CTFC Mediated HSV-1 Gene Expression in Latency and Reactivation

CTFC 介导的 HSV-1 潜伏期和重新激活基因表达

• 批准号: 10516765
• 负责人: DONNA M NEUMANN
• 金额: $ 7.24万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-11-15 至 2022-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10516765
• 关键词: 3-Dimensional; Address; Adopted; Affect; Afferent Neurons; Antiviral Agents; Architecture; Area; Binding; Binding Sites; Blindness; CCCTC-binding factor; Cells; Chromatin; Chromatin Loop; Chromatin Structure; DNA; DNA Viruses; Data; Defect; Deposition; Disease; Enhancers; Epigenetic Process; Epithelial Cells; Euchromatin; Eukaryota; Evaluation; Forms Controls; Future; Ganglia; Gene Delivery; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; Herpesviridae; Herpesvirus 1; Herpetic Keratitis; Heterochromatin; Higher Order Chromatin Structure; Immediate-Early Genes; Infection; Infectious Agent; Knowledge; Life Cycle Stages; Location; Lytic; Lytic Phase; Maintenance; Mediating; Methods; Molecular; Molecular Biology; Molecular Conformation; Mus; Neurons; Physiological; Proteins; Publishing; Recurrence; Research; Research Personnel; Role; Site; Space Perception; Structure; Techniques; Testing; Therapeutic Intervention; Time; Transcription Coactivator; VP 16; Viral; Viral Genome; Virus; Virus Latency; acute infection; base; chromosome conformation capture; deep sequencing; epigenetic regulation; human pathogen; in vivo; in vivo Model; latency associated transcript; latent gene expression; latent infection; lytic gene expression; lytic replication; mammalian genome; mutant; new therapeutic target; next generation sequencing; novel; prevent; programs; promoter; reactivation from latency; recruit; three dimensional structure

Herpes Simplex Virus 1 (HSV-1) is a significant human pathogen, and is the leading cause of blindness in the US due to an infectious agent. HSV-1 establishes a lifelong latent infection in host sensory neurons, where lytic gene expression is repressed. Latent HSV-1 periodically reactivates, and recurrences are often associated with HSV-1-related disease. It is now widely recognized that the LAT promoter and enhancer and the immediate early (IE) genes of HSV-1 are regulated through the deposition and maintenance of chromatin. However, the mechanisms involved in directing chromatin deposition during latency have not been defined, and the identification of the mechanism regulating the latent-lytic switch during reactivation remains elusive. A major focus of our research is to overcome these significant gaps in our knowledge so that novel HSV-1 therapeutic interventions can be developed in the future. Recently, we identified seven binding motifs for the cellular insulating protein CTCF. The genomic locations of each motif in HSV-1 show that CTCF binding domains flank the LAT and each IE region of the genome separately, providing evidence that CTCF insulators could control latent chromatin recruitment and HSV-1 gene expression. We showed that site was occupied by the insulator protein CTCF during latency, three sites function as enhancer-blocking insulators, and that CTCF was evicted at early times in reactivation. We subsequently pioneered a gene delivery method to show that CTCF depletion drives HSV-1 reactivation in vivo. CTCF insulators regulate gene expression through multiple mechanisms, one of which is the formation of higher order chromatin structures known as chromatin loops. Chromatin loops bring enhancers and promotors into close spatial proximity to regulate gene expression. Using deep sequencing on latently infected mouse neurons (3C-seq) we found 3 distinct chromatin loops. We are the first investigators to discover chromatin loops in an alpha-herpes virus. Consequently, each of these loops adopt a 3D conformation that orient the IE genes as “off” or “poised for reactivation”. We hypothesize that the three loops in HSV-1 1) control the chromatin architecture by recruitment of co-regulating proteins, and; 2) populate neuronal subtypes that can (or cannot) reactivate, based on the loop conformation. The aims of this project methodically test how the 3D orientation of these CTCF loops in HSV-1 control lytic, latent and reactivating genomes. We have pioneered novel techniques to test this hypothesis using in vivo models of HSV-1 infection, making our study not only relevant on a molecular level, but physiologically relevant as well, and our expertise in epigenetic regulation of DNA viruses make us the ideal investigators to carry out these studies. In summary, we will push the field of epigenetic control of herpesviruses into areas that have never been explored by combining genetics, molecular biology and in vivo models together to achieve comprehensive, methodical and meticulous evaluations of how the viral transcriptional program is controlled by CTCF-chromatin loops at all stages of the virus life cycle.

单纯疱疹病毒 1 (HSV-1) 是一种重要的人类病原体，在美国是导致失明的主要原因，因为 HSV-1 会在宿主感觉神经元中建立终生潜伏感染，其中裂解基因表达受到抑制。潜伏的HSV-1周期性地重新激活，并且复发通常与HSV-1相关疾病有关。现在人们广泛认识到HSV-1的LAT启动子和增强子以及立即早期（IE）基因。然而，潜伏期间指导染色质沉积的机制尚未明确，并且重新激活期间调节潜伏裂解开关的机制仍然难以确定。克服我们知识中的这些重大空白，以便在未来开发新的 HSV-1 治疗干预措施。最近，我们确定了细胞绝缘蛋白 CTCF 的七个结合基序。HSV-1 中每个基序的基因组位置表明。 CTCF 结合域分别位于基因组的 LAT 和每个 IE 区域的侧翼，这提供了 CTCF 绝缘体可以控制潜伏染色质招募和 HSV-1 基因表达的证据，我们表明该位点在潜伏期间被绝缘体蛋白 CTCF 占据，三个位点发挥作用。我们随后开创了一种基因传递方法，以证明 CTCF 耗尽可驱动 HSV-1 在体内重新激活。 CTCF 绝缘体通过多种机制调节基因表达，其中之一是形成称为染色质环的高级染色质结构，使增强子和启动子在空间上紧密相连，从而利用对潜伏感染的小鼠神经元进行深度测序来调节基因表达。 -seq）我们发现了 3 个不同的染色质环，我们是第一个在测试的 α-疱疹病毒中发现染色质环的研究人员。将 IE 基因定位为“关闭”或“准备重新激活”的 3D 构象我们发现 HSV-1 中的三个环 1) 通过招募共调节蛋白来控制染色质结构，2) 填充神经亚型该项目的目的是系统地测试这些 CTCF 环在 HSV-1 中的 3D 方向如何控制裂解、潜伏。我们开创了利用 HSV-1 感染体内模型来检验这一假设的新技术，使我们的研究不仅在分子水平上具有相关性，而且在生理学上也具有相关性，而且我们在 DNA 病毒表观遗传调控方面的专业知识使得我们的研究变得更加重要。总之，我们将通过将遗传学、分子生物学和体内模型结合在一起，将疱疹病毒的表观遗传控制领域推向从未探索过的领域，以实现全面、系统和细致的评估。如何病毒式传播转录程序在病毒生命周期的所有阶段均由 CTCF-染色质环控制。