Human p32 is a novel FOXC1-interacting protein that regulates FOXC1 transcriptional activity in ocular cells.
OBJECTIVE
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.
Huang
Lijia
L
Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.
Chi
Jonathan
J
Berry
Fred B
FB
Footz
Timothy K
TK
Sharp
Michael W
MW
Walter
Michael A
MA
D000818:Animals; D000869:Anterior Eye Segment / Q000002:abnormalities*; D019556:COS Cells; D002352:Carrier Proteins / Q000502:physiology*; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D005124:Eye Abnormalities / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D051858:Forkhead Transcription Factors / Q000235:genetics*; D005901:Glaucoma / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D024101:Mitochondrial Proteins / Q000502:physiology*; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology*; D014162:Transfection; D020798:Two-Hybrid System Techniques
C087299:C1QBP protein, human; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C112743:FOXC1 protein, human; D051858:Forkhead Transcription Factors; D024101:Mitochondrial Proteins