Regulation of HSV-1 gene expression and reactivation by insulator protein CTCF

绝缘体蛋白 CTCF 对 HSV-1 基因表达和再激活的调节

• 批准号: 8710795
• 负责人: DONNA M NEUMANN
• 金额: $ 34.68万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-15 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8710795
• 关键词: Affect; Afferent Neurons; Antiviral Agents; Binding; Biological Assay; Blindness; CCCTC-binding factor; Chromatin; Chromatin Loop; Chromosomes; DNA Binding; DNA Sequence Rearrangement; Data; Development; Disease; Disease Outbreaks; Distant; Elements; Enhancers; Enzymes; Epigenetic Process; Episome; Eye; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Goals; Health Care Costs; Herpesvirus 1; Herpetic Keratitis; Histones; Individual; Infection; Infectious Agent; Insulator Elements; Introns; Life; Maps; Mediating; Methodology; Modeling; Molecular; Molecular Conformation; Mus; Mutation; Occupations; Oryctolagus cuniculus; Pathogenesis; Pathway interactions; Phenotype; Play; Publishing; Recurrence; Recurrent disease; Regulation; Repressor Proteins; Research; Role; Site; Testing; Transcription Coactivator; United States; Viral Genes; Virus; Work; chromatin immunoprecipitation; chromatin remodeling; effective therapy; histone modification; in vivo; innovation; insight; latency associated transcript; novel; prevent; promoter; recombinant virus; response; stressor

DESCRIPTION (provided by applicant): Herpes Simplex Virus 1 (HSV-1) affects millions of individuals, and is the single largest cause of infectious blindness in the U.S. HSV-1 establishes life-long latency in sensory neurons, where abundant viral gene expression is limited to the latency associated transcript (LAT). HSV-1 remains latent as a circular episome associated with histones in sensory neurons, but has the ability to reactivate in response to stressors. This reactivation causes recurrent outbreaks, which is responsible for the majority of HSV-1-related disease and the associated health care costs. Unfortunately, the molecular mechanisms that regulate latency and HSV-1 reactivation are not well defined. A major focus of our research is to determine the molecular mechanisms that control HSV-1 latency and contribute to recurrent HSV-1 infections. Recently, we found that epigenetic marks present on the HSV-1 genome change as the virus reactivates from latency. We have also identified clusters of DNA binding motifs for the cellular insulating protein, CTCF around genomic regions of HSV-1 that are critical for both the establishment of latency as well as in reactivation. These binding motifs are enriched in CTCF during latency and we have already characterized three CTCF sites as chromatin insulators. The overall hypothesis to be tested in this proposal is that the cellular insulating protein, CTCF, regulates HSV-1 gene expression during latency through long-range chromatin interactions that can be destabilized to facilitate HSV-1 reactivation. In Aim 1, we will characterize the insulator function of the CTCF binding motifs in HSV-1 using chromatin immunoprecipitation assays, and map the histone profiles around these CTCF binding motifs. This will allow us to characterize additional elements within the HSV-1 genome that might be responsible for facilitating HSV-1 gene expression during latency. We will also use chromatin conformation capture (3-C) assays to determine whether CTCF regulates HSV-1 gene expression through classic insulator function (in a linear manner) or indirectly through the formation of chromatin loop domains. Finally, we will determine whether mutations to the CTCF binding domains in HSV-1 affect HSV-1 gene expression during latency. In Aim 2 of this proposal, we will be investigating the role of CTCF-mediated gene expression during in vivo reactivation using the highly efficient rabbit ocular model to characterize ocular pathogenesis and CTCF-mediated HSV-1 gene expression during reactivation in vivo. The proposed studies will provide key insight into how the HSV-1 genome is regulated epigenetically, as well as define the role of CTCF and chromatin insulators in HSV-1 gene regulation during latency and in reactivation. In addition, this work will likely increase our understanding of how CTCF regulates cellular transcription from chromosomal elements. .

描述（由申请人提供）：单纯疱疹病毒 1 (HSV-1) 影响数百万人，是美国感染性失明的最大单一原因。HSV-1 在感觉神经元中建立终生潜伏期，其中丰富的病毒基因表达仅限于潜伏相关转录本 (LAT)。 HSV-1 作为与感觉神经元中的组蛋白相关的圆形附加体保持潜伏状态，但具有响应应激源而重新激活的能力。这种重新激活会导致疾病反复爆发，这是造成大部分 HSV-1 相关疾病和相关医疗费用的原因。不幸的是，调节潜伏期和 HSV-1 重新激活的分子机制尚不明确。我们研究的一个主要重点是确定控制 HSV-1 潜伏期和导致 HSV-1 复发感染的分子机制。最近，我们发现 HSV-1 基因组上存在的表观遗传标记随着病毒从潜伏期重新激活而发生变化。我们还鉴定了 HSV-1 基因组区域周围细胞绝缘蛋白 CTCF 的 DNA 结合基序簇，这些基序对于潜伏期的建立以及重新激活都至关重要。这些结合基序在潜伏期在 CTCF 中富集，我们已经将三个 CTCF 位点定性为染色质绝缘子。该提案要测试的总体假设是，细胞绝缘蛋白 CTCF 在潜伏期通过长程染色质相互作用调节 HSV-1 基因表达，这种相互作用可被破坏以促进 HSV-1 重新激活。在目标 1 中，我们将 使用染色质免疫沉淀测定来表征 HSV-1 中 CTCF 结合基序的绝缘体功能，并绘制这些 CTCF 结合基序周围的组蛋白谱。这将使我们能够表征 HSV-1 基因组中可能负责促进潜伏期 HSV-1 基因表达的其他元件。我们还将使用染色质构象捕获 (3-C) 测定来确定 CTCF 是否通过经典绝缘子功能（以线性方式）或通过染色质环结构域的形成间接调节 HSV-1 基因表达。最后，我们将确定 HSV-1 中 CTCF 结合域的突变是否会影响潜伏期 HSV-1 基因的表达。在本提案的目标 2 中，我们将使用高效的兔眼模型来研究 CTCF 介导的基因表达在体内再激活过程中的作用，以表征眼部发病机制和 CTCF 介导的 HSV-1 基因表达在体内再激活过程中的特征。拟议的研究将提供关于 HSV-1 基因组如何进行表观遗传调控的关键见解，并定义 CTCF 和染色质绝缘子在 HSV-1 潜伏期和重新激活期间基因调控中的作用。此外，这项工作可能会增加我们对 CTCF 如何调节染色体元件的细胞转录的理解。 。