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

从头胞嘧啶 DNA 甲基转移酶 3a 的作用

• 批准号: 8595152
• 负责人: Ivo Teneng
• 金额: $ 5.66万
• 依托单位: LOVELACE BIOMEDICAL & ENVIRONMENTAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-19 至 2014-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8595152
• 关键词: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Address; Adenocarcinoma; Affect; Agar; Benzo(a)pyrene; Bioinformatics; Biological Assay; Biometry; Carcinogen exposure; Carcinogens; Cell Line; Cells; Cessation of life; ChIP-on-chip; Chromatin; Custom; Cyclin-Dependent Kinase 4; Cytosine; DNA Methyltransferase; DNA Modification Methylases; DNA Repair; DNMT3B gene; DNMT3a; Data; Development; Disease; Down-Regulation; Education; Epigenetic Process; Epithelial Cells; Exposure to; Family; Fellowship; Fostering; Gene Mutation; Gene Silencing; Gene Targeting; Genes; Genetic Predisposition to Disease; Genome; Growth; Heterochromatin; Histologic; Human; In Vitro; Injection of therapeutic agent; Knowledge; Laboratories; Link; Lung; Lung Neoplasms; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Methylation; Methylnitrosourea; Methyltransferase; MicroRNAs; Modeling; Molecular; Molecular Biology; Nude Mice; Pathway interactions; Phenotype; Prevalence; Primary Neoplasm; Promoter Regions; Proteins; Public Health; Role; Sampling; Smoker; Telomerase; Time; Tobacco-Associated Carcinogen; Training; Tumor Suppressor Genes; Tumor-Derived; Tumor-Suppressor Gene Inactivation; Tumorigenicity; cancer cell; cancer diagnosis; cell transformation; chromatin remodeling; epigenome; epithelial to mesenchymal transition; in vitro Model; neoplastic cell; never smoker; next generation sequencing; overexpression; prevent; promoter; public health relevance; statistics; transcription factor

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.

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。改变的基因和 microRNA (miR) 以及它们在腺癌早期癌前病变中相互作用所调节的途径的阐明在很大程度上尚不清楚。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 表达增加。虽然众所周知 DNMT 在从头甲基化和基因沉默中发挥作用，但人们对 DNMT3a 和 DNMT3b 的靶点或其对腺癌发展的影响知之甚少。我们的小组开发了一种使用永生化支气管上皮细胞系（HBEC）的体外转化模型来研究肺癌的癌前病变。接触烟草致癌物会诱导形态转变，包括诱导 HBEC 中的上皮细胞向间质细胞转变，这通过 microRNA (miR)-200 家族和 miR-205 的下调、胞嘧啶 DNMT 表达的变化以及肿瘤抑制基因的甲基化来体现。该博士后奖学金提出了这样的假设：DNMT3a和DNMT3b（从头DNMT）在HBEC转化过程中针对特定基因和miR进行甲基化和染色质沉默，并且DNMT3a和DNMT3b的过度表达将加速致癌物处理的HBEC向完全恶性肿瘤的转化。将过表达DNMT3a或DNMT3b的HBEC暴露于烟草致癌物，并确定对转化时间、转化效率、EMT和裸鼠生长的影响。 [由于我们的初步数据表明 HBEC2 细胞中 DNMT3b 的过表达使转化效率提高了 2 倍，因此我们现在将重点关注 DNMT3b 调控基因]。 Illumina Infinium Human Mmethylation27 BeadChip 阵列将用于比较过表达 DNMT3b 的转化 HBEC 与亲本 HBEC 或具有正常 DNMT3b 背景的转化 HBEC，以鉴定受 DNMT3b 调节的甲基化基因。使用 Ilumina 平台和 ChIP 芯片的定制平铺 miR 启动子阵列的下一代测序将用于评估与具有正常 DNMT3b 表达的转化 HBEC 和亲本 HBEC 相比，过度表达 DNMT3b 的转化 HBEC 中存在的 miR 中染色质标记的分布。将在 100 个原发性腺癌样本和 17 个肺肿瘤衍生细胞系中测定 8-10 个 DNMT3b 调节基因和 2-3 个 miR 的启动子甲基化状态。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来探究表观基因组，通过下一代测序来探究微小RNA组，并通过ChIP分析来探究染色质重塑。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短期课程和一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。