Oxidation of 5-methylcytosine: DNA damage and epigenetic reprogramming

5-甲基胞嘧啶的氧化：DNA 损伤和表观遗传重编程

基本信息

批准号：
8845531
负责人：
Lawrence C Sowers
金额：
$ 31.89万
依托单位：
UNIVERSITY OF TEXAS MED BR GALVESTON
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-01 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8845531
关键词：
Affect Affinity Base Excision Repairs Binding Binding Proteins Biology Cancer Etiology Cancer cell line Cells Chemicals Cultured Cells DNA DNA Damage DNA Maintenance DNA Modification Methylases DNA Modification Process DNA Repair DNA Sequence Alteration DNA biosynthesis DNA-Protein Interaction Data Deamination Digestion Electrophoretic Mobility Shift Assay Epigenetic Process Excision Gel Gene Expression Genetic Transcription High Pressure Liquid Chromatography Human Hydroxylation Incubated Isotopes Label Light Mass Spectrum Analysis Measures Mediating Methionine Methods Methylation Methyltransferase Modification Monitor Mus Mutation Oligonucleotide Microarrays Oligonucleotides Organism Pathway interactions Pattern Positioning Attribute Process Protein Binding Proteins Pyrimidine Pyrimidines Radio Regenerative Medicine Relative (related person)Series Signal Transduction Specificity Stable Isotope Labeling Stem cells Testing Uridine Work base cancer cell cancer stem cell cancer therapy cell type chemotherapy demethylation detector embryonic stem cell established cell line innovation insight nerve stem cell novel novel strategies oxidation oxidative damage public health relevance repaired stable isotope stem cell differentiation stem cell fate

Affect Affinity Base Excision Repairs Binding Binding Proteins Biology Cancer Etiology Cancer cell line Cells Chemicals Cultured Cells DNA DNA Damage DNA Maintenance DNA Modification Methylases DNA Modification Process DNA Repair DNA Sequence Alteration DNA biosynthesis DNA-Protein Interaction Data Deamination Digestion Electrophoretic Mobility Shift Assay Epigenetic Process Excision Gel Gene Expression Genetic Transcription High Pressure Liquid Chromatography Human Hydroxylation Incubated Isotopes Label Light Mass Spectrum Analysis Measures Mediating Methionine Methods Methylation Methyltransferase Modification Monitor Mus Mutation Oligonucleotide Microarrays Oligonucleotides Organism Pathway interactions Pattern Positioning Attribute Process Protein Binding Proteins Pyrimidine Pyrimidines Radio Regenerative Medicine Relative (related person)Series Signal Transduction Specificity Stable Isotope Labeling Stem cells Testing Uridine Work base cancer cell cancer stem cell cancer therapy cell type chemotherapy demethylation detector embryonic stem cell established cell line innovation insight nerve stem cell novel novel strategies oxidation oxidative damage public health relevance repaired stable isotope stem cell differentiation stem cell fate

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项目摘要

DESCRIPTION (provided by applicant): The DNA of all organisms undergoes persistent damage that can result in genetic mutations or epigenetic changes unless the damage is correctly repaired. The modified base, 5-methylcytosine (5mC), can undergo both deamination and oxidation resulting in both genetic mutations and epigenetic perturbation. We have demonstrated that the oxidation damage product, 5-hydroxymethylcytosine (5hmC), inhibits the binding of proteins that selectively bind to methylated DNA and that the maintenance methyltransferase DNMT1 does not recognize 5hmC when methylating DNA following DNA replication. Therefore, the formation of 5hmC could heritably alter epigenetic patterns in replicating cells. Emerging evidence indicates, however, that the conversion of 5mC to 5hmC could also be part of an enzymatic epigenetic reprogramming pathway essential for stem cell differentiation and that 5hmC is absent from most if not all human cancer cells. In this application, we propose a series of studies to further understand this putative DNA demethylation pathway in human neural stem cells, mouse embryonic stem cells and in a series of human cancer stem cells and established cell lines. We wish to understand how DNA demethylation relies upon DNA damage repair pathways, and how DNA damage repair and DNA demethylation might become entangled. In the first aim, we propose a novel stable isotope labeling method that will allow us to follow the dynamic processes of DNA replication, methylation and hydroxylation and excision of possible intermediates by the base excision repair pathway. In the second aim, we propose to use a series of innovative new methods to identify and quantify enzymatic activities that might act on pathway intermediates, further defining as yet unknown parts of this pathway. In the third aim, we propose some novel mass spectrometry approaches to identify proteins that bind to intermediates of the pathway and to measure how specific DNA intermediates, including 5hmC, affect the specificity and magnitude of the DNA-protein interactions. The proposed studies will allow examination of this important demethylation pathway at an unprecedented level. The information obtained is essential for understanding the biology of normal stem cells within the context of regenerative medicine, and understanding how the pathway becomes defective in human cancer cells could provide new insights into novel targeted chemotherapy.

描述（由申请人提供）：所有生物体的DNA遭受持续损害，除非正确修复损害，否则可能导致基因突变或表观遗传变化。改良的基碱（5MC）可以同时进行脱氨酸和氧化，从而导致遗传突变和表观遗传扰动。我们已经证明，氧化损伤产物，5-羟基环霉素（5HMC），抑制了选择性结合与甲基化DNA的蛋白质的结合，并且在DNA重复后甲基化DNA时，维持甲基转移酶DNMT1识别5HMC。因此，5HMC的形成可能可以遗传地改变复制细胞中的表观遗传模式。然而，新兴的证据表明，5MC向5HMC的转化也可能是干细胞分化必不可少的酶学表观遗传重编程途径的一部分，并且大多数人（如果不是全部）人类癌细胞不存在5HMC。在此应用中，我们提出了一系列研究，以进一步了解人类神经干细胞，小鼠胚胎干细胞以及一系列人类癌症干细胞和已建立的细胞系中的这种假定的DNA去甲基化途径。我们希望了解DNA脱甲基化如何依赖DNA损伤修复途径，以及DNA损伤修复和DNA脱甲基化如何纠缠。在第一个目标中，我们提出了一种新型的同位素标记方法，该方法将使我们能够遵循DNA复制，甲基化和羟基化的动态过程，并通过基础切除修复途径对可能的中间体进行切除。在第二个目标中，我们建议使用一系列创新的新方法来识别和量化可能作用于途径中间体的酶活性，进一步定义该途径的未知部分。在第三个目标中，我们提出了一些新型的质谱方法，以鉴定与途径中间体结合的蛋白质，并测量包括5HMC在内的特定DNA中间体如何影响DNA蛋白相互作用的特异性和幅度。拟议的研究将允许在前所未有的水平上检查这一重要的脱甲基化途径。获得的信息对于在再生医学的背景下了解正常干细胞的生物学至关重要，并且了解该途径在人类癌细胞中如何有缺陷可以为新的靶向化疗提供新的见解。