The Role of DNMT3B in the DNA Methylation of Cancer Cells

DNMT3B 在癌细胞 DNA 甲基化中的作用

基本信息

批准号：
7458216
负责人：
LUCY Ann GODLEY
金额：
$ 30.63万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2013-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7458216
关键词：
Address Affect Aging Binding Biological Assay Breast Cancer Cell Cancer Model Catalytic Domain Cell Line Cell physiology Cells Characteristics Chromatin Chromatin Structure Chromosomes Co-Immunoprecipitations DNA DNA Methylation DNA Methyltransferase DNA Modification Methylases DNA Sequence DNMT3B gene Development Embryo Embryonic Development Enzymes Epigenetic Process Exhibits Gene Expression Genes Genetic Transcription Genomic Imprinting Goals Hematopoietic Neoplasms Histone Deacetylase Inhibitor Histones Human Immunofluorescence Immunologic In Situ Hybridization Kidney Knowledge Laboratories Learning Length Location Malignant Neoplasms Measures Mediating Methylation Modeling Modification Molecular Molecular Profiling Mus Mutation Neuroblastoma Nonsense Codon Normal Cell Pathway interactions Pattern Phenotype Physiological Process Protein Isoforms Proteins Public Health RNA Splicing Reaction Reverse Transcriptase Polymerase Chain Reaction Role Sampling Structure Testing Therapeutic Transcript Transgenes Transgenic Mice Tumor Cell Line Tumor Suppressor Genes Work X Inactivation Yeasts Zinc Fingers base cancer cell cell growth human DNMT3B protein interest leukemia novel novel diagnostics nucleophosmin programs promoter research study small hairpin RNA tissue/cell culture transgene expression tumorigenesis yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Epigenetic changes, such as DNA methylation and histone modifications, alter chromatin structure and regulate gene transcription. Cancer cells are characterized by abnormal DNA methylation: Repetitive DNA sequences and some gene promoters are hypomethylated and transcriptionally active, whereas many tumor suppressor gene promoters are hypermethylated and transcriptionally inactive without the presence of mutations. My laboratory discovered that cancer cells exhibit aberrant splicing of the DNMT3B gene, which encodes one of the three DNA methyltransferases. The aberrant splicing produces DNMT3B transcripts containing premature stop codons and encoding truncated proteins lacking the catalytic domain. Tissue culture cells expressing DNMT3B7, the most frequently observed aberrant DNMT3B transcript in cancer cells, show DNA methylation changes that correlate with altered gene expression. Furthermore, transgenic mice that express DNMT3B7 display disrupted embryonic development and changes in DNA methylation that are dependent on DNMT3B7 transgene levels. We hypothesize that truncated DNMT3B proteins influence DNA methylation in cancer cells, and we propose to test this idea using three Specific Aims: (1) To examine the effect of DNMT3B7 on mouse development by: (A) determining the pattern of DNMT3B7 transgene expression within embryos by in situ hybridization; and (B) examining the effects of DNMT3B7 transgene expression on DNA methylation, histone modifications, and gene expression; (2) To study the effect of DNMT3B7 expression on the DNA methylation patterns and phenotypes of cancer cells by: (A) inhibiting DNMT3B7 expression in breast cancer cells via shRNA and examining the effects on DNA methylation; (B) correlating DNMT3B7 expression with particular phenotypes in two distinctive types of neuroblastoma cell lines; and (C) quantifying DNMT3B7 levels in primary leukemia samples and correlating those with DNA methylation levels; and (3) To determine how DNMT3B7 could alter DNA methylation by: (A) testing the predictions of our models, and (B) further characterizing the interactions between DNMT3B/DNMT3B7 and three exciting interacting proteins, ZNF445, CHTF18, and NPM. Our proposed studies address the mechanism by which epigenetic alterations originate within cancer cells. The knowledge gained from the proposed work is likely to provide a basis for novel diagnostic and therapeutic strategies applicable to virtually all forms of cancer. Moreover, the pathways found to mediate the effects of DNMT3B7 are likely to reveal paradigms common to other processes that use DNA methylation to control gene expression. PUBLIC HEALTH RELEVANCE: The DNA within a cell can be modified by methylation to alter its structure and affect gene expression. DNA methylation is involved in many normal cellular processes and is abnormally distributed in cancer cells, leading to some of the phenotypes of cancer cells. The mechanism by which cancer cells acquire and maintain abnormal DNA methylation is not understood. We have discovered that cancer cells express shortened forms of DNMT3B, one of the enzymes that carries out the DNA methylation reaction, and we hypothesize that truncated DNMT3B proteins contribute to abnormal DNA methylation patterns in cancer cells. The knowledge gained from the proposed experiments is likely to provide a basis for novel diagnostic and therapeutic strategies that will be applicable to virtually all forms of cancer. Furthermore, the cellular pathways found to mediate the effects of truncated DNMT3B proteins are likely to reveal paradigms common to other processes that involve DNA methylation, such as mammalian embryonic development, X-chromosome inactivation, genomic imprinting, and aging.

