The Role of de novo Cytosine DNA Methyltransferases 3a & 3b in Gene Methylation

描述（由申请人提供）：肿瘤抑制基因的失活是所有形式的人类癌症发展中的重要步骤。阐明改变的基因和microRNA（miR）和他们调节的途径，在腺癌的早期癌前病变的相互作用在很大程度上是未知的。恶性肿瘤和细胞系的特征在于胞嘧啶DNA甲基转移酶（DNMT）1、DNMT 3a和DNMT 3b的表达增加。虽然已知DNMT在从头甲基化和基因沉默中合作，但关于DNMT 3a和DNMT 3b的靶点或它们对腺癌发展的影响知之甚少。本课题组利用永生化支气管上皮细胞系（HBECs）建立了一种体外转化模型，用于肺癌癌前病变的研究。暴露于烟草致癌物诱导形态学转化，包括诱导HBEC中的上皮向间充质转化，其通过下调microRNA（miR）- 200家族和miR-205、胞嘧啶DNMT表达的变化和肿瘤抑制基因的甲基化来表现。这个博士后奖学金解决了DNMT 3a和DNMT 3b（从头DNMTs）靶向特定基因和miR的甲基化和染色质沉默在HBEC的转化过程中的假设，以及DNMT 3a和DNMT 3b的过度表达将加速致癌剂治疗的HBEC转化为完全恶性肿瘤。将过表达DNMT 3a或DNMT 3b的HBEC暴露于烟草致癌物，并测定对转化时间、转化效率、EMT和裸鼠生长的影响。[由于我们的初步数据表明DNMT 3b在HBEC 2细胞中的过表达使转化效率提高了2倍，我们现在将关注DNMT 3b调节的基因]。Illumina Infinium Human Methylation 27 BeadChip阵列将用于询问过表达DNMT 3b的转化HBEC与亲本HBEC或具有正常DNMT 3b背景的转化HBEC，以鉴定由DNMT 3b调节的甲基化基因。使用Ilumina平台和用于ChIP芯片的定制平铺miR启动子阵列的下一代测序将用于评价与具有正常DNMT 3b表达的转化HBEC和亲本HBEC相比，过表达DNMT 3b的转化HBEC中存在的miR中染色质标记的分布。将在100个原发性腺癌样品和17个肺肿瘤衍生细胞系中确定8-10个DNMT 3b调节基因和2-3个miR的启动子甲基化状态。在原发性肿瘤中最常沉默的两个基因和两个miR将在肺肿瘤衍生的细胞系中过表达，并确定对表型的影响。在该奖学金的三年期间，申请人将使用全基因组方法通过启动子甲基化阵列询问表观基因组，通过下一代测序询问microRNAome，并通过ChIP测定询问染色质重塑。为了促进他在生物信息学和统计学方面的教育，他将参加生物信息学的短期课程和生物统计学的一个学期课程。这些活动沿着通过本申请中概述的研究接受的培训将为Ivo提供独特的优势，以及整合分子生物学、生物信息学和统计学的能力。

DESCRIPTION (provided by applicant): Inactivation of tumor suppressor genes is an important step in the development of all forms of human cancer. The elucidation of altered genes and microRNAs (miRs) and the pathways they regulate that interact in early premalignancy of adenocarcinoma are largely unknown. Malignant tumors and cell lines are characterized by increased expression of cytosine DNA methyltransferase (DNMT) 1, DNMT3a and DNMT3b. While it is known that DNMTs cooperate in de novo methylation and gene silencing, very little is known about targets for DNMT3a and DNMT3b or their impact on the development of adenocarcinoma. Our group has developed an in vitro transformation model using immortalized bronchial epithelial cell lines (HBECs) to study pre-malignancy of lung cancer. Exposure to tobacco carcinogens induces morphological transformation involving induction of epithelial to mesenchymal transition in HBECs that manifests through down regulation of the microRNA (miR)- 200 family and miR-205, changes in expression of cytosine DNMTs and methylation of tumor suppressor genes. This Postdoctoral fellowship addresses the hypothesis that DNMT3a and DNMT3b (de novo DNMTs) target specific genes and miRs for methylation and chromatin silencing during transformation of HBECs, and that overexpression of DNMT3a and DNMT3b will accelerate transformation of carcinogen-treated HBECs to full malignancy. HBECs overexpressing DNMT3a or DNMT3b will be exposed to tobacco carcinogens, and the effect on time to transformation, transformation efficiency, EMT and growth in nude mice will be determined. [Since our preliminary data indicates that overexpression of DNMT3b in HBEC2 cells increased transformation efficiency by 2-fold, we will now focus on DNMT3b regulated genes]. The Illumina Infinium Human Methylation27 BeadChip array will be used to interrogate transformed DNMT3b overexpressing HBECs vs. parental HBECs or transformed HBECs with normal DNMT3b background to identify methylated genes regulated by DNMT3b. Next generation sequencing using the Ilumina platform and a custom tiling miR promoter array for ChIP-chip will be used to evaluate the distribution of chromatin marks in miRs present in transformed HBECs overexpressing DNMT3b compared to transformed HBECs with normal DNMT3b expression and parental HBECs. Promoter methylation status of 8-10 DNMT3b - regulated genes and 2-3 miRs will be determined in 100 primary adenocarcinoma samples and 17 lung tumor-derived cell lines. Two genes and two miRs that are most commonly silenced in primary tumors will be overexpressed in lung tumor-derived cell lines and the effect on phenotype determined. During the three years of this fellowship, the applicant will use whole genome approaches to interrogate the epigenome through promoter methylation arrays, the microRNAome through Next Generation sequencing, and chromatin remodeling through ChIP assays. To foster his education in bioinformatics and statistics, he will take a short course in bioinformatics and a one-semester course in Biostatistics. These activities along with the training received through the studies outlined in this application will provide Ivo with unique strengths, and the ability to integrate molecular biology, bioinformatics, and statistics.