Factors affecting tooth and B cell development

Lef-1 and Pitx2 are expressed in the dental epithelium and both factors show overlapping expression in the tooth bud and cap stages. My RT-PCR and quantitative RT-PCR experiments show that Pitx2-/- mutant mice have reduced Lef-1 expression in facial tissues. I also show that the human 2.5kb LEF-1 promoter is activated by PITX2. PITX2 isoforms are co-expressed in dental epithelium and LEF-1 promoter is differentially activated by these isoforms. The 2.5kb LEF-1 promoter has two inhibitory regions that inhibit its transcription in concert with PITX2. The LEF-1 downstream promoter contains a Wnt-responsive element (WRE) that attenuates PITX2 activation. Truncated Lef-1 from Lef-1DeltaN113 cannot auto-regulate LEF-1 promoter expression, however co-transfection of PITX2 and Lef-1DeltaN113 result in a synergistic activation of the 2.5kb LEF-1 promoter. Lef-1 interacts with the C-terminal tail of PITX2. Deletion of distal 800 bp of the LEF-1 promoter shows an increase in PITX2 activation, and synergistic activation with Lef-1DeltaN113. I show that beta-catenin in combination with PITX2 synergistically activates the LEF-1 promoter and this activation is independent of the Wnt-responsive element. beta-catenin directly interacts with PITX2. PITX2 and PITX2/beta-catenin interact with Lef-1 at the common activation domain and this interaction has an inhibitory element at the C-terminal of Lef-1. My research provides a new mechanism for the regulation of LEF-1 expression by PITX2, Lef-1 and beta-catenin by direct physical interactions. LEF-1 and beta-catenin interactions with PITX2 provide new mechanisms for the regulation of PITX2 transcriptional activity.; ST6Gal I sialyltransferase activity is required for a proper immune response evidenced by compromised B cell function seen in ST6Ga1 I knock out mice (Hennet et al.; 1997). The mouse ST6Gal I has eight different mRNA isoforms which are expressed in a developmental and tissue specific manner (Dalziel et al.; 2001). The present research establishes an immature B cell line which is responsive to IL-4 stimulation and transfectable with standard plasmids to study the differential response to stimulus of different ST6Gal I promoters.; I also generated 5'-UTR-containing promoter constructs to study tissue specific expression driven by the information within the ST6Gal I 5'-UTR sequences. Protocol for RNA extraction from transfected WEHI-231 cells with minimum or no plasmid DNA contamination was also established as a part of this research.