HISTONE MODIFICATIONS GUIDING HIV INTEGRATION

组蛋白修饰指导HIV整合 项目摘要/摘要 人类免疫缺陷病毒(HIV-1)是获得性免疫缺陷综合征(AIDS)的病原体 目前全球约有3670万人感染。病毒基因组的整合建立了一个不可逆转的 将前病毒序列插入宿主染色质。整合的基因组确保有效的HIV基因 表达并最终产生病毒或建立潜伏期，其中前病毒保持休眠 一段很长的时间。逆转录病毒整合是由病毒整合酶(IN)蛋白介导的，IN蛋白与 病毒DNA基因组的直接重复末端，以及其他细胞和病毒辅助因子 整合到宿主染色质部位。这项提案的首要目标是了解病毒整合是如何 通过组蛋白翻译后修饰(PTM)靶向染色质，以及细胞因子如何紧密地 控制这些PTM的本地化影响目标地点的选择。 LEDGF/p75是一个关键的细胞转录共激活因子，通过C端整合酶结合与HIV-1 IN结合 域名(IBD)。LEDGF/p75的N-末端含有一个可识别组蛋白的PWWP结构域 甲基化赖氨酸PTMS。HIV-1整合复合体(内含体)与转录相关基因的双峰连接 组蛋白PTM被认为可以解释观察到的~76%的染色体整合 活跃地转录基因。LEDGF/p75据称针对HIV-1到含有三甲基化的核小体 组蛋白H3赖氨酸36个残基(H3K36me3)。然而，H3K36me3最常在3‘端发现 转录的基因，而HIV-1更频繁地向基因的5‘端整合。 HIV-1整合位点与组蛋白PTMS的生物信息学相关性受到 DNA元素百科全书(ENCODE)数据库。值得注意的是，ENCODE中没有这种基因的基因组图谱 H3K36me2 PTM在任何细胞类型中。我们对H3K36me2进行了芯片序列分析，并确定它是 通常发现在基因的5‘转录起始点附近。此外，我们发现HIV-1整合位点 与H3K36me3相比，与H3K36me2的位置相关性更好。我们建议研究H3K36me2的作用 和H3K36me3，有两个特定的目标。目标1将扩大目前对H3K36甲基化的理解和 它与HIV-1整合效率和体内部位选择的关系。目标2将探讨 H3K36me2和H3K36me3对HIV-1体外整合的影响。我们的方法将包括先进的技术 质谱学、整合位点图谱和单分子荧光显微镜。这些研究的结果 研究将明确确定H3K36me2和H3K36me3在HIV-1整合过程中的作用。

HISTONE MODIFICATIONS GUIDING HIV INTEGRATION PROJECT SUMMARY / ABSTRACT Human immunodeficiency virus (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS) with ~36.7 million people currently infected worldwide. Integration of the viral genome establishes an irreversible insertion of the proviral sequence into the host chromatin. The integrated genome ensures effective HIV gene expression and ultimately virus production or the establishment of latency where the provirus remains dormant for an extended time. Retroviral integration is mediated by the viral integrase (IN) protein that is bound to the direct-repeated ends of the viral DNA genome, along with additional cellular and viral co-factors that direct integration to host chromatin sites. The overarching goal of this proposal is to understand how viral integration is targeted to chromatin by histone post translational modifications (PTMs), and how cellular factors that tightly control the localization of these PTMs influence target site choice. LEDGF/p75 is a key cellular transcription co-activator that binds to HIV-1 IN via a C-terminal integrase binding domain (IBD). The N-terminus of LEDGF/p75 contains a PWWP domain that is expected to recognize histone methylated lysine PTMs. Bimodal tethering of the HIV-1 integration complex (intasome) to a transcription-related histone PTM has been proposed to account for the observed ~76% of chromosomal integrations that occur in actively transcribed genes. LEDGF/p75 purportedly targets HIV-1 to nucleosomes containing trimethylation of histone H3 lysine 36 residues (H3K36me3). However, H3K36me3 is most often found at the 3’ ends of transcribed genes, whereas HIV-1 integrates more frequently toward the 5’ ends of genes. Bioinformatic correlations of HIV-1 integration sites with histone PTMs is limited by the data available in the Encyclopedia of DNA Elements (ENCODE) database. Notably absent from ENCODE are genomic maps of the H3K36me2 PTM in any cell type. We performed a ChIP-Seq analysis of H3K36me2 and determined that it was commonly found near the 5’ transcription start site of genes. Moreover, we found that HIV-1 integration sites correlate better with the location of H3K36me2 than H3K36me3. We propose to examine the role of H3K36me2 and H3K36me3 with two Specific Aims. Aim 1 will expand the current understanding of H3K36 methylation and its connection to HIV-1 integration efficiency and site selection in vivo. Aim 2 will probe the influence of H3K36me2 and H3K36me3 on HIV-1 integration in vitro. Our approaches will include technologically advanced mass spectrometry, integration site mapping, and single molecule fluorescence microscopy. The results of these studies will clearly determine the role of H3K36me2 and H3K36me3 during HIV-1 integration.