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）的酶赋予。有三种具有催化活性的DNMT：DNMT 1在细胞分裂期间充当维持甲基转移酶，而DNMT 3A和DNMT 3B是负责在发育早期建立甲基化模式的从头甲基转移酶。DNMT 3L是调节从头甲基转移酶活性的辅因子，并且在母体基因组印记中具有重要意义。DNA甲基化和DNMT在癌症中特别令人感兴趣，因为通常观察到异常甲基化，例如肿瘤抑制基因启动子中的基因特异性高甲基化和重复和转座DNA元件处的整体低甲基化。目前还不清楚异常甲基化是如何在癌症基因组中发生的，本研究的总体目标是确定每个DNMT的独特和重叠的靶位点，以更好地了解DNA甲基化的异常调节，并最终开发治疗策略以靶向癌症中改变的DNMT活性。该项目将集中于两个目标：（1）定义DNMT个体和组合丢失的全基因组DNA甲基化和组蛋白修饰模式;（2）识别和表征每个DNMT的独特和重叠靶位点。第一个目的将通过经由siRNA介导的敲低以单独和组合方式耗尽不同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微阵列分析全基因组DNA甲基化、与DNA甲基化相关的重要标志物的组蛋白修饰和基因表达。这三个全基因组检测将使用生物信息学数据分析进行整合，以构建代表每个DNMT耗尽场景的综合表观基因组景观。将通过比对不同的表观基因组景观来确定DNMT的独特和重叠的靶位点。将使用HCT 116 1 KO（DNMT 1敲除）和3BKO（DNMT 3B敲除）细胞系评价DNMT的募集和已知DNMT 1和DNMT 3B靶位点的时间表观遗传调控，这些细胞系已被修饰为在四环素存在下分别诱导表达DNMT 1和DNMT 3B。将在12小时时间点评价DNA甲基化（亚硫酸氢盐基因组测序）、组蛋白修饰（ChIP-定量PCR）和基因表达（逆转录-定量PCR）。最后，该相同的HCT 116细胞系系统将用于表征和评价DNMT的鉴定的独特和重叠靶位点的表观遗传调控。总之，这些实验将确定每个DNMT的靶位点，并提供对癌症中这些位点的表观遗传调控的更好理解。

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.