Targeting lysine methylation for latency reversal in HIV-infected drug users

项目摘要 兴奋剂在大脑中引起广泛的表观遗传扰动，但它们对表观遗传的影响， 潜伏的HIV前病毒的调节和对针对该调节的潜伏逆转疗法的反应是 未知本申请的中心假设是SET和MYND结构域的组蛋白甲基化- 含有酶Smyd 2（也称为KMT 3C）的蛋白质是一种强大的潜伏诱导机制，也是一种新的靶点。 HIV+可卡因使用者的潜伏期逆转疗法。这一假设是在初步研究的基础上提出的。 由申请人产生的结果，在45种细胞赖氨酸甲基转移酶中鉴定Smyd 2为最高的赖氨酸甲基转移酶。 抑制HIV在潜伏的CD 4 + T细胞中转录的酶。它也是基于其他人发表的作品 显示G9 a的表达，另一种与HIV潜伏期相关的甲基转移酶，在大脑中减少 慢性可卡因暴露后，Smyd 2的表达没有变化。中心假设将被检验 在三个具体的目标：1）定义Smyd 2如何作为转录抑制因子在HIV潜伏期的功能。工作 假设Smyd 2通过在体内与潜伏HIV启动子结合，诱导持久的转录 尽管可卡因暴露。申请人将通过CRISPR/Cas9基因编辑来验证这一假设。 技术和研究模式Smyd 2招募与可卡因暴露。她还将 在从HIV+可卡因中分离的CD 4 + T细胞中响应小分子Smyd 2抑制剂的测试潜伏期逆转 用户. 2)确定潜伏HIV启动子处由Smyd 2设定的赖氨酸甲基化标记。工作 假设组蛋白H4（H4 K20 me 1）赖氨酸20的单甲基化是Smyd 2在 潜伏的病毒启动子申请人将通过确定 使用质谱法和染色质分析法， 在有和没有可卡因的情况下，对潜伏细胞中Smyd 2组蛋白甲基标记的免疫沉淀（ChIP） 治疗3)确定H4 K20 me 1如何诱导HIV潜伏期的机制。工作假设 潜伏原病毒上的H4 K20 me 1与含MBT的蛋白L3 MBTL 1结合， 染色质致密化，因此，持久的转录抑制和可卡因暴露的潜伏期。的 申请人将通过进行ChIP和组蛋白可及性测定（ATAC-Seq）来测试该假设， L3 MBTL 1募集和染色质压实，分别在有和没有可卡因的潜伏细胞 exposure.成功完成这一提案将大大提高对表观基因组的理解 艾滋病毒/艾滋病感染与药物滥用相结合的监管机制与该RFA保持一致。 拟议的研究是创新的，因为它代表了一个新的和实质性的偏离的地位， 通过将HIV潜伏期研究的重点转移到组蛋白H4的单甲基化，L3 MBTL 1的募集， 和诱导组蛋白致密化。基于这项工作，申请人希望在以下方面取得重要进展： 艾滋病毒阳性吸毒者的治疗和提高对吸毒的表观遗传学的理解。

PROJECT ABSTRACT Stimulant drugs induce wide-spread epigenetic perturbations in the brain, but their effects on the epigenetic regulation of the latent HIV provirus and responses to latency-reversing therapies targeting this regulation are unknown. The central hypothesis of this application is that histone methylation by the SET and MYND domain- containing enzyme Smyd2 (also called KMT3C) is a robust latency-inducing mechanism and a new target for latency-reversing therapy in HIV+ cocaine users. This hypothesis was formulated on the basis of preliminary results generated by the applicant identifying Smyd2, among 45 cellular lysine methyltransferases, as a top enzyme that suppresses HIV transcription in latent CD4+ T cells. It is also based on published work by others showing that expression of G9a, another methyltransferase linked to HIV latency, is decreased in the brain after chronic cocaine exposure, while expression of Smyd2 is unchanged. The central hypothesis will be tested in three specific aims: 1) Define how Smyd2 functions as transcriptional repressor in HIV latency. The working hypothesis is that Smyd2, by associating with the latent-HIV promoter in vivo, induces durable transcriptional repression despite cocaine exposure. The applicant will test this hypothesis with CRISPR/Cas9 gene–editing technology and by examining the mode of Smyd2 recruitment with and without cocaine exposure. She will also test latency reversal in response to small-molecule Smyd2 inhibitors in CD4+ T cells isolated from HIV+ cocaine users. 2) Determine the lysine methylation mark(s) set by Smyd2 at the latent HIV promoter. The working hypothesis is that monomethylation of lysine 20 at histone H4 (H4K20me1) is the main mark set by Smyd2 at the latent viral promoter during cocaine exposure. The applicant will test this hypothesis by determining the methyl marks introduced by Smyd2 using mass spectrometry and by performing chromatin immunoprecipitations (ChIP) for Smyd2 histone–methyl marks in latent cells with and without cocaine treatment. 3) Identify the mechanism underlying how H4K20me1 induces HIV latency. The working hypothesis is that H4K20me1 at the latent provirus is bound by the MBT-containing protein L3MBTL1, which induces chromatin compaction and, thus, durable transcriptional repression and latency with cocaine exposure. The applicant will test this hypothesis by performing ChIP and histone accessibility assays (ATAC-Seq) to measure L3MBTL1 recruitment and chromatin compaction, respectively, in latent cells with and without cocaine exposure. Successful completion of this proposal will significantly enhance the understanding of epigenomic regulatory mechanisms in HIV/AIDS infection in combination with substance abuse in alignment with this RFA. The proposed research is innovative because it represents a new and substantive departure from the status quo by shifting the focus in HIV latency research to monomethylation of histone H4, recruitment of L3MBTL1, and induction of histone compaction. Based on this work, the applicant expects to make important advances in therapies for HIV+ drug users and in improving the understanding of the epigenetics of drug use.