title: Analysis of mutations of the PITX2 transcription factor found in patients with Axenfeld-Rieger syndrome.
abstract: OBJECTIVE
  To assess the effects of previously uncharacterized PITX2 missense mutations found in patients with Axenfeld-Rieger syndrome and to determine the functional roles of the C-terminal region of PITX2.
  
  
  METHODS
  Recombinant PITX2 proteins were analyzed with the use of cellular immunofluorescence, electrophoretic mobility shift, reporter transactivation, and protein half-life assays in human trabecular meshwork cells.
  
  
  RESULTS
  Two homeobox mutations, R43W and R90C, resulted in severely reduced DNA-binding and transcriptional activation despite normal nuclear localization. L105V, located C-terminal to the homeodomain, resulted in normal localization, reporter gene transactivation, and protein half-life, but with an altered mobility shift pattern of protein-DNA complexes. N108T, also located C-terminal to the homeodomain, resulted in an altered mobility shift pattern and with slightly increased reporter transactivation and shortened protein half-life. The PITX2 C-terminal region contains at least three domains, each with distinct modulating effects on reporter transactivation.
  
  
  CONCLUSIONS
  PITX2 homeobox mutations predictably resulted in decreased function of the protein. However, the two C-terminal mutations exhibited only subtle defects on PITX2 transactivation and protein-DNA binding, suggesting that ocular development is sensitive to even slight alterations of PITX2 function. The C-terminal mutations L105V and N108T lie in a domain that inhibits PITX2 transcriptional activation. These two mutations produce electrophoretic mobility shift assay patterns representing altered protein-DNA interactions that may be important for accurate target gene selection. Additionally, N108T resulted in a less stable PITX2 mutant protein with elevated activity that may result in stochastic dysregulation during critical stages of development. Together, the results clearly indicate that stringent control of PITX2 is required for normal ocular development and function.
authors.affiliation: Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Tim
authors.initials: T
authors.lastname: Footz
authors.forename: Faisal
authors.initials: F
authors.lastname: Idrees
authors.forename: Moulinath
authors.initials: M
authors.lastname: Acharya
authors.forename: Kathy
authors.initials: K
authors.lastname: Kozlowski
authors.forename: Michael A
authors.initials: MA
authors.lastname: Walter
mesh_terms: D000015:Abnormalities, Multiple / Q000235:genetics*; D000818:Animals; D000869:Anterior Eye Segment / Q000002:abnormalities*; D015153:Blotting, Western; D019556:COS Cells / Q000378:metabolism; D002522:Chlorocebus aethiops; D024202:Electrophoretic Mobility Shift Assay; D005124:Eye Abnormalities / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D017930:Genes, Reporter / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D020125:Mutation, Missense; D013577:Syndrome; D014129:Trabecular Meshwork / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation / Q000502:physiology*; D014162:Transfection; D000097577:Homeobox Protein PITX2
chemical_list: D018398:Homeodomain Proteins; D014157:Transcription Factors; D000097577:Homeobox Protein PITX2