The Role of DNMT3B in the DNA Methylation of Cancer Cells

DNMT3B 在癌细胞 DNA 甲基化中的作用

• 批准号: 8223291
• 负责人: LUCY Ann GODLEY
• 金额: $ 35.02万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 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/8223291
• 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; Cultured Tumor Cells; DNA; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA Sequence; DNMT3B gene; Embryo; Embryonic Development; Enzymes; Epigenetic Process; Exhibits; Gene Expression; Genes; Genetic Transcription; Genomic Imprinting; Goals; Hematopoietic Neoplasms; Histone Deacetylase Inhibitor; Human; Immunofluorescence Immunologic; In Situ Hybridization; Kidney; Knowledge; Laboratories; Learning; Length; Location; Malignant Neoplasms; Measures; Mediating; Methylation; Modeling; Molecular; Molecular Profiling; Mus; Mutation; Nonsense Codon; Normal Cell; Pathway interactions; Pattern; Phenotype; Physiological; Process; Protein Isoforms; Proteins; RNA Splicing; Reaction; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Structure; Testing; Transcript; Transgenes; Transgenic Mice; Tumor Cell Line; Tumor Suppressor Genes; Work; X Inactivation; Yeasts; Zinc Fingers; abstracting; base; cancer cell; cell growth; histone modification; human DNMT3B protein; interest; leukemia; mouse development; neuroblastoma cell; novel; novel diagnostics; novel therapeutics; nucleophosmin; programs; promoter; research study; small hairpin RNA; tissue/cell culture; transgene expression; tumorigenesis; yeast two hybrid system

Abstract 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. Project Narrative 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.

摘要 表观遗传变化，如DNA甲基化和组蛋白修饰，改变染色质结构和 调节基因转录。癌细胞的特征是DNA甲基化异常：重复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 抑制基因启动子在没有存在的情况下是高度甲基化和转录无效的 突变。我的实验室发现癌细胞表现出Dnmt3b基因的异常剪接，这是 编码三种DNA甲基转移酶之一。异常剪接产生Dnmt3b转录本 含有过早终止密码子，编码缺乏催化结构域的截短蛋白质。组织 培养表达DNMT3B7的细胞，DNMT3B7是癌细胞中最常见的异常DNMT3B转录本， 显示与基因表达变化相关的DNA甲基化变化。此外，转基因小鼠 表达DNMT3B7表现出扰乱胚胎发育和DNA甲基化的变化 依赖于DNMT3B7转基因水平。 我们假设截短的Dnmt3b蛋白影响癌细胞中的DNA甲基化，并且我们 建议通过三个具体目标来验证这一想法：(1)检测DNMT3B7对小鼠的影响 通过以下方式发育：(A)原位测定DNMT3B7转基因在胚胎中的表达模式 以及(B)检测DNMT3B7转基因表达对DNA甲基化、组蛋白的影响 (2)研究DNMT3B7表达对DNA甲基化的影响 (A)通过以下途径抑制DNMT3B7在乳腺癌细胞中的表达 ShRNA和检测对DNA甲基化的影响；(B)DNMT3B7的表达与特定的 两种不同类型的神经母细胞瘤细胞系的表型；以及(C)对原代细胞中DNMT3B7水平的量化 白血病样本及其与DNA甲基化水平的相关性；以及(3)确定DNMT3B7如何 可以通过以下方式改变DNA甲基化：(A)测试我们模型的预测，以及(B)进一步表征 DNMT3b/DNMT3B7与ZNF445、CHTF18和NPM三种激动型相互作用蛋白的相互作用 我们提出的研究解决了表观遗传改变在癌症中起源的机制。 细胞。从拟议的工作中获得的知识很可能为新的诊断和 治疗策略适用于几乎所有形式的癌症。此外，发现的调解 DNMT3B7的影响可能揭示出其他使用DNA甲基化来 控制基因表达。项目叙事 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甲基化参与了许多正常的细胞过程，并在癌细胞中异常分布， 导致了癌细胞的某些表型。癌细胞获得和转移的机制 维持异常的DNA甲基化还不清楚。我们已经发现癌细胞表达 Dnmt3b的缩写形式，它是执行DNA甲基化反应的酶之一，我们 假设截短的Dnmt3b蛋白导致癌细胞中异常的DNA甲基化模式。 从拟议的实验中获得的知识很可能为新的诊断和 治疗策略将适用于几乎所有形式的癌症。此外，细胞通路 被发现介导截短的Dnmt3b蛋白的影响可能揭示出与其他 涉及DNA甲基化的过程，如哺乳动物胚胎发育、X染色体 失活、基因组印记和老化。

项目成果

Recurrent somatic TET2 mutations in normal elderly individuals with clonal hematopoiesis.

• DOI: 10.1038/ng.2413
• 发表时间: 2012-11
• 期刊: Nature genetics
• 影响因子: 30.8
• 作者: Busque L;Patel JP;Figueroa ME;Vasanthakumar A;Provost S;Hamilou Z;Mollica L;Li J;Viale A;Heguy A;Hassimi M;Socci N;Bhatt PK;Gonen M;Mason CE;Melnick A;Godley LA;Brennan CW;Abdel-Wahab O;Levine RL
• 通讯作者: Levine RL

DNMT3B7, a truncated DNMT3B isoform expressed in human tumors, disrupts embryonic development and accelerates lymphomagenesis.

• DOI: 10.1158/0008-5472.can-10-0847
• 发表时间: 2010-07-15
• 期刊: Cancer research
• 影响因子: 11.2
• 作者: Shah MY;Vasanthakumar A;Barnes NY;Figueroa ME;Kamp A;Hendrick C;Ostler KR;Davis EM;Lin S;Anastasi J;Le Beau MM;Moskowitz IP;Melnick A;Pytel P;Godley LA
• 通讯作者: Godley LA

Epigenetic Control of Apolipoprotein E Expression Mediates Gender-Specific Hematopoietic Regulation.

载脂蛋白 E 表达的表观遗传控制介导性别特异性造血调节。

• DOI: 10.1002/stem.2214
• 发表时间: 2015
• 期刊: Stem cells (Dayton, Ohio)
• 作者: Vasanthakumar,Aparna;Zullow,Hayley;Lepore,JanetB;Thomas,Kenya;Young,Natalie;Anastasi,John;Reardon,CatherineA;Godley,LucyA
• 通讯作者: Godley,LucyA

Hydroxymethylation at gene regulatory regions directs stem/early progenitor cell commitment during erythropoiesis.

• DOI: 10.1016/j.celrep.2013.11.044
• 发表时间: 2014-01-16
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Madzo J;Liu H;Rodriguez A;Vasanthakumar A;Sundaravel S;Caces DBD;Looney TJ;Zhang L;Lepore JB;Macrae T;Duszynski R;Shih AH;Song CX;Yu M;Yu Y;Grossman R;Raumann B;Verma A;He C;Levine RL;Lavelle D;Lahn BT;Wickrema A;Godley LA
• 通讯作者: Godley LA