The Role of DNMT3B in the DNA Methylation of Cancer Cells

描述（由申请人提供）：表观遗传变化，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序列和一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和转录失活的，不存在突变。我的实验室发现，癌细胞表现出DNMT3B基因的异常剪接，该基因编码三种DNA甲基转移酶之一。异常剪接产生含有提前终止密码子和编码缺乏催化结构域的截短蛋白质的DNMT3B转录物。表达DNMT3B7（癌细胞中最常观察到的异常DNMT3B转录物）的组织培养细胞显示与改变的基因表达相关的DNA甲基化变化。此外，表达DNMT3B7的转基因小鼠显示出依赖于DNMT3B7转基因水平的胚胎发育中断和DNA甲基化的变化。我们假设截短的DNMT 3B蛋白影响癌细胞中的DNA甲基化，并且我们提出使用三个特定目的来测试这个想法：（1）通过以下方法来检测DNMT 3B7对小鼠发育的影响：（A）通过原位杂交确定DNMT 3B7转基因在胚胎中的表达模式;和（B）检测DNMT 3B7转基因表达对DNA甲基化、组蛋白修饰和基因表达的影响;（2）研究DNMT 3B7表达对乳腺癌细胞DNA甲基化模式和表型的影响：（A）通过shRNA抑制乳腺癌细胞DNMT 3B7表达，检测其对DNA甲基化的影响;（B）在两种不同类型的神经母细胞瘤细胞系中将DNMT 3 B7表达与特定表型相关联;和（C）定量原发性白血病样品中的DNMT 3 B7水平并将其与DNA甲基化水平相关联;和（3）通过以下方法确定DNMT 3 B7如何改变DNA甲基化：（A）测试我们的模型的预测，以及（B）进一步表征DNMT 3 B/DNMT 3 B7与三种激动剂相互作用蛋白ZNF 445、CHTF 18和NPM之间的相互作用。我们提出的研究解决了表观遗传改变起源于癌细胞的机制。从拟议的工作中获得的知识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基础。此外，发现的介导DNMT3B7作用的途径可能揭示了使用DNA甲基化控制基因表达的其他过程的共同范例。 公共卫生相关性：细胞内的DNA可以通过甲基化进行修饰，以改变其结构并影响基因表达。DNA甲基化参与许多正常的细胞过程，并在癌细胞中异常分布，导致癌细胞的一些表型。癌细胞获得和维持异常DNA甲基化的机制尚不清楚。我们已经发现癌细胞表达DNMT3B的缩短形式，DNMT3B是进行DNA甲基化反应的酶之一，我们假设截短的DNMT3B蛋白有助于癌细胞中异常的DNA甲基化模式。从拟议实验中获得的知识可能为新型诊断和治疗策略提供基础，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涉及DNA甲基化的其他过程的共同范例，例如哺乳动物胚胎发育，X染色体失活，基因组印记和衰老。

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.