精氨酸甲基转移酶1介导的组蛋白精氨酸甲基化修饰在可卡因成瘾中的作用

Currently, the mechanism of epigenetic in cocaine-induced addiction has been one of the research focuses. It is well known that histone modification plays an importamt role in chromosome remodelling and gene transcription regulation. Histone modification mainly includes two types: histone lysine methylation and arginine methylation. The former has been widely studied like G9a; however, the latter in drug addiction remains unknown. Protein arginine N-methyltransferase 1（PRMT1） is thought to contribute to most of all cellular PRMT activity.It influences gene transcription through catalyzing the addition of two methyl groups to the same terminal nitrogen group of histone arginine.Our previous research showed that PRMT1 expression was significantly increased in the nucleus accubens of cocaine-addicted mice.Moreover, administration of PRMT1 inhibitor could inhibited cocaine CPP.These results strongly hinted that PRMT1 may anticipate cocaine addiction as a histone modifying enzyme.In this study,we are going to study the expression of PRMT1 in different brain area. The effect of PRMT1 on histone arginine methylation is to be investigated. Moreover,the roles of such histone arginine methylation in transcrption factor recuitment and addcitive gene expression are to be studied. Using gene silience technology the inhibitory effect of cocaine addition after PRMT1 is inhibited is to be studied, including cocaine behavior and reward. Our study would reveal a new epigenetic mechanism of histone methtlation modification in drug addction.

成瘾表观遗传机制是成瘾研究的前沿领域。组蛋白修饰对染色质结构重塑和基因表达调控起关键作用，其修饰模式包括赖氨酸和精氨酸位点的甲基化，前者在成瘾领域已有较大突破，如G9a、HDAC等，但后者在成瘾中的作用尚不清楚。精氨酸甲基化修饰主要依赖精氨酸甲基转移酶1（PRMT1），通过调控精氨酸非对称二甲基化影响染色质结构，从而调节靶基因转录。我们前期发现：可卡因成瘾小鼠脑伏隔核PRMT1表达显著升高，而PRMT1抑制剂MTA可抑制成瘾，提示PRMT1介导的组蛋白精氨酸修饰机制参与成瘾。本课题在前期基础上，研究伏隔核PRMT1在可卡因成瘾过程中的动态表达；基因沉默定位敲除伏隔核PRMT1，观察对成瘾行为及奖赏效应的影响；探索PRMT1对组蛋白精氨酸甲基化修饰模式的影响，以及这种修饰特征如何调节成瘾基因启动子对转录复合物的招募，最终调控基因转录。本课题将揭示一种新型组蛋白修饰模式的成瘾机制及关键分子。

组蛋白修饰对染色质结构重塑和基因表达调控起关键作用，其修饰模式包括赖氨酸和精氨酸位点的甲基化，前者在成瘾领域已有较大进展，如G9a、HDAC等，但后者在成瘾中的作用尚不清楚。精氨酸甲基化修饰主要依赖精氨酸甲基转移酶1(PRMT1)，通过调控精氨酸非对称二甲基化影响染色质结构从而调节靶基因转录。本课题采用多种可卡因成瘾模型，采用RT-qPCR、Western blot和免疫荧光等技术检测伏隔核脑区PRMT1表达，发现可卡因可以特异性增高伏隔核PRMT1表达，但不影响纹状体、海马和前额皮质PRMT1表达。伏隔核内注射PRMTs非特异性抑制剂MTA和AMI-1，均可抑制可卡因诱导的奖赏行为。利用计算机辅助药物设计，筛选和化学合成了一个结构全新的化合物SKLB-639，该化合物在体外和体内实验中均特异性抑制PRMT1活性；伏隔核内注射SKLB-639能够明显减弱可卡因诱导的奖赏行为。PRMT1小分子抑制剂通过抑制H4R3me2a修饰从而弱化可卡因奖赏行为。伏隔核定位注射LV-shPRMT1慢病毒显著降低可卡因诱导的小鼠成瘾行为及奖赏效应；PRMT1通过降低H4R3me2a和acH3K9/K14表达的机制调控成瘾基因启动子对转录复合物的招募，从而调节FosB、CaMKII和CREB等基因的转录。. 本课题揭示了PRMT1参与调节可卡因成瘾效应的神经生物学机制，发现了一种新的可卡因成瘾的表观调控机制——组蛋白精氨酸甲基化，丰富了现有的成瘾表观机制，为成瘾治疗提供新思路与新策略。获得具有自主知识产权的PRMT1特异性小分子抑制剂SKLB-639中国发明专利2项（中国发明专利ZL 201510458757.X、ZL 201510466750.2），为成瘾治疗提供新的候选化合物分子。