Arruri Vijay, Morris-Blanco Kahlilia C, Mehta Suresh L, Kaur Mehakpreet, Vemuganti Raghu
Department of Neurological Surgery, University of Wisconsin-Madison (V.A., K.C.M.-B., S.L.M., M.K., R.V.).
William S. Middleton Veterans Administration Hospital, Madison, WI (R.V.).
Stroke. 2025 Jul 16. doi: 10.1161/STROKEAHA.125.052347.
Epigenetic modifications 5-methylcytosine and 5-hydroxymethylcytosine in DNA regulate neuronal survival under ischemic stress. We previously showed that TET3 (ten-eleven translocation 3)-mediated 5-methylcytosine to 5-hydroxymethylcytosine conversion induces neuroprotective gene transcription after stroke. As TET3 neuronal isoform lacks the DNA-binding domain, how TET3 drives 5-hydroxymethylcytosine-mediated transcriptional induction in the ischemic brain remains unclear. Long noncoding RNAs (lncRNAs) act as structural scaffolds to recruit chromatin-modifying proteins and other RNAs to specific genomic loci. However, whether TET3 requires an lncRNA to drive DNA hydroxymethylation in the ischemic brain is unknown.
Adult male and female mice were subjected to transient middle cerebral artery occlusion. TET3-bound lncRNAs were immunoprecipitated from peri-infarct cortex, and TILR (TET3-interacting lncRNA; AK020504) identified was inhibited with small interfering RNA injected at 5 minutes of reperfusion. Ascorbate was administered at 30 minutes of reperfusion to induce TET3 activity. Poststroke DNA hydroxymethylation was assessed with hydroxymethylation DNA immunoprecipitation sequencing, and sensorimotor deficits, and infarct volume were evaluated between days 1 and 7 of reperfusion.
TILR binds to TET3 with high affinity and was significantly upregulated in the peri-infarct cortex at 12 hours of reperfusion. Knockdown of TILR increased the infarct volume and reduced the motor function recovery after transient middle cerebral artery occlusion, in a TET3-dependent manner. On contrary, TET3 activation by ascorbate decreased brain damage and improved motor function recovery after ischemia. However, ascorbate-induced postischemic protection was abrogated by TILR knockdown. Genome-wide profiling showed that ascorbate increases the number of differentially hydroxymethylated regions in the poststroke genome, a neuroprotective effect that is reversed by TILR knockdown. Moreover, TILR inhibition significantly reduced the DNA hydroxymethylation in the intergenic regions associated with enhancers, super enhancers, and the promoters of other lncRNAs, microRNAs, and PIWI-interacting RNAs.
These findings highlight the essential role of TILR in TET3-mediated 5-hydroxymethylcytosine-dependent epigenetic reprogramming in the ischemic brain.
DNA中的表观遗传修饰5-甲基胞嘧啶和5-羟甲基胞嘧啶在缺血应激下调节神经元存活。我们之前表明，TET3（十一易位蛋白3）介导的5-甲基胞嘧啶向5-羟甲基胞嘧啶的转化在中风后诱导神经保护基因转录。由于TET3神经元亚型缺乏DNA结合结构域，TET3如何在缺血性脑中驱动5-羟甲基胞嘧啶介导的转录诱导仍不清楚。长链非编码RNA（lncRNA）作为结构支架，将染色质修饰蛋白和其他RNA募集到特定的基因组位点。然而，TET3在缺血性脑中驱动DNA羟甲基化是否需要lncRNA尚不清楚。
成年雄性和雌性小鼠接受短暂性大脑中动脉闭塞。从梗死灶周围皮质免疫沉淀与TET3结合的lncRNA，并在再灌注5分钟时注射小干扰RNA抑制鉴定出的TILR（与TET3相互作用的lncRNA；AK020504）。在再灌注30分钟时给予抗坏血酸以诱导TET3活性。用羟甲基化DNA免疫沉淀测序评估中风后的DNA羟甲基化，并在再灌注第1天至第7天评估感觉运动功能缺陷和梗死体积。
TILR与TET3高亲和力结合，在再灌注12小时时梗死灶周围皮质中显著上调。敲低TILR以TET3依赖的方式增加了短暂性大脑中动脉闭塞后的梗死体积并降低了运动功能恢复。相反，抗坏血酸激活TET3减少了脑损伤并改善了缺血后的运动功能恢复。然而，TILR敲低消除了抗坏血酸诱导的缺血后保护作用。全基因组分析表明，抗坏血酸增加了中风后基因组中差异羟甲基化区域的数量，TILR敲低可逆转这种神经保护作用。此外，TILR抑制显著降低了与增强子、超级增强子以及其他lncRNA、微小RNA和PIWI相互作用RNA的启动子相关的基因间区域的DNA羟甲基化。
这些发现突出了TILR在缺血性脑中TET3介导的5-羟甲基胞嘧啶依赖性表观遗传重编程中的重要作用。
