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. Overall design: 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).

DNA甲基化（DNA methylation）是调控多种生理过程的关键表观遗传事件，可对基因转录产生显著影响。甲基化胞嘧啶（5mC）可被TET家族酶氧化为5-羟甲基胞嘧啶（5-hmC）——后者是去甲基化循环中的关键中间产物——而在急性髓系白血病（AML）、黑色素瘤等恶性肿瘤中，5-hmC水平显著降低。
本研究构建了人皮肤鳞状细胞癌（cutaneous Squamous Cell Carcinoma，SCC）组织微阵列，发现相较于癌旁正常皮肤，肿瘤组织中5-hmC水平呈全局下降趋势。
我们利用小鼠K14-CreER系统开展研究，发现Tet2缺失可促进致癌物诱导的皮肤鳞状细胞癌发生，且与Tp53缺失协同驱动上皮组织自发性鳞状细胞癌形成。正常表皮中Tet2缺失会引发5-hmC水平的位点特异性改变，众多基因的5-hmC水平呈现上调或下调态势。
值得注意的是，这些改变富集于调控角质形成细胞分化与自我更新的基因中，这与此前报道的TET家族酶在造血干细胞（hematopoietic stem cells）、胚胎干细胞（ES cells）中调控谱系定型的功能一致。本研究明确了Tet2在表皮5-hmC调控中的功能性作用，并证实Tet2是皮肤鳞状细胞癌中确凿的肿瘤抑制基因。
整体实验设计如下：我们采用带条形码标记的羟甲基化DNA免疫沉淀（hydroxymethylated DNA immunoprecipitation，hMeDIP）联合深度测序技术（hMeDIP-seq），定量比较野生型小鼠与表皮特异性敲除Tet2的小鼠（K14-CreER Tet2Flox）表皮组织中特定基因组位点的5-hmC标签密度。我们分别对溶剂对照组（n=3）和他莫昔芬处理组（n=3）的K14-CreER Tet2L/L小鼠表皮组织进行了RNA测序（RNA-seq）。