Mechanisms of DNMT3B-mediated DNA Methylation Maintenance

DNMT3B 介导的 DNA 甲基化维持机制

• 批准号: 10652551
• 负责人: Bailey Michelle Tibben
• 金额: $ 2.11万
• 依托单位: VAN ANDEL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-08-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10652551
• 关键词: Aberrant DNA Methylation; Area; Binding; Biochemical; Body Regions; CRISPR/Cas technology; Cancer Etiology; Cancer cell line; Cell Differentiation process; Cells; Centromere; Chemicals; Chromatin; Complement; Complex; DNA; DNA Binding; DNA Methylation; DNA Modification Methylases; DNA biosynthesis; DNMT3B gene; DNMT3a; Data; Deposition; Development; Elongation Factor; Embryo; Embryonic Development; Enzymes; Epigenetic Process; Face; Fellowship; Gene Expression; Gene Expression Regulation; Gene Mutation; Gene Silencing; Genes; Genetic; Genetic Diseases; Genetic Engineering; Genetic Transcription; Germ cell tumor; Goals; HCT116 Cells; Histone H3; Histones; Human; Hypermethylation; Immunologic Deficiency Syndromes; Intercistronic Region; Knock-out; Lead; Literature; Lysine; Maintenance; Malignant Neoplasms; Measures; Mediating; Methylation; Methyltransferase; Modeling; Mutation; Pattern; Post-Translational Protein Processing; Process; RNA Polymerase II; Regulation; Reporting; Research; Role; Site; Somatic Cell; Syndrome; Tail; Testing; The Cancer Genome Atlas; Transcription Elongation; Tumor Cell Line; cancer cell; cancer genetics; cancer genome; cancer type; cell type; clinically relevant; colon cancer cell line; defined contribution; experimental study; functional outcomes; genetic manipulation; genome editing; inhibitor; insight; methylation pattern; mutant; oncohistone; promoter; protein protein interaction; recruit; self-renewal; stem cells; tool; transcriptome sequencing; tumor

PROJECT SUMMARY DNA methylation (5mC) is catalyzed by three DNA methyltransferases (DNMTs): DNMT1, DNMT3A, and DNMT3B. DNMT3A and 3B are often referred to as de novo DNMTs, functioning to establish 5mC patterns during early embryonic development. DNMT1 is referred to as the maintenance methyltransferase, functioning to copy established 5mC patterns on newly replicated DNA in differentiated and self-renewing stem cells. Despite this simplified division of labor, DNMT3A and 3B also contribute to 5mC maintenance, though the regulatory mechanisms involved are incompletely understood. A key 5mC regulatory mechanism is the interaction between DNMTs and histone post-translational modifications (PTMs). For example, lysine methylation on H3K4 and H3K36 has been reported to repel or recruit DNMT3B binding, respectively. The functional outcomes of these interactions aid in the regulation of gene expression and transcription fidelity. Specifically, DNMT3B-mediated methylation of actively transcribed gene bodies was shown to protect against spurious transcription, and this function is dependent on tri-methylation of H3K36 (H3K36me3). However, my preliminary data, as well as data from prior literature, show that H3K36me3 is dispensable for gene body 5mC maintenance. These results challenge the established model and suggest that there are alternative mechanisms which regulate DNMT3B-mediated 5mC at intragenic regions. The overarching goal of this fellowship proposal is to define mechanisms of DNMT3B recruitment and activity in gene bodies of embryonic and differentiated cells. In addition to histone H3, DNMT3B was reported to directly interact with the transcription elongation-associated hPAF1C complex in embryonic development. Further, hPAF1C components are required for deposition of H3K36me3 across gene bodies, and hPAF1C mutation results in aberrant expression of genes silenced by DNA hypermethylation. Taken together, these data lead me to hypothesize that DNMT3B is recruited to chromatin through multivalent engagement of the N-terminus of histone H3 and hPAF1C to establish and maintain gene body 5mC. This hypothesis will be tested with two Specific Aims. In Aim 1, I will utilize oncohistone mutations, CRISPR/Cas9 genome editing, and genetic complementation experiments to rigorously define the contribution of H3K4 and H3K36 methylation in the regulation of DNMT3B-mediated 5mC maintenance. Additionally, I will use publicly available cancer genome data to understand the clinical relevance of DNMT3B- mediated gene body 5mC. In Aim 2, I will use chemical inhibitors of transcription elongation, protein-protein interaction analysis, and deep RNA-sequencing to investigate the interaction between DNMT3B and hPAF1C and its role in co-transcriptional deposition of 5mC in gene bodies. Collectively, these studies will lead to a deeper understanding of mechanisms of epigenetic and transcriptional crosstalk that contribute to the propagation of aberrant 5mC patterning in human cancers.

项目摘要 DNA甲基化（5 mC）由三种DNA甲基转移酶（DNMT）催化：DNMT 1，DNMT 3A和DNMT 3B。 DNMT 3B。DNMT 3A和3B通常被称为从头DNMT，用于建立5 mC模式 在早期胚胎发育过程中。DNMT 1被称为维持甲基转移酶， 在分化和自我更新的干细胞中，在新复制的DNA上复制已建立的5 mC模式。 尽管这种简化的劳动分工，DNMT 3A和3B也有助于5 mC的维护，虽然 所涉及的调节机制还不完全清楚。一个关键的5 mC调节机制是 DNMT和组蛋白翻译后修饰（PTM）之间的相互作用。例如赖氨酸 据报道，H3 K4和H3 K36上的甲基化分别排斥或募集DNMT 3B结合。的 这些相互作用的功能结果有助于调节基因表达和转录保真度。 具体地说，DNMT 3B介导的活跃转录基因体的甲基化显示出保护免受 假转录，并且该功能依赖于H3 K36的三甲基化（H3 K36 me 3）。不过我 初步数据以及来自先前文献的数据显示，H3 K36 me 3与基因体5 mC无关 上维护这些结果对已建立的模型提出了挑战，并建议存在替代方案。 在基因内区域调节DNMT 3B介导的5 mC的机制。这个项目的首要目标是 奖学金的建议是确定DNMT 3B的招聘和胚胎细胞基因体的活性机制， 和分化的细胞。除组蛋白H3外，据报道DNMT 3B直接与组蛋白H3相互作用。 转录延长相关hPAF 1C复合物在胚胎发育中的作用。此外，hPAF 1C H3 K36 me 3跨基因体沉积所需的组分，hPAF 1C突变导致 基因的异常表达被DNA超甲基化沉默。综合起来，这些数据让我 假设DNMT 3B通过多价接合的N-末端被募集到染色质， 组蛋白H3和hPAF 1C共同参与基因体5 mC的建立和维持。这一假设将用两个 具体目标。在目标1中，我将利用癌组蛋白突变，CRISPR/Cas9基因组编辑和遗传学， 互补实验，以严格定义H3 K4和H3 K36甲基化在H3 K36甲基化中的贡献。 DNMT 3B介导的5 mC维持的调节。另外，我将使用公开的癌症基因组 数据以了解DNMT 3B介导的基因体5 mC的临床相关性。在目标2中，我将使用化学 转录延伸抑制剂、蛋白质-蛋白质相互作用分析和深度RNA测序， 研究DNMT 3B和hPAF 1C之间的相互作用及其在5 mC共转录沉积中的作用 在基因体中。总的来说，这些研究将导致对表观遗传机制的更深入理解。 和转录串扰，其有助于人类癌症中异常5 mC模式的传播。