Regulation of 5-hydroxymethylcytosine by TET2 contributes to Squamous Cell Carcinoma tumorigenesis

DNA methylation is a key regulatory event controlling a variety of physiological processes and can have dramatic effects on gene transcription. Methylated Cytosine (5mC) can be oxidized by the TET family of enzymes to 5-hydroxymethylcytosine (5-hmC), a key intermediate in the de-methylation cycle, and 5-hmC levels are reduced in malignancies such as AML and melanoma. We constructed a tissue microarray of human cutaneous Squamous Cell Carcinoma (SCC) tumors and found a global reduction in 5-hmC levels compared to adjacent skin. Using a murine K14-CreER system, we have found that loss of Tet2 promotes carcinogen-induced SCC, and cooperates with loss of Tp53 in to drive spontaneous SCC tumors in epithelial tissues. Loss of Tet2 in the normal epidermis leads to site-specific changes in 5-hmC levels, with many genes showing both increased and decreased levels of 5-hmC. Importantly, many of these changes were in genes regulating keratinocyte differentiation and self-renewal, consistent with reported roles for Tet enzymes in controlling lineage commitment in hematopoietic stem cells and ES cells. These results establish a functional role for Tet2 in the regulation of 5-hmC in the epidermis and demonstrate that Tet2 is a bone fide tumor suppressor in SCC. We used ba barcoded hydroxymethylated DNA immunoprecipitation (hMeDIP) method coupled with deep sequencing (hMeDIP-seq) for quantitative comparison of 5-hmC tag density at specific genomic loci in the epidermis of wild-type mice compared to mice which had epidermal specific deletion of Tet2 (K14-CreER Tet2Flox). We performed RNA-seq on the epidermis of K14-CreER Tet2L/L mice treated with vehicle (n=3) or tamoxifen (n=3).