DNA hydroxymethylation and post stroke brain damage

DNA羟甲基化与中风后脑损伤

• 批准号: 10001037
• 负责人: Raghu VEMUGANTI
• 金额: $ 41.54万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001037
• 关键词: Adult; Affect; Affinity; Age; Antioxidants; Astrocytes; Azacitidine; Binding; Brain; Brain Injuries; Cell Survival; Chromatin; Chromatin Structure; CpG dinucleotide; Cytosine; DNA; DNA Binding Domain; DNA Methylation; DNA Modification Methylases; Data; Disease; Epigenetic Process; Female; Functional disorder; Gene Expression; Genes; Genetic Transcription; Genomics; Goals; High-Throughput Nucleotide Sequencing; Histones; Infarction; Ischemia; Ischemic Brain Injury; Knock-out; Location; Mammals; Maps; Massive Parallel Sequencing; Mediating; Methods; Methylation; Mixed Function Oxygenases; Modification; Molecular; Mus; Neurologic Dysfunctions; Neurons; Organ; Outcome; Oxides; Pathology; Play; Protein Isoforms; Proteins; RNA; RNA purification; Recovery of Function; Rodent; Rodent Model; Role; Site; Structure; Testing; Untranslated RNA; Zebularine; ascorbate; chromatin immunoprecipitation; derepression; functional outcomes; gain of function; inhibitor/antagonist; interest; knock-down; loss of function; male; mortality; neuronal survival; neuroprotection; novel therapeutics; post stroke; prevent; recruit; scaffold; sex; stroke outcome

Epigenetic changes in DNA and histones are known to significantly influence the gene expression and outcome after many diseases. The role of epigenetics in ischemic brain damage is not yet fully understood. Of particular interest, the cytosine in DNA undergoes methylation to form 5-methylcytosine (5mC) which is known to be a transcriptional silencer. Recent studies showed that 5mC will be oxidized by ten-eleven translocation (TET) hydroxylases to form 5-hydroxymethylcytosine (5hmC). This epigenetic change is considered as a transcriptional derepression mark that increases cell survival under adverse conditions. In particular, brain contains ~10 fold higher 5hmC levels than other organs of the body. Preliminary studies showed that transient focal ischemia in adult rodents significantly increase the genomic 5hmC levels in the peri-infarct cortex. TET3 knockdown decreased 5hmC levels, and exacerbated post-ischemic mortality and infarction in both male and female mice. On the other hand, increasing 5hmC levels by treatment with ascorbate (a TET inducer) significantly protected the brain after focal ischemia in a TET3-dependent manner. Hence, we hypothesize that “Tet3 mediated induction of 5hmC is a neuroprotective adaptation that can be potentiated to protect brain after stroke.” The major neuronal isoform of TET3 lacks DNA binding domains. Our preliminary data show that TET3 binds to lncRNAs with high affinity. The lncRNAs are known to act as scaffolds to bring DNA/RNA/protein together enabling their action. LncRNAs are also known to modulate post-stroke outcome. Hence, we further hypothesize that “lncRNAs play a vital role in scaffolding and guiding TET3 to specific genomic sites, and thus modulate 5hmC levels and functional outcome after stroke.” Aim 1: To evaluate if DNA hydroxymethylation is neuroprotective after stroke. We will test the functional significance of 5hmC in post-stroke pathophysiology by loss of function and gain of function of TET3. Genomic sites where 5hmC is increased after stroke will be mapped by chromatin immunoprecipitation combined with massively parallel DNA sequencing (ChIP-seq) following TET3 knockdown and induction. Aim 2: To study if lncRNAs regulate TET3-mediated DNA hydroxymethylation and the ensuing neuroprotection after stroke. We will determine the genomic locations modulated by the TET3-interacting lncRNAs by high throughput sequencing method chromatin isolation by RNA purification (ChiRP-seq). We will further study if lncRNA function is essential for TET3/5hmC mediated neuroprotection after stroke. The long-term goal is to define the role of 5hmC in post-ischemic pathology and to test if increasing 5hmC levels is beneficial after stroke.

已知DNA和组蛋白的表观遗传变化会显着影响基因表达和结果 经过多种疾病。表观遗传学在缺血性脑损伤中的作用尚未完全理解。特别 兴趣，DNA中的胞嘧啶经历甲基化以形成5-甲基环肽（5MC），已知为一种 转录消音器。最近的研究表明，5MC将被十个易位（TET）氧化 羟基酶形成5-羟基甲苯肽（5HMC）。这种表观遗传变化被认为是转录 消除标记，可在不良条件下增加细胞存活。特别是，大脑包含〜10倍 比人体其他器官高的5HMC水平更高。初步研究表明，短暂的局灶性缺血 成年啮齿动物显着提高了侵入周期皮层中的基因组5HMC水平。 TET3敲低 雄性和雌性小鼠的缺血后死亡率和梗塞加剧了5HMC的水平，并加剧了病后。 另一方面，通过用抗坏血酸（TET引起的TET）治疗可显着保护5HMC水平 局灶性缺血后的大脑以Tet3依赖性方式。因此，我们假设“ TET3介导 5HMC的诱导是一种神经保护适应，可以在中风后保护大脑。” TET3的主要神经元同工型缺乏DNA结合结构域。我们的初步数据表明TET3与 具有高亲和力的lncRNA。 lncrNA被称为脚手架，将DNA/RNA/蛋白融合在一起 实现他们的行动。 LNCRNA也已知可以调节势后结果。因此，我们进一步假设 “ lncrnas在脚手架和引导TET3中起着至关重要的作用，因此 调节中风后的5HMC水平和功能结果。” 目的1：评估中风后DNA羟甲基化是否为神经保护作用。我们将测试功能 5HMC通过功能丧失和TET3功能增长而在势后病理生理学中的重要性。基因组 中风后增加5HMC的位置将通过染色质免疫沉淀与 TET3敲低和诱导后，大规模平行的DNA测序（CHIP-SEQ）。 目的2：研究LNCRNA是否调节TET3介导的DNA羟基化和随后的神经保护作用 中风后。我们将确定由TET3相互作用LNCRNA调制的基因组位置 通过RNA纯化（CHIRP-SEQ）通过RNA纯化分离吞吐量测序方法。我们将进一步研究 LNCRNA功能对于中风后TET3/5HMC介导的神经保护至关重要。 长期目标是定义5HMC在缺血后病理学中的作用，并测试是否增加5HMC水平 中风后是有益的。