DNA hydroxymethylation and post stroke brain damage

DNA羟甲基化与中风后脑损伤

基本信息

批准号：
9757829
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
项目类别：
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/9757829
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

项目摘要

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.

DNA 和组蛋白的表观遗传变化会显着影响基因表达和结果在许多疾病之后，表观遗传学在缺血性脑损伤中的作用尚未完全了解。感兴趣的是，DNA 中的胞嘧啶经历甲基化形成 5-甲基胞嘧啶 (5mC)，已知它是最近的研究表明，5mC 会被 10-11 易位（TET）氧化。羟化酶形成 5-羟甲基胞嘧啶 (5hmC)，这种表观遗传变化被认为是转录过程。去抑制标记可提高细胞在不利条件下的存活率，特别是大脑中含有约 10 倍。初步研究表明，5hmC 水平高于身体其他器官。成年啮齿类动物梗塞周围皮质中的基因组 5hmC 水平显着增加。 5hmC 水平下降，并加剧雄性和雌性小鼠的缺血后死亡率和梗塞。另一方面，通过抗坏血酸（TET 诱导剂）治疗增加 5hmC 水平可显着保护因此，我们认为“Tet3 介导”。 5hmC 的诱导是一种神经保护性适应，可以增强其在中风后保护大脑。” TET3 的主要神经亚型缺乏 DNA 结合域。我们的初步数据表明 TET3 结合。具有高亲和力的 lncRNA 已知可充当将 DNA/RNA/蛋白质结合在一起的支架。 LncRNA 也可以调节中风后的结果，因此我们进一步进行了试点。 “lncRNA 在支架和引导 TET3 到达特定基因组位点方面发挥着至关重要的作用，因此调节 5hmC 水平和中风后的功能结果。” 目标 1：评估 DNA 羟甲基化在中风后是否具有神经保护作用，我们将测试其功能。 TET3 基因组功能丧失和功能增强表明 5hmC 在中风后病理生理学中的重要性。中风后 5hmC 增加的位点将通过染色质免疫沉淀结合 TET3 敲低和诱导后的大规模平行 DNA 测序 (ChIP-seq)。目标 2：研究 lncRNA 是否调节 TET3 介导的 DNA 羟甲基化以及随之而来的神经保护作用中风后，我们将确定由高 TET3 相互作用的 lncRNA 调节的基因组位置。通过 RNA 纯化进行染色质分离的通量测序方法 (ChiRP-seq) 我们将进一步研究是否。 lncRNA 功能对于 TET3/5hmC 介导的中风后神经保护至关重要。长期目标是确定 5hmC 在缺血后病理学中的作用并测试是否增加 5hmC 水平对中风后有益。