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 在经历了许多疾病之后。表观遗传学在缺血性脑损伤中的作用尚不完全清楚。特别的 有趣的是，DNA中的胞嘧啶经历甲基化形成5-甲基胞嘧啶(5mC)，这是一种已知的 转录消音器。最近的研究表明，5 mC会被10-11易位(Tet)氧化。 羟基酶形成5-羟甲基胞嘧啶(5hmC)。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率的去阻抑标记。特别是，大脑包含~10倍 5hmC水平高于身体其他器官。初步研究表明，短暂性局灶性脑缺血 成年啮齿动物显著增加梗死灶周围皮质的基因组5hmC水平。TET3基因敲除 降低5hmC水平，并加重雄性和雌性小鼠的缺血后死亡率和脑梗塞。 另一方面，用抗坏血酸(一种Tet诱导剂)处理增加5hmC水平显著保护 局灶性脑缺血后以TET3依赖的方式表达。因此，我们假设Tet3介导了 5hmC的诱导是一种神经保护性适应，可以增强以保护中风后的大脑。 TET3的主要神经元亚型缺乏DNA结合域。我们的初步数据显示，TET3与 具有高亲和力的IncRNAs。众所周知，lncRNAs作为支架将dna/rna/蛋白质连接在一起。 使他们能够采取行动。众所周知，lncRNAs也可以调节中风后的结局。因此，我们进一步假设 LncRNAs在支撑和引导TET3到特定的基因组位置上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：评价DNA羟甲基化对卒中后的神经保护作用。我们将测试功能 5hmC在卒中后TET3功能丧失和功能恢复中的意义基因组学 卒中后5hmC升高的部位将通过染色质免疫沉淀结合 TET3基因敲除和诱导后的大规模平行DNA测序(CHIP-SEQ)。 目的2：研究lncRNAs是否调节TET3介导的DNA羟甲基化及随后的神经保护作用 中风后。我们将确定由TET3相互作用的lncRNAs调控的基因组位置。 吞吐量测序法RNA纯化染色质分离(chirp-seq)。我们会进一步研究是否 LncRNA功能在TET3/5hmC介导的卒中后神经保护中起重要作用。 长期目标是确定5hmC在缺血后病理中的作用，并测试是否增加5hmC水平 对中风后是有益的。

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.