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. 2011 Aug 16;39(21):9194–9205. doi: 10.1093/nar/gkr658

Figure 1.

Figure 1.

Disruption of PCNA and DMAP1-binding domains of DNMT1 results in E-cadherin downregulation, nuclear translocation of β-catenin and activation of β-catenin-dependent transcriptional signaling. (A) Schematic representation of full-length DNMT1 protein domain structure and deletion mutants used in this study. DMAP1 domain, amino acids 1–120; PCNA domain, amino acids 163–174; NLS, nuclear localization signal domain, amino acids 177–205; DNA replication foci-targeting domain, amino acids 331–550; ZnF, zinc finger region, amino acids 646–692; KEN, KEN box (KENxxxR), amino acids 644–650; BAH1 and BAH2, bromo-adjacent homology domains, amino acids 755–880 and 972–1100, respectively; catalytic domain, amino acids 1139–1616. Pictures are not drawn to scale. (B) Phase-contrast images of living cultured cells (left panels) and Ca2+-dependent fast cell–cell aggregation assays in the presence or absence of a functional antibody against E-cadherin (Decma1) and the Ca2+-chelating agent EDTA (right panel). These indicate a strong reduction of cell–cell adhesiveness in human HCT116 cells lacking PCNA and DMAP1-binding domains of DNMT1 (DNMT1ΔE3−6), but not in parental cells (DNMT1+/+), or those lacking DNMT3b (DNMT3b−/−). The mean ± SD of results from three experiments are shown. **Student's t-test, P < 0.001. (C) Immunolocalization (red signal) of E-cadherin (upper panels) and β-catenin (lower panels) in HCT116 DNMT1+/+, DNMT1ΔE3−6 and DNMT3b−/− cells showing the extensive disorganization of E-cadherin cell–cell contacts and nuclear translocation of β-catenin in DNMT1ΔE3−6 cells. Chromatin is counterstained with DAPI (blue signal). Bars: 10 μm. (D) Semiquantitative analysis of protein expression by immunoblot showing total levels of E-cadherin, β-catenin, and SNAIL1 in HCT116 DNMT1+/+, DNMT1ΔE3−6, and DNMT3b−/− cells. β-tubulin protein content is included as a loading control. (E) Normalized Luciferase/Renilla activities of reporter vectors transiently transfected in HCT116 DNMT1+/+, DNMT1ΔE3−6 and DNMT3b−/− cells containing either TOP multimerized promoter sequences recognized by β-catenin-Lef/Tcf complexes (upper panel), or the human cyclinD1 promoter (lower panel). The activity of both reporters is significantly higher in DNMT1ΔE3−6 cells. Analyses were performed in triplicate and the mean ± SD are shown. **P < 0.001.