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

靶向赖氨酸甲基化以逆转感染艾滋病毒的吸毒者的潜伏期

基本信息

批准号：
9236043
负责人：
Melanie Maria Ott
金额：
$ 59.84万
依托单位：
J. DAVID GLADSTONE INSTITUTES
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9236043
关键词：
AIDS/HIV problem ATAC-seq Acquired Immunodeficiency Syndrome Address Affect Amphetamines Autopsy Binding Biological Assay Brain CD4 Positive T Lymphocytes CRISPR/Cas technology Cells Chromatin Chronic Clinical Cocaine Cocaine Users Development Drug Targeting Drug usage Drug user Enzymes Epigenetic Process Failure Funding Future Genes Genetic Transcription Goals HIV HIV therapy Histone Acetylation Histone H3 Histone H4 Histones Individual Infection Knowledge Laboratories Lead Link Lysine Malignant Neoplasms Maps Mass Spectrum Analysis Measures Methamphetamine Methylation Methyltransferase Microarray Analysis Midbrain structure Mission Modification Mus Pharmaceutical Preparations Pharmacologic Substance Play Post-Translational Protein Processing Protein Acetylation Protein Methylation Proteins Proviruses Public Health Publishing Regulation Research Research Support Role Substance abuse problem Technology Testing Therapeutic Transcription Repressor/Corepressor Transcriptional Regulation Transposase Viral Work base chromatin immunoprecipitation clinical application cocaine exposure cocaine use epigenetic drug epigenetic regulation epigenomics gene repression histone methylation improved in vivo inhibitor/antagonist innovation interest neglect personalized approach pre-clinical promoter research study response small molecule small molecule inhibitor targeted treatment

项目摘要

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.

项目摘要兴奋剂会引起大脑中广泛的表观遗传扰动，但它们对表观遗传的影响潜伏 HIV 原病毒的调节以及针对该调节的潜伏期逆转疗法的反应该应用的中心假设是 SET 和 MYND 结构域的组蛋白甲基化。含有酶 Smyd2（也称为 KMT3C）是一种强大的潜伏期诱导机制，也是 HIV+可卡因使用者的潜伏期逆转疗法是在初步研究的基础上提出的。申请人生成的结果将 Smyd2 在 45 种细胞赖氨酸甲基转移酶中识别为顶级抑制潜伏 CD4+ T 细胞中 HIV 转录的酶它也基于其他人发表的工作。显示大脑中另一种与 HIV 潜伏期相关的甲基转移酶 G9a 的表达下降长期接触可卡因后，Smyd2 的表达没有变化。中心假设将得到检验。三个具体目标： 1) 定义 Smyd2 如何在 HIV 潜伏期发挥转录抑制因子的作用。假设 Smyd2 通过与体内潜在的 HIV 启动子结合，诱导持久的转录尽管接触可卡因仍会受到抑制，申请人将使用 CRISPR/Cas9 基因编辑来测试这一假设。技术并通过检查 Smyd2 招募模式（有或没有可卡因暴露）她还将了解。测试从 HIV+ 可卡因中分离出的 CD4+ T 细胞对小分子 Smyd2 抑制剂反应的潜伏期逆转 2) 确定 Smyd2 在潜在 HIV 启动子上设置的赖氨酸甲基化标记。假设是组蛋白 H4 (H4K20me1) 上赖氨酸 20 的单甲基化是 Smyd2 在申请人将通过确定可卡因暴露期间的潜伏病毒启动子来检验该假设。 Smyd2 使用质谱分析和染色质分析引入甲基标记使用和不使用可卡因的情况下，对潜伏细胞中的 Smyd2 组蛋白甲基标记进行免疫沉淀 (ChIP) 3) 确定 H4K20me1 诱导 HIV 潜伏期的机制。是潜伏原病毒处的 H4K20me1 与含有 MBT 的蛋白 L3MBTL1 结合，从而诱导染色质压缩，从而导致持久的转录抑制和可卡因暴露的潜伏期。申请人将通过进行 ChIP 和组蛋白可及性测定 (ATAC-Seq) 来测试这一假设在有和没有可卡因的潜伏细胞中，L3MBTL1 分别招募和染色质压缩该提案的成功完成将显着增强对表观基因组的理解。艾滋病毒/艾滋病感染与药物滥用的监管机制与本 RFA 一致。拟议的研究具有创新性，因为它代表了对现状的新的实质性偏离通过将 HIV 潜伏期研究的重点转向组蛋白 H4 的单甲基化、L3MBTL1 的招募，基于这项工作，申请人期望在以下方面取得重要进展。 HIV+吸毒者的治疗方法以及提高对吸毒表观遗传学的了解。