描述（由申请人提供）：表观遗传变化，例如DNA甲基化和组蛋白修饰，改变染色质结构并调节基因转录。癌细胞的特征是异常的DNA甲基化：重复的DNA序列，某些基因启动子是低甲基化和转录活性的，而许多肿瘤抑制基因启动子是高甲基化的，并且在不存在突变的情况下进行了转录非活性。我的实验室发现，癌细胞表现出DNMT3B基因的异常剪接，该基因编码了三种DNA甲基转移酶之一。异常的剪接产生包含过早停止密码子的DNMT3B转录本，并编码缺乏催化结构域的截短蛋白质。表达DNMT3B7的组织培养细胞是癌细胞中最常观察到的异常DNMT3B转录物，显示出与基因表达改变的DNA甲基化变化。此外，表达DNMT3B7的转基因小鼠表现出破坏的胚胎发育和DNA甲基化的变化，这取决于DNMT3B7转基因水平。我们假设截断的DNMT3B蛋白会影响癌细胞中的DNA甲基化，我们建议使用三个特定目的来测试这一想法：（1）检查DNMT3B7对小鼠发育的影响：（a）确定DNMT3B7的胚胎在胚胎中的模式，通过现场通过现场杂交胚胎中的胚胎表达; （b）检查DNMT3B7转基因表达对DNA甲基化，组蛋白修饰和基因表达的影响；（2）通过以下方法研究DNMT3B7表达对癌细胞DNA甲基化模式和表型的影响：（b）将DNMT3B7表达与两种独特类型的神经母细胞瘤细胞系中的特定表型相关联；（c）量化原发性白血病样品中的DNMT3B7水平，并与DNA甲基化水平相关；（3）为了确定DNMT3B7如何通过：（a）测试我们的模型预测，以及（b）进一步表征DNMT3B/DNMT3B7之间的相互作用和三种令人兴奋的相互作用蛋白，Znf445，CHTF18和NPM。我们提出的研究探讨了表观遗传改变起源于癌细胞内的机制。从拟议的工作中获得的知识很可能为适用于几乎所有形式的癌症的新型诊断和治疗策略提供了基础。此外，发现介导DNMT3B7影响的途径可能揭示了其他使用DNA甲基化来控制基因表达的过程常见的范例。公共卫生相关性：细胞内的DNA可以通过甲基化来改变其结构并影响基因表达。 DNA甲基化参与许多正常细胞过程，并异常分布在癌细胞中，导致癌细胞的某些表型。癌细胞获得和维持异常DNA甲基化的机制尚不清楚。我们发现癌细胞表达缩短的DNMT3B形式，DNMT3B是进行DNA甲基化反应的酶之一，我们假设截短的DNMT3B蛋白截断有助于癌细胞中异常的DNA甲基化模式。从拟议的实验中获得的知识很可能为新颖的诊断和治疗策略提供了基础，这些策略将适用于几乎所有形式的癌症。此外，发现介导截短的DNMT3B蛋白的作用的细胞途径可能揭示了涉及DNA甲基化的其他过程常见的范式，例如哺乳动物胚胎发育，X-Chromosoms Inactivation，X-Chromoson Inactivation，Genomic Princomic Inglinting和Agining。