Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing
Summary
Methylation and demethylation of cytosines in DNA are believed to act as keystones of cell-specific gene expression through controlling chromatin structure and accessibility to transcription factors. Cancer cells have their own transcriptional programs and we sought to alter such a cancer-specific program by enforcing expression of the catalytic domain (CD) of the methylcytosine dioxygenase TET2 in breast cancer cells. TET2 CD decreased the tumorigenic potential of cancer cells through both activation and repression of a repertoire of genes. In addition to promoting the establishment of an antiviral state, TET2 activated 5mC turnover at thousands of MYC binding motifs and down-regulated a panel of known MYC-repressed genes involved in lysosome biogenesis and function.
Overall design
MCF-7 cells stably transfected with an empty vector or with plasmids encoding either wild type mouse TET2 catalytic domain (CD - aa916-1921, pcDNA3-Flag-TET2 CD, addgene #72219) or the catalytically dead mutant H1304Y, D1306A mutant (pcDNA3-Flag-TET2 mCD, addgene #72220) were analyzed for gene expression by RNA-seq and for chromatin marks by ChIP-seq (H3K4me3 and H3K27me3) and selective chemical labeling (5hmC).