PITX2 and β-Catenin Interactions Regulate Lef-1 Isoform Expression

Abstract

Lef-1 and PITX2 function in the Wnt signaling pathway by recruiting and interacting with ␤-catenin to activate target genes. Chromatin immunoprecipitation (ChIP) assays identified the Lef-1 promoter as a PITX2 downstream target. Transgenic mice expressing LacZ driven by the 2.5-kb LEF-1 promoter demonstrated expression in the tooth epithelium correlated with endogenous Lef-1 FL epithelial expression. PITX2 isoforms regulate the LEF-1 promoter, and ␤-catenin synergistically enhanced activation of the LEF-1 promoter in combination with PITX2 and Lef-1 isoforms. PITX2 enhances endogenous expression of the full-length ␤-catenin-dependent Lef-1 isoform (Lef-1 FL) while decreasing expression of the N-terminally truncated ␤-catenin-independent isoform. Our research revealed a novel interaction between PITX2, Lef-1, and ␤-catenin in which the Lef-1 ␤-catenin binding domain is dispensable for its interaction with PITX2. PITX2 interacts with two sites within the Lef-1 protein. Furthermore, ␤-catenin interacts with the PITX2 homeodomain and Lef-1 interacts with the PITX2 C-terminal tail. Lef-1 and ␤-catenin interact simultaneously and independently with PITX2 through two different sites to regulate PITX2 transcriptional activity. These data support a role for PITX2 in cell proliferation, migration, and cell division through differential Lef-1 isoform expression and interactions with Lef-1 and ␤-catenin.Pitx2 and Lef-1 encode two transcription factors whose expression can be regulated by early signaling events involved in numerous developmental programs. Pitx2 and Lef-1 are differentially expressed in many tissues, and they demonstrate overlapping expression during tooth development. Lef-1 can be activated by BMP, Wnt, Smads, and transforming growth factor ␤ signaling (18,32,33). Furthermore, Lef-1 transcriptional activity is regulated by its interaction with ␤-catenin. Secreted

Hierarchical Interactions of Homeodomain and Forkhead Transcription Factors in Regulating Odontogenic Gene Expression

“…Pitx2 binds to the 5Ј-TAATCC-3Ј DNA sequence to regulate gene expression. Chromatin immunoprecipitation assays have previously demonstrated that Pitx2 binds to the Lef-1 promoter and synergistically regulates Lef-1 isoform expression in concert with Lef-1 and ␤-catenin (25,32). In this report we have identified a new transcriptional mechanism, where the Dlx2 protein has a repressive effect on PITX2 activation of the Dlx2 promoter.…”

“…Chromatin Immunoprecipitation (ChIP) Assay-The ChIP assays were performed as previously described using the ChIP assay kit (Upstate Biotechnology) with the following modifications (24,25). LS-8 cells were fed for 24 h, harvested, and plated in 60-mm dishes.…”

“…Electroporation of CHO cells were performed at 380 V and 950 microfarads (Gene Pulser XL, Bio-Rad). Electroporation of LS-8 cells has been previously described (25). Transfected cells were incubated for 24 h in 60-mm culture dishes and fed with 5% FBS and DMEM and then lysed and assayed for reporter activities and protein content by Bradford assay (Bio-Rad).…”

Wnt signaling and adipocyte lineage commitment

“…Of interest, paired-like homeodomain transcription factor-2 (Pitx2) is known to upregulate the expression of Lef1, hence the decrease in Pitx2 expression (Table 1) appears to explain the decrease in Lef1 expression. 35 The differential expression of Lef/Tcf family members in 10T1/2 stem cells and committed A33 preadipocytes is likely to be important in determining their respective phenotypes.…”

Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal

“…Pitx2 is the first transcriptional marker of tooth development and Pitx2 null mice have tooth development arrest at E12.5 (Liu et al, 2003;Lu et al, 1999). We have previously shown that Pitx2 regulates Lef-1 expression during odontogenesis (Amen et al, 2007;Vadlamudi et al, 2005). RNAsequencing data showed that Sox2 expression was upregulated in the mandibles of Pitx2 overexpression mice and downregulated in the mandibles of Pitx2 null mice (B.A.…”