Identification of Promoter and Other Regulatory Regions of Pitx3 Gene

Abstract

Abstract: : Purpose: To identify sequences involved in regulation of expression of the Pitx3 gene. Pitx3 gene plays important role during the development of the anterior segment of the eye. Mutations in the Pitx3 gene result in anterior segment dysgenesis, Peters' anomaly and cataract in humans and aphakia in mice. Studies of regions responsible for specific expression of Pitx3 gene will facilitate identification of other factors involved in development of anterior segment structures and provide new targets for mutation screening in human patients. Methods: In order to locate potential regulatory regions, human, mouse and rat Pitx3 genomic sequences were obtained and compared with each other to identify conserved elements. A transcription initiation site has been determined by 5'RACE experiments. MatInspector and Softberry software have been utilized to perform promoter searches and to identify binding sites for transcription factors/potential regulators of Pitx3 activity. DNA fragments containing Pitx3 genomic sequences were cloned into pGL3 luciferase reporter vectors and tested for transcriptional activity by transient transfections in different cell lines including human and mouse lens epithelial cell lines. Results: Seven highly conserved regions have been identified in the genomic sequences of human , mouse and rat Pitx3 genes. Potential binding sites for such transcription factors as PAX6, forkhead family, Ap1 and Ap2, deltaEF1, GATA1, MYOD, Mafs and many others have been identified within these regions. DNA fragment encompassing nucleotides at positions from -301 to +31 to the transcription initiation site caused 5-10 fold activation of the reporter gene expression in different cell lines, while DNA fragments containing an additional ~700- 3000 nt of 5' sequence demonstrated significant reduction in reporter activity indicating to the presence of negative regulatory elements in these regions. Co-transfection of Pitx3 reporter plasmids with PAX6 expression plasmids resulted in x2-3 increase in reporter gene activity in correlation with the presence of potential PAX6 binding sites. Further characterization of these sequences and their role in Pitx3 gene expression is underway. Conclusions: Identification of promoter and other regulatory regions of Pitx3 gene provides a framework for further studies of gene hierarchy during development of the anterior segment of the eye.

Keywords: anterior segment • gene/expression • cataract