Molecular characterization of the PITX2 homeodomain protein.

摘要

PITX2 is a bicoid type homeobox gene that is mutated in patients with Axenfeld-Rieger syndrome (ARS). ARS is an autosomal dominant disorder characterized by ocular, dental, and umbilical abnormalities. The objective of my thesis was to characterize the functional consequences of ARS mutations and to identify novel PITX2-interacting factors.; I first showed that an ARS homeodomain mutation (K88E) resulted in a dominant negative protein that had defective DNA binding, transactivation, and inhibited wildtype PITX2 activity. To determine the dominant-negative mechanism, I assessed PITX2 dimerization. Wildtype PITX2 formed dimers with itself and with the K88E mutant. However, the wildtype-K88E heterodimer showed greater interaction than the wildtype homodimer. The significance of wildtype dimerization was that it allowed cooperative binding to a DNA element containing tandem bicoid (TAATCC) sites. While the PITX2-K88E heterodimer could bind the tandem sites, it did so without cooperativity and less activation of a reporter gene. I propose that the PITX2-K88E heterodimer is in a ‘constrained’ state that has reduced activity. I also characterized two C-terminal truncation mutants (W133Stop and D122FS), which showed increased DNA binding and cell-line-specific transactivation relative to wildtype. This confirmed the presence of a C-terminal inhibitory domain and suggests that optimal PITX2 function requires cell-specific protein interactions with the C-terminal tail.; Finally, I identified two novel PITX2 interacting factors, MEF2A and PIASy. I showed that PITX2 and MEF2A could synergistically activate the ANF promoter and that p38 MAP kinases enhanced PITX2 activation. These data implicate PITX2 in ANF regulation during development and possibly cardiac hypertrophy. PIASy was identified from a yeast two-hybrid screen of a mouse E17 cDNA library. PIASy is an E3 SUMO-ligase known to sumoyiate and inhibit transcription factors, including Lef1, which is expressed with Pitx2 in the developing tooth. PIASy and PITX2 cotransfection studies revealed that PIASy specifically inhibits PITX2 activity. This suggests coordinate regulation of Pitx2 and Lefl in the dental epithelium.; These studies have provided a new perspective on the pathogenesis of ARS mutations and the mechanism by which PITX2 may function in development.