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的重要步骤。在腺癌早期恶性前病变中，改变的基因和microrna （miRs）及其调控的相互作用途径的阐明在很大程度上是未知的。恶性肿瘤和细胞系的特征是胞嘧啶DNA甲基转移酶（DNMT） 1、DNMT3a和DNMT3b的表达增加。虽然已知dnmtts参与从头甲基化和基因沉默，但对于DNMT3a和DNMT3b的靶点及其对腺癌发展的影响知之甚少。本小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来研究肺癌的恶性前病变。烟草致癌物暴露诱导HBECs的形态学转变，包括诱导上皮向间质转化，表现为microRNA (miR)- 200家族和miR-205的下调、胞嘧啶dnmt表达的改变和肿瘤抑制基因的甲基化。本博士后研究项目研究了DNMT3a和DNMT3b （de novo DNMTs）在HBECs转化过程中靶向特定基因和miRs进行甲基化和染色质沉默的假设，并且DNMT3a和DNMT3b的过表达将加速致癌物质治疗的HBECs向完全恶性肿瘤的转化。过表达DNMT3a或DNMT3b的HBECs将暴露于烟草致癌物中，并测定其对裸鼠转化时间、转化效率、EMT和生长的影响。[由于我们的初步数据表明，在HBEC2细胞中过表达DNMT3b可使转化效率提高2倍，因此我们现在将重点研究DNMT3b调节基因]。Illumina Infinium Human Methylation27 BeadChip阵列将用于询问转化过表达HBECs的DNMT3b与亲本HBECs或具有正常DNMT3b背景的转化HBECs，以鉴定DNMT3b调节的甲基化基因。下一代测序使用illumina平台和自定义平纹miR启动子阵列ChIP-chip将用于评估转化的过表达DNMT3b的HBECs与正常表达DNMT3b的转化HBECs和亲本HBECs中存在的miRs中染色质标记的分布。将在100个原发性腺癌样本和17个肺肿瘤来源细胞系中测定8-10个DNMT3b调节基因和2-3个miRs的启动子甲基化状态。在原发肿瘤中最常沉默的两个基因和两个mir将在肺肿瘤来源的细胞系中过度表达，并确定对表型的影响。在三年的研究期间，申请人将使用全基因组方法通过启动子甲基化阵列，通过下一代测序的microRNAome，以及通过ChIP分析的染色质重塑来询问表观基因组。为了加强他在生物信息学和统计学方面的教育，他将学习生物信息学的短期课程和一个学期的生物统计学课程。这些活动以及通过本申请中概述的研究所接受的培训将为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.