Novel forms of Paired-like homeodomain transcription factor 2 (PITX2):: Generation by alternative translation initiation and mRNA splicing

Background: Members of the Paired-like homeodomain transcription factor ( PITX) gene family, particularly PITX1 and PITX2, play important roles in normal development and in differentiated cell functions. Three major isoforms of PITX2 were previously reported to be produced through both alternative mRNA splicing (PITX2A and PITX2B) and alternative promoter usage (PITX2C). The proteins derived from these mRNAs contain identical homeodomain and carboxyl termini. Differences in the amino-termini of the proteins may confer functional differences in some contexts. Results: Here, we report the identification of two novel PITX2 isoforms. First, we demonstrate that the Pitx2c mRNA generates two protein products, PITX2C alpha and PITX2C beta, via alternative translation initiation. Second, we identified a novel mRNA splice variant, Pitx2b2, which uses the same 5' splice donor in intron 2 as Pitx2b (hereafter referred to as Pitx2b1), but employs an alternative 3' splice acceptor, leading to an in-frame deletion of 39 base pairs relative to Pitx2b1. Pitx2b2 mRNA is expressed in both murine and human pituitary. The data show that in a murine gonadotrope cell line and adult murine pituitary what was previously thought to be PITX2B1 is actually PITX2C beta, or perhaps PITX2B2. PITX2B1 is expressed at lower levels than previously thought. PITX2C beta and PITX2B2 activate gonadotrope-specific gene promoter-reporters similarly to known PITX2 isoforms. Conclusion: We have identified and characterized two novel isoforms of PITX2, generated by alternative translation initiation (PITX2C beta) and alternative mRNA splicing (PITX2B2). These proteins show similar DNA binding and trans-activation functions as other PITX2 isoforms in vitro, though their conservation across species suggests that they may play distinct, as yet unidentified, roles in vivo.