Human p32 is a novel FOXC1-interacting protein that regulates FOXC1 transcriptional activity in ocular cells

¹ Medical Genetics, University of Alberta, Edmonton, Canada
² Department of Ophthalmology & Medical Genetics, University of Alberta, 832 Medical Sciences Building, Edmonton, T6G 2H7, Canada

^* To whom correspondence should be addressed. E-mail: mwalter{at}ualberta.ca.

	Abstract

PURPOSE. Mutations in the human forkhead box C1 gene (FOXC1) cause Axenfeld-Rieger (AR) malformations, often leading to glaucoma. Understanding the function of FOXC1 necessitates characterizing the proteins that interact with FOXC1. We therefore endeavored to isolate FOXC1-interacting proteins and determine their effects on FOXC1. METHODS. To identify FOXC1-interacting proteins, we screened a human trabecular meshwork (HTM) yeast two-hybrid (Y2H) cDNA library. The interaction and co-localization between FOXC1 and its putative protein partner were confirmed by Ni2+ pull-down assays, immunoprecipitation, and immunofluorescence, respectively. The electrophoretic mobility shift assay (EMSA) was used to study the effect of the interacting protein on FOXC1 DNA-binding ability. Dual-luciferase assays using FOXC1 reporter plasmids in HTM cells were performed to determine the effect of the interaction on FOXC1 transcription activity. RESULTS. The human p32 protein was isolated as a putative FOXC1-interacting protein from a Y2H screen. The interaction of FOXC1 with p32 was confirmed by Ni-pull down assays and immunoprecipitation. While p32 is predominantly cytoplasmic, the portion of p32 that is within the nucleus co-localizes with FOXC1. The FOXC1 forkhead domain (FHD) was identified as the p32 interaction domain. p32 significantly inhibited FOXC1-mediated transcription activation in a dose dependent manner but didn't affect FOXC1 DNA-binding ability. Interestingly, a FOXC1 mutation F112S displayed an impaired interaction with p32. CONCLUSIONS. Our study identified the human p32 protein as a novel regulator of FOXC1-mediated transcription activation. Failure of p32 to interact with FOXC1 containing the disease-causing F112S mutation indicates that impaired protein interaction may be a disease mechanism for AR malformations

Key Words: transcription factors, genetic diseases, mutation screening

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