DNA methyltransferase I plays the central role in maintenance of CpG DNA methylation patterns across the genome and alteration of CpG methylation patterns is a frequent and significant occurrence across many cancers. Cancer cells carrying hypomorphic alleles of Dnmt1 have become important tools for understanding Dnmt1 function and CpG methylation. In this study, we analyse colorectal cancer cells with a homozygous deletion of exons 3 to 5 of Dnmt1, resulting in reduced Dnmt1 activity. Although this cell model has been widely used to study the epigenome, the effects of the Dnmt1 hypomorph on cell signalling pathways and the wider proteome are largely unknown. In this study, we perform the first quantitative proteomic analysis of this important cell model and identify multiple signalling pathways and processes that are significantly dysregulated in the hypomorph cells. In Dnmt1 hypomorph cells, we observed a clear and unexpected signature of increased Epithelial-to-Mesenchymal transition (EMT) markers as well as reduced expression and sub-cellular re-localization of Beta-Catenin. Expression of wild-type Dnmt1 in hypomorph cells or knock-down of wild-type Dnmt1 did not recapitulate or rescue the observed protein profiles in Dnmt1 hypomorph cells suggesting that hypomorphic Dnmt1 causes changes not solely attributable to Dnmt1 protein levels. In summary, we present the first comprehensive proteomic analysis of the widely studied Dnmt1 hypomorph colorectal cancer cells and identify redistribution of Dnmt1 and its interaction partner Beta-Catenin.

BackgroundDNA methyltransferase I is the primary eukaryotic DNA methyltransferase engaged in maintenance of CpG 5 DNA methylation patterns across the genome. Alteration of CpG methylation patterns and levels is a frequent and significant occurrence across many cancers, and targeted inhibition of Dnmt1 has become an approach of choice for select malignancies. There has been significant interest both in the methyltransferase activity as well as methylation-independent functions of Dnmt1. A previously generated hypomorphic allele of Dnmt1 in HCT116 colorectal cancer cells has become an important tool for understanding Dnmt1 function and how CpG 10 methylation patterns are modulated across the genome. Colorectal cancer cells with the Dnmt1 hypomorphic allele carry a homozygous deletion of exons 3 to 5 of Dnmt1, resulting in greatly reduced Dnmt1 protein expression whilst still exhibiting a limited functional activity and methyltransferase ability. Although this cell model of reduced Dnmt1 levels and function have been used to study the downstream effects on the epigenome and transcriptome, the broader effects of the Dnmt1 hypomorph on the proteome and wider cell 15 signalling are largely unknown. ResultsIn this study, we used quantitative proteomic analysis of nuclear-enriched samples of HCT116 Dnmt1 hypomorph cells to identify signalling pathways and processes dysregulated in the hypomorph cells as compared to wild-type HCT116 cells. Unexpectedly, we observed a clear signature of increased expression of 20 Epithelial-to-Mesenchymal (EMT) in Dnmt1 hypomorph cells. We also observed reduced expression and subcellular re-localization of Beta-Catenin in Dnmt1 hypomorph cells. Expression of wild-type Dnmt1 in hypomorph cells or knock-down of wild-type Dnmt1 did not recapitulate or rescue the observed protein profiles in Dnmt1 hypomorph cells suggesting that hypomorphic Dnmt1 causes changes not solely attributable 4 to Dnmt1 protein levels. ConclusionsIn summary we present the first comprehensive proteomic analysis of the widely studied Dnmt1 hypomorph colorectal cancer cells and identify redistribution of Dnmt1 and its interaction partner Beta-Catenin as well as the dysregulation of EMT related processes and signalling pathways related to the development of a cancer 5 stem cell phenotype.