/2mouse, and was previously shown by IHC. These data demonstrate that Pitx2 and Dact2 are co-expressed in the dental epithelium during development. We next asked if Dact2 was part of the Wnt/Pitx2 transcriptional activator mechanism. 9 Dact2 Regulates PITX2 and Wnt Signaling PITX2 activates Dact2 expression We analyzed the sequence of the 59 flanking region of the Dact2 gene and found several potential Pitx2 binding sites. After an evolutional conservation screening a putative binding site at 6142 bp was found with a high degree of conservation among mouse, rat, human and chimp. Chromatin immunoprecipitation assays performed in LS-8 cells demonstrate endogenous Pitx2 binding to this site on the Dact2 promoter. A set of primers flanking this Pitx2 binding site were able to amplify the Dact2 promoter from chromatin input, as well as from the Pitx2 immunoprecipitated chromatin, demonstrating Pitx2 specifically binds to the element in the Dact2 promoter. A PCR with DNA pulled down by normal IgG was examined as a negative control. 18790636 Since Pitx2 does not regulate Msx2, a negative control experiment was done in parallel using the same ChIP DNA pulled down by Pitx2 antibody and control IgG to amplify Msx2 promoter region with specific Msx2 primers.. In addition, we performed another control experiment by testing a Dact2 promoter fragment containing a putative binding site with no significant conservation. The result shows no enrichment in Pitx2 antibody pull down DNA. To verify whether this binding is functional, we performed Sodium laureth sulfate site transient co-transfection in cells with PITX2 expression and a luciferase expression plasmid driven by the 10 kb Dact2 promoter. Cells transfected with PITX2 activated the Dact2 promoter about 8-fold. A 66 bp DNA segment of Dact2 promoter containing the PITX2 binding site in Fig. 2A at was cloned into TK-Luc reporter in tandem. A similar reporter was constructed with the same tandem flanking promoter sequence, except the binding motif GGATTA in this reporter was mutated into scrambled motif AGTTCG. Luciferase assays in 19276073 Fig. 3B conducted on CHO cells transfected with PITX2A and each of these two reporters showed a loss of PITX2 activation when the binding motif was mutated. Furthermore, endogenous Dact2 expression was increased in PITX2C transgenic mouse embryonic fibroblast cells compared to wild type MEF cells. Furthermore, Dact2 expression was very low in Pitx2 knockout MEF cells. These data strongly suggest that endogenous Pitx2 activates Dact2 expression. Dact2 represses PITX2 transcriptional activity To understand the function of Dact2, cell transfections were performed 
with Dact2 and PITX2 over expression plasmids and luciferase reporter constructs under the control of Dlx2 and amelogenin promoters. Dlx2 is an ameloblast differentiation marker under Pitx2 regulation in the transcriptional hierarchy of tooth development. Amelogenin is a structural protein that contributes to the enamel formation during the late stage of tooth development, which is also under transcriptional control of Pitx2. The luciferase reporters driven by Amelx and Dlx2 promoters were analyzed for Dact2 function. PITX2A activates the Amelx promoter at,22-fold and Dact2 alone does not activate the Amelx promoter in transfected cells. However, Dact2 represses PITX2 activation of the Amelx promoter, from 22-fold to 14-fold activation. Because Dact2 is involved in Wnt/b-catenin signaling we asked if Dact2 repression of PITX2 transcriptional activity was
