Dact2 Represses PITX2 Transcriptional Activation and Cell Proliferation through Wnt/beta-Catenin Signaling during Odontogenesis
Figure 2
Endogenous Pitx2 binds to a conservative region on the Dact2 promoter.
(A) Schematic of Dact2 10 kb promoter with six PITX2 binding motifs (TAATCC) indicated by arrowheads. The red arrowhead indicates the site verified by ChIP assay. The location of the sense primer and the antisense primer are shown for amplification of the immunoprecipitated chromatin. Blue arrowheads are putative binding sites with less conservation. The white arrowhead indicate a non-conserved Pitx2 binding motif that we tested in ChIP experiment as negative control shown in Figure S3. (B) Endogenous ChIP assay was performed in LS-8 cells. Lane 1 contains the PCR marker. Lane 2 shows the Dact2 primers-only control. Lane 3 is the immunoprecipitation using normal rabbit IgG and Dact2 primers. Lane 4 is the Pitx2 immunoprecipitated chromatin amplified using the specific Dact2 promoter primers. Lane 5 is the chromatin input amplified using the Dact2 primers. Lane 6 shows the Msx2 promoter primers-only control. Lane 7 is the immunoprecipitation using normal rabbit IgG and Msx2 primers. Lane 8 is the Pitx2 immunoprecipitated chromatin amplified using the specific Msx2 promoter primers. Lane 9 is the chromatin input amplified using the Msx2 primers. The amplified region of Msx2 promoter is −632 to −359 bp relative to transcription start site. All PCR products were sequenced to confirm their identity. (C) The PITX2 binding element on mouse Dact2 promoter verified by ChIP was mapped to a highly conserved (>70%) region among Mouse, Human, Chimpanzee, Rhesus macaque and Rat. The blue box indicates the PCR amplified region on Dact2 promoter in (B).