DNA hydroxymethylation and post stroke brain damage

已知DNA和组蛋白的表观遗传变化显著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。特别 有趣的是，DNA中的胞嘧啶经历甲基化形成5-甲基胞嘧啶（5 mC），这是已知的一种 转录沉默子最近的研究表明，5 mC会被10 - 11易位（泰特）氧化， 羟化酶形成5-羟甲基胞嘧啶（5 hmC）。这种表观遗传变化被认为是一种转录 在不利条件下增加细胞存活的去抑制标记。特别是，大脑含有约10倍 5 hmC水平高于身体其他器官。初步研究表明， 成年啮齿类动物显著增加梗塞周围皮质中的基因组5 hmC水平。TET 3敲除 降低5 hmC水平，并加剧缺血后死亡率和梗死的雄性和雌性小鼠。 另一方面，通过抗坏血酸（一种泰特诱导剂）处理增加5 hmC水平显著保护了 TET 3依赖性局灶性脑缺血后的脑组织。因此，我们假设“Tet 3介导 5 hmC的诱导是一种神经保护性适应，可以加强中风后的大脑保护。 TET 3的主要神经元同种型缺乏DNA结合结构域。我们的初步数据显示TET 3与 高亲和力的lncRNA。已知lncRNA作为支架将DNA/RNA/蛋白质结合在一起 使他们的行动。还已知lncRNA调节中风后结果。因此，我们进一步假设 lncRNA在支架和引导TET 3到特定的基因组位点中起着至关重要的作用， 调节中风后5 hmC水平和功能结果。 目的1：评估DNA羟甲基化是否对中风后具有神经保护作用。我们将测试功能 通过TET 3的功能丧失和功能获得，5 hmC在卒中后病理生理学中的意义。基因组 卒中后5 hmC增加的位点将通过染色质免疫沉淀结合 TET 3敲低和诱导后的大规模平行DNA测序（ChIP-seq）。 目的2：研究lncRNA是否调节TET 3介导的DNA羟甲基化和随后的神经保护作用 中风后我们将确定由TET 3相互作用lncRNA调节的基因组位置， 通过RNA纯化的染色质分离（ChiRP-seq）的通量测序方法。我们会进一步研究， lncRNA功能对于TET 3/5 hmC介导的脑卒中后神经保护是必不可少的。 长期目标是确定5 hmC在缺血后病理学中的作用，并测试增加5 hmC水平是否 对中风后有好处

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.