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Functional Cloning Using a Xenopus Oocyte Expression System
Published on: January 30, 2016
Human p32 is a novel FOXC1-interacting protein that regulates FOXC1 transcriptional activity in ocular cells
Lijia Huang1, Jonathan Chi, Fred B Berry
1Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.
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. This study was undertaken to isolate FOXC1-interacting proteins and determine their effects on FOXC1.
Methods:
To identify FOXC1-interacting proteins, a human trabecular meshwork (HTM) yeast two-hybrid (Y2H) cDNA library was screened. The interaction and colocalization between FOXC1 and its putative protein partner were confirmed by Ni(2+) 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. Although p32 is predominantly cytoplasmic, the portion of p32 that is within the nucleus colocalizes 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 did not affect FOXC1 DNA-binding ability. Of interest, a FOXC1 mutation F112S displayed an impaired interaction with p32.
Conclusions:
In the study, 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.
Insights
Human p32 protein regulates FOXC1 transcription, a gene linked to Axenfeld-Rieger malformations. Impaired interaction between mutated FOXC1 and p32 may contribute to disease development.
Area of Science:
- Molecular Biology
- Genetics
- Ophthalmology
Background:
- Mutations in the human forkhead box C1 gene (FOXC1) are associated with Axenfeld-Rieger malformations and glaucoma.
- Understanding FOXC1 function requires identifying its interacting proteins.
Purpose of the Study:
- To isolate proteins that interact with FOXC1.
- To characterize the functional impact of these interactions on FOXC1 activity.
Main Methods:
- Yeast two-hybrid screening of a human trabecular meshwork cDNA library to identify FOXC1 interactors.
- Confirmation of protein interactions using Ni(2+) pull-down and immunoprecipitation assays.
- Assessment of FOXC1 transcriptional activity and DNA-binding using dual luciferase and EMSA assays.
Main Results:
- Human p32 protein was identified as a FOXC1-interacting protein.
- p32 inhibits FOXC1-mediated transcription activation but does not affect FOXC1 DNA binding.
- A disease-associated FOXC1 mutation (F112S) showed impaired interaction with p32.
Conclusions:
- Human p32 is a novel regulator of FOXC1 transcription.
- Impaired interaction between FOXC1 and p32 due to mutations may be a mechanism underlying Axenfeld-Rieger malformations.
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