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）会影响数百万个个体，并且是美国HSV-1在感官神经元中建立终身延迟的最大原因，其中丰富的病毒基因表达仅限于潜伏相关的成绩单（LAT）。 HSV-1仍然是与感觉神经元中组蛋白相关的圆形综合体的潜在，但具有响应压力源的重新激活的能力。这种重新激活导致反复爆发，这导致了大多数与HSV-1相关疾病和相关的医疗保健费用。不幸的是，调节潜伏期和HSV-1重新激活的分子机制尚未很好地定义。我们研究的主要重点是确定控制HSV-1潜伏期的分子机制，并导致复发性HSV-1感染。最近，我们发现随着病毒从潜伏期重新激活，HSV-1基因组变化上存在表观遗传标记。我们还确定了针对HSV-1基因组区域围绕的细胞绝缘蛋白，CTCF的DNA结合基序的簇，这对于建立潜伏期和重新激活至关重要。这些结合基序在延迟期间富含CTCF，我们已经将三个CTCF位点表征为染色质绝缘子。在该提案中要检验的总体假设是，通过远距离染色质相互作用，在潜伏期期间调节了细胞绝缘蛋白CTCF，可以不稳定以促进HSV-1的重新激活。在AIM 1中，我们将 使用染色质免疫沉淀测定法表征了HSV-1中CTCF结合基序的绝缘函数，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基因组中的其他元素，这些元素可能负责促进延迟期间的HSV-1基因表达。我们还将使用染色质构象捕获（3-C）测定来确定CTCF是通过经典的绝缘体功能（以线性方式）或通过染色质环域的形成来调节HSV-1基因表达。最后，我们将确定HSV-1中CTCF结合结构域的突变是否会影响潜伏期期间HSV-1基因的表达。在该提案的AIM 2中，我们将使用高效的兔眼模型研究CTCF介导的基因表达在体内重新激活过程中的作用，以表征眼部发病机理和CTCF介导的HSV-1基因在体内重新激活过程中的作用。拟议的研究将提供有关如何表观遗传调节HSV-1基因组，并定义CTCF和染色质绝缘子在潜伏期和重新激活过程中HSV-1基因调节中的作用的关键见解。此外，这项工作可能会增加我们对CTCF如何调节染色体元素细胞转录的理解。 。