Analysis of mutations of the PITX2 transcription factor found in Axenfeld-Rieger Syndrome patients

¹ Department of Medical Genetics, University of Alberta, Edmonton, Canada
² Royal Eye Unit, Kingston Hospital, Kingston upon Thames, Surrey, United Kingdom
³ Department of Medicine (Division of Nephrology), University of Alberta, Edmonton, Canada

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

	Abstract

Purpose: To assess the effects of previously uncharacterized PITX2 missense mutations found in ARS patients, and to determine the functional roles of the C-terminal region of PITX2. Methods: Recombinant PITX2 proteins were analyzed via cellular immunofluorescence, electrophoretic mobility shift, reporter transactivation and protein half-life assays in human trabecular meshwork cells. Results: Two homeobox mutations, R43W and R90C, both 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 C-terminal, also resulted in an altered mobility shift pattern, but additionally 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 EMSA patterns that represent altered protein-DNA interactions which 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.

Key Words: transcription factors, genetic diseases, molecular biology, PITX2, protein function

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