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C.-D. Hsu, G.-H. Peng, A. Onishi, U. Alexis, S. Blackshaw, S. Chen; Pias3 Regulates Opsin Gene Expression by Interacting With Photoreceptor Transcription Factors Crx and Nr2e3. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2924.
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Protein Inhibitor of Activated Stat3 (Pias3) is a transcription co-regulator that modulates the activity, function or subcellular localization of target proteins via direct interaction or catalyzing covalent modifications such as SUMOylation. Previous serial analysis of gene expression (SAGE) and in situ analyses on the mouse retina showed that Pias3 expression predominates in post-mitotic photoreceptor precursors. This study was designed to examine whether the developmentally important photoreceptor transcription factors Crx and Nr2e3 are targets of Pias3 and how the consequences of the interaction affect photoreceptor differentiation.
Epitope-tagged proteins were made by in vitro translation or transient expression in HEK293 cells and used for in vitro co-immunoprecipitation (co-ip) assays to reveal direct interactions between Pias3 and known photoreceptor-specific transcription factors. To determine Pias3 domains critical for these interactions, deletions were made in the coding sequence of the plasmid used for expressing the Pias3 protein. Genes regulated by Pias3 in vivo were identified by chromatin immunoprecipitation (ChIP) from P14 mouse retina using an anti-Pias3 antibody. Transient co-transfection assays with opsin-luciferase reporters in HEK293 cells revealed functional interactions among the three proteins.
An antibody against Pias3 co-precipitates either Crx or Nr2e3 in vitro. Reciprocally, antisera to Crx or Nr2e3 co-precipitate Pias3. Deletion analysis of Pias3 demonstrated that interaction with Nr2e3 is mediated by the Pias3 N-terminal domain and RING finger responsible for the SUMOylase activity. The RING finger appears important for Pias3-Crx interaction as well. ChIP assays showed that, in the developing mouse retina, Pias3 is associated with the promoter/enhancer regions of Crx/Nr2e3-regulated photoreceptor genes including rod and cone opsins, but not with those genes expressed by bipolar (mGlu6) or ganglion cells (Thy-1). Co-expression of Pias3 with Crx and Nr2e3 in HEK293 cells altered their activity to regulate rhodopsin or M-cone opsin promoter.
Pias3 appears to directly interact with Crx or Nr2e3 to modify their activity in regulating photoreceptor gene expression, implicating a role of Pias3 in photoreceptor development and disease.
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