Mechanisms of DNMT3B-mediated DNA Methylation Maintenance

DNMT3B 介导的 DNA 甲基化维持机制

• 批准号: 10447002
• 负责人: Bailey Michelle Tibben
• 金额: $ 4.58万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10447002
• 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; Conflict (Psychology); DNA; DNA Binding; DNA Methylation; DNA Modification Methylases; 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; Recruitment Activity; 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.

项目总结 DNA甲基化(5mC)由三种DNA甲基转移酶(DNMT)催化：DNMT1、DNMT3A和 DNMT3b。DNMT3A和3B通常被称为新的DNMT，其功能是建立5mC模式 在早期胚胎发育过程中。DNMT1被称为维持甲基转移酶，起作用 在分化和自我更新的干细胞中复制新复制的DNA上已建立的5mC模式。 尽管简化了分工，DNMT3A和3B也为5mC的维护做出了贡献，尽管 其中涉及的监管机制还不完全清楚。一个关键的5mC监管机制是 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，H3K4和H3K36上的甲基化分别排斥或招募Dnmt3b结合。这个 这些相互作用的功能结果有助于基因表达和转录保真度的调节。 具体地说，Dnmt3b介导的活跃转录基因体的甲基化被证明对 虚假转录，这一功能依赖于H3K36(H3K36me3)的三甲基化。然而，我的 初步数据以及来自先前文献的数据表明，H3K36me3对于基因体5mC来说是必不可少的 维修。这些结果挑战了已建立的模型，并表明还有其他选择 基因内区调控Dnmt3b介导的5mC的机制。这件事的首要目标是 联谊会的建议是确定Dnmt3b在胚胎基因体中的招募和活性机制 和分化的细胞。除了组蛋白H3，Dnmt3b还被报道直接与组蛋白H3相互作用 胚胎发育中转录延伸相关的hPAF1C复合体。此外，hPAF1C H3K36me3跨基因体沉积需要成分，hPAF1C突变导致 因DNA高甲基化而沉默的基因异常表达。综上所述，这些数据让我发现 假设Dnmt3b是通过N端的多价结合而招募到染色质的 组蛋白H3和hPAF1C建立和维持基因体5mC。这一假设将通过两个例子进行检验 明确的目标。在目标1中，我将利用癌组蛋白突变、CRISPR/Cas9基因组编辑和基因 补充实验严格定义H3K4和H3K36甲基化在 Dnmt3b介导的5mC维持的调节。此外，我将使用公开的癌症基因组 资料了解Dnmt3b介导的基因体5mC的临床相关性。在《目标2》中，我将使用化学物质 转录延伸抑制物、蛋白质相互作用分析和深度RNA测序 Dnmt3b与hPAF1C的相互作用及其在5mC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层次的理解 以及转录串扰，这有助于在人类癌症中传播异常的5mC模式。