Purchase this article with an account.
L. Huang, M.A. Walter; The Splicing Factor Sf2–Associated Protein (p32) Is a Novel FOXC1–Interacting Protein That Inhibits FOXC1 Transcription Activation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3117.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Mutations in the forkhead transcription factor FOXC1 cause Axenfeld–Rieger malformations, often leading to glaucoma. Understanding the function of FOXC1 necessitates characterizing the proteins that interact with FOXC1. These interactions may enhance or inhibit transcriptional activity of FOXC1 and thus help regulate important gene expression in the eye. We therefore endeavored to isolate FOXC1–interacting proteins and determine their effects on FOXC1.
To identify FOXC1–interacting proteins, we created a human trabecular meshwork yeast two– hybrid (TM Y2H)cDNA library. Approximately 1.9x10e6 clones from the TM Y2H cDNA library were subjected to an interaction screen with FOXC1. Clones supporting growth in four yeast reporter assays were selected for further analyses. Seven such clones corresponded to cDNAs encoding the splicing factor SF2–associated protein (p32). The interaction and co–localization between FOXC1 and p32 were confirmed in mammalian cells by Ni–pull down assay and immunofluorescence, respectively. Dual–luciferase assays using FOXC1 reporter plasmid were used to determine the effect of the interaction on FOXC1 transcription activity.
The splicing factor SF2–associated protein (p32) was isolated as a putative FOXC1–interacting protein from an Y2H screen. Ni–pull down assays were used to confirm the interaction of FOXC1 with p32 in mammalian cells. Further analyses, using immunofluorescence, determined that while p32 is predominantly cytoplasmic, the portion of p32 that is within the nucleus co–localized with FOXC1. Interestingly, in transactivation assays, p32 significantly inhibited FOXC1–mediated transcription activation in a dose dependent manner. Furthermore, using Ni–pull down assays, the FOXC1 forkhead domain (FHD) was identified as the p32 interaction domain. Our previous analyses of missense mutations within the FOXC1 FHD revealed that the P79L, P79T, S112F, and G165R mutant proteins could bind to DNA but not activate transcription, suggesting that these mutations may alter FOXC1’s ability to interact with other proteins. Ni–pull down assays demonstrated that F112S displayed an impaired interaction with p32.
Our study identified the splicing factor SF2–associated protein (p32) as a novel and specific co–repressor of FOXC1–mediated transcription activation. Importantly, we show here, for the first time, that impaired protein interaction is a disease mechanism for Axenfeld–Rieger malformations.
This PDF is available to Subscribers Only