from nuclear extracts of P14 mouse retina, 2) immunostaining of Crx, Cbp and p300 in mouse retinal sections, and 3) chromatin immunoprecipitation (ChIP) assays to detect binding of Crx and Cbp/p300 to the opsin genes in the retinae of wild-type and Crx knockout mice.
An anti-Crx antibody immunoprecipited in vitro translated Cbp or p300 with Crx, and an anti-Cbp (or p300) antibody also co-immunoprecipitated Crx, suggesting a direct interaction between Crx and Cbp or p300 in vitro. Co-ip with deleted/mutated forms of Crx showed that Cbp interacts with the Crx region spanning amino acids 1-107 that contains the homeodomain, while p300 interacts with Crx transactivation domain 2 (AD2). The anti-Crx antibody also co-immunoprecipited Cbp/p300 from retinal nuclear extracts of P14 wild-type mice, but not Crx-/- mice, suggesting interactions between these proteins in vivo. Immunohistochemistry studies showed that Cbp and p300 are present in all the nuclear layers of the mouse retina including the outer nuclear layer where Crx is expressed. ChIP demonstrated that Cbp and p300 are recruited to the opsin gene chromatin in the retina. Cpb binding to the opsin genes is defective in Crx-/- mouse retina, consistent with defects in histone acetylation and transcription of the opsin genes.
These results suggest that Crx can simultaneously recruit Cbp and p300 to its target genes. These Crx-coactivator interactions may play an important role in regulating photoreceptor gene expression in retinal development and diseases.
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