Q 1 2 A Protein Interaction between b-Catenin and 3 Dnmt 1 Regulates Wnt Signaling Q 2 and DNA 4 Methylation in Colorectal Cancer Cells 5

8 Aberrant activation of the Wnt signaling pathway is an 9 important step in the initiation and progression of tumor 10 development in diverse cancers. The central effector of canon11 ical Wnt signaling, b-catenin (CTNNB1), is a multifunctional 12 protein, and has been extensively studied with respect to its 13 roles in cell–cell adhesion and in regulation of Wnt-driven 14 transcription. Here, a novel mass spectrometry–based proteo15 mics technique in colorectal cancer cells expressing stabilized 16 b-catenin, was used to identify a protein–protein interaction 17 between b-catenin and DNA methyltransferase I (Dnmt1) pro18 tein, the primary regulator of DNA methylation patterns in 19 mammalian cells. Dnmt1 and b-catenin strongly colocalized in 20 the nuclei of colorectal cancer cells, and the interaction is 21 mediated by the central domain of the Dnmt1 protein. Dnmt1 22 protein abundance is dependent upon the levels of b-catenin, 24 and is increased in cells expressing stabilized mutant b-catenin. 25 Conversely, the Dnmt1 regulates the levels of nuclear b-catenin 26 and b-catenin/TCF–driven transcription. In addition, lysine27 specific demethylase 1 (LSD1/KDM1A), a regulator of DNMT1 28 stability, was identified as a component of the Dnmt1–b-cate29 nin protein complex and perturbation of the Dnmt1–b-catenin 30 interaction altered DNA methylation. In summary, a functional 31 protein–protein interaction was identified between two criti32 cally important oncoproteins, in turn revealing a link between 33 Wnt signaling and downstream nuclear functions mediated by 34 Dnmt1.