DNA Methyltransferase Target Sites in Cancer

描述(由申请人提供)：DNA甲基化是一种可遗传的表观遗传标记，由一类称为DNA甲基转移酶(DNMT)的酶授予。有三种催化活性的DNMT：DNMT1是细胞分裂过程中的维持甲基转移酶，DNMT3a和Dnmt3b是从头开始的甲基转移酶，负责在发育早期建立甲基化模式。DNMT3L是一种调节从头甲基转移酶活性的辅因子，在母体基因组印记中具有重要意义。DNA甲基化和DNMT在癌症中特别重要，因为常见的异常甲基化，如肿瘤抑制基因启动子中的基因特异性高甲基化，以及重复和转座的DNA元件的全局低甲基化。目前还不清楚癌症基因组中异常甲基化是如何发生的，本研究提案的总体目标是确定每个DNMT的独特和重叠的靶点，以更好地了解DNA甲基化的异常调节，并最终开发针对癌症中改变的DNMT活性的治疗策略。该项目将集中于两个目标：(1)定义基因组范围的DNA甲基化和组蛋白修饰模式，针对个体和组合丢失的DNMT和(2)识别和表征每个DNMT的独特和重叠的靶点。第一个目标将通过siRNA介导的敲除来单独和以组合的方式耗尽不同DNMT的胚胎癌细胞(NCCIT)来实现。在DNMT耗尽后，将分别使用甲基-CpG结合结构域(MBD)捕获技术结合大规模平行测序(MBD-SEQ)、染色质免疫沉淀(CHIP-SEQ)和RNA微阵列来分析全基因组DNA甲基化、与DNA甲基化相关的重要标记的组蛋白修饰以及基因表达。这三种全基因组分析将利用生物信息学数据分析进行整合，以构建代表每一种DNMT枯竭情景的综合表观基因组图景。DNMT的独特和重叠的靶点将通过比对不同的表观基因组景观来确定。将利用HCT116 1KO(DNMT1基因敲除)和3BKO(Dnmt3b基因敲除)细胞株来评估DNMT的招募和已知DNMT1和DNMT3b靶点的时间表观遗传调节，这些细胞株已被修饰为在四环素存在下分别诱导表达DNMT1和DNMT3b。DNA甲基化(亚硫酸氢盐基因组测序)、组蛋白修饰(CHIP-定量聚合酶链式反应)和基因表达(逆转录-定量聚合酶链式反应)将在12小时时间点进行评估。最后，这个相同的HCT116细胞系系统将被用来表征和评估已识别的DNMT的独特和重叠的靶点的表观遗传调节。总之，这些实验将确定每个DNMT的目标位置，并提供更好的了解这些位置在癌症中的表观遗传调节。

DESCRIPTION (provided by applicant): DNA methylation is a heritable, epigenetic mark that is conferred by a class of enzymes referred to as the DNA methyltransferases (DNMTs). There are three catalytically active DNMTs: DNMT1 serves as the maintenance methyltransferase during cell division, and DNMT3A and DNMT3B are the de novo methyltransferases that are responsible for establishing methylation patterns early in development. DNMT3L is a cofactor that modulates the activity of the de novo methyltransferases and has important implications in maternal genomic imprinting. DNA methylation and the DNMTs are of particular interest in cancer as aberrant methylation such as gene- specific hypermethylation in tumor suppressor gene promoters and global hypomethylation at repetitive and transposable DNA elements is commonly observed. It is not well understood how aberrant methylation occurs in the cancer genome, and it is the overall goal of this research proposal to determine the unique and overlapping target sites for each of the DNMTs to better understand aberrant regulation of DNA methylation and ultimately develop therapeutic strategies to target altered DNMT activity in cancer. This project will focus on two aims: (1) To define genome-wide DNA methylation and histone modification patterns specific for individual and combinatorial loss of the DNMTs and (2) To identify and characterize unique and overlapping target sites for each DNMT. The first aim will be accomplished by depleting embryonic carcinoma cells (NCCIT) of the different DNMTs both individually and in a combinatorial fashion via siRNA-mediated knockdown. Following depletion of the DNMTs, genome-wide DNA methylation, histone modifications for important marks associated with DNA methylation, and gene expression will be assayed using the methyl-CpG binding domain (MBD) capture technique coupled with massively parallel sequencing (MBD-seq), chromatin immunoprecipitation (ChIP-seq), and RNA microarrays, respectively. These three genome-wide assays will be integrated using bioinformatic data analysis to construct comprehensive epigenomic landscapes representative of each DNMT-depletion scenario. Unique and overlapping target sites of the DNMTs will be determined by aligning the different epigenomic landscapes. Recruitment of DNMTs and temporal epigenetic regulation of known DNMT1 and DNMT3B target sites will be evaluated by utilizing HCT116 1KO (DNMT1 knockout) and 3BKO (DNMT3B knockout) cell lines that have been modified to inducibly express DNMT1 and DNMT3B, respectively in the presence of tetracycline. DNA methylation (bisulfite genomic sequencing), histone modifications (ChIP-quantitative PCR) and gene expression (reverse transcription-quantitative PCR) will be evaluated at 12 hour time points. Finally, this same HCT116 cell line system will be used to characterize and evaluate epigenetic regulation of the identified unique and overlapping target sites of the DNMTs. All together, these experiments will identify target sites for each of the DNMTs as well as provide a better understanding of the epigenetic regulation of these sites in cancer.