Dissecting Gene Regulatory Roles of TET Enzymes and 5-hydroxymethylcytosine in Mammalian Active DNA Demethylation

解析 TET​​ 酶和 5-羟甲基胞嘧啶在哺乳动物活性 DNA 去甲基化中的基因调控作用

• 批准号: 10413217
• 负责人: Alex Tianjiun Wei
• 金额: $ 2.62万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2023-02-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10413217
• 关键词: Base Excision Repairs; Binding; Biological Process; Cell Lineage; Cell division; Cell physiology; Cells; Chromatin; Chromatin Structure; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Comprehension; Coupled; Cytosine; DNA; DNA Methylation; DNA Modification Process; DNA biosynthesis; Deposition; Development; Dioxygenases; Disease; ES Cell Line; Enzymes; Epigenetic Process; Etiology; Excision Repair; Exhibits; Family; Foundations; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Genetically Engineered Mouse; Genomic DNA; Human; Inherited; Kinetics; Knock-out; Knockout Mice; Mammalian Cell; Mammals; Measures; Mediating; Methods; Methylation; Methyltransferase; Mitotic; Modification; Mus; Natural regeneration; Nucleosomes; Oxides; Pathology; Pathway interactions; Physiologic pulse; Play; Positioning Attribute; Process; Proliferating; Protein translocation; Regulator Genes; Regulatory Element; Resolution; Role; Sampling; Somatic Cell; Structure of primordial sex cell; System; Technology; Testing; Thymine DNA Glycosylase; Time; Transcriptional Activation; Transcriptional Regulation; Variant; Work; base; bisulfite; bisulfite sequencing; blastocyst; chromatin protein; chromatin remodeling; demethylation; embryonic stem cell; epigenome editing; established cell line; experimental study; genome-wide; genome-wide analysis; human disease; in vivo; insight; mammalian genome; novel; novel therapeutics; nucleobase; overexpression; oxidation; pluripotency; promoter; recruit; restoration; targeted treatment; transcription factor; tumorigenesis

Project Summary Epigenetic modifications have important roles in cellular functions and in specialization of cell lineages. On DNA, epigenetic modification occurs on the 5-position of cytosine nucleobases. The most common modification is 5- methylcytosine (5mC), and TET-Eleven-Translocation (TET) proteins enzymatically remove DNA methylation by iteratively oxidizing 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). In mammals, TET-mediated active DNA demethylation can be achieved by replication-dependent dilution of 5hmC, or thymine DNA glycosylase (TDG) mediated base excision repair (BER) of 5fC/5caC to regenerate unmodified C. However, the functional significance of these oxidized modifications (ox-mC) and mechanistically distinct active DNA demethylation pathways remains poorly understood, precluding our comprehension on how dysregulated DNA methylation contributes to disease. Indeed, ox-mC has been implicated in crucial biological processes such as gene transcription. However, it has been challenging to ascribe functional roles to ox-mC as it was technically challenging to decouple generation of 5hmC from 5fC/5caC, and unmodified C. Elucidating functional roles of ox-mC will establish a foundational understanding for developing novel therapeutics for various pathologies. To this end, I developed a CRISPR/dCas9 platform that recruits 5hmC-stalling TET-variants to interrogate gene regulatory roles of 5hmC, 5fC/5caC, and TDG/BER in mammalian systems. Preliminary results generated from comprehensive epigenetic sequencing (bisulfite sequencing (BS- Seq)/Bisulfite-assisted APOBEC-Coupled Epigenetic sequencing (bACE-Seq)/Methylase-Assisted Bisulfite sequencing (MAB-Seq)) revealed 5hmC alone could not reactivate a hypermethylated gene promoter in proliferative human cells, and generation of 5fC/5caC was requisite. These results show for the first time, functional distinction between ox-mC. It remains ambiguous how downstream higher ox-mC pathways could reactivate gene expression. I hypothesize 5fC/5caC deposition depletes nucleosome occupancy to facilitate transcription. In aim 1, I will evaluate the role of 5hmC/5fC/5caC/C and TDG in Tet1-3 triple knock out (TKO) and Tet1-3/Tdg quadruple knockout (QKO) mouse embryonic stem cells (mESCs), on gene expression and local chromatin structure. My results also reveal 5hmC alone could not restore unmodified C by replication-dependent dilution of 5hmC suggesting this mechanism of active DNA demethylation is more tightly regulated than previously anticipated. Quantitative genome-wide analysis of how ox-mC bases is mitotically inherited across division is currently lacking. I hypothesize 5hmC is mitotically inherited to nascent strands, while 5fC/5caC is rapidly removed by TDG/BER. In aim 2, I will develop technologies to quantify and profile ox-mC mitotic inheritance at single-base resolution in genetically engineered mESCs. By completing the proposed aims, I will afford unprecedented insight into active DNA demethylation pathways and functional roles of ox-mC that will contribute to our understanding of disease inception.

项目总结