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: 10295772
• 负责人: DONNA M NEUMANN
• 金额: $ 38.61万
• 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 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10295772
• 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型(HSV-1)是一种重要的人类病原体，是美国因感染性因素导致失明的主要原因。HSV-1在宿主感觉神经元中建立了终生潜伏感染，在那里裂解基因的表达受到抑制。潜伏的HSV-1周期性地重新激活，复发通常与HSV-1相关疾病有关。目前，人们普遍认为HSV-1的LAT启动子和增强子以及即刻早期(IE)基因是通过染色质的沉积和维持来调节的。然而，在潜伏期指导染色质沉积的机制还没有确定，在重新激活期间调节潜伏裂解开关的机制仍然难以确定。我们研究的一个主要重点是克服我们知识中的这些重大差距，以便在未来开发新的HSV-1治疗干预措施。最近，我们确定了细胞隔离蛋白CTCF的七个结合基序。对HSV-1中每个基序的基因组定位表明，CTCF结合区分别位于基因组的LAT和每个IE区的两侧，这为CTCF绝缘体能够控制潜在的染色质募集和HSV-1基因的表达提供了证据。我们发现，在潜伏期，CTCF被绝缘蛋白CTCF占据，有三个位点起到增强子阻断绝缘子的作用，而CTCF在重新激活的早期被驱逐。随后，我们开创了一种基因传递方法，表明CTCF耗尽在体内驱动HSV-1的重新激活。CTCF绝缘体通过多种机制调节基因表达，其中之一是形成更高阶染色质结构，称为染色质环。染色质环使增强子和启动子在空间上接近，从而调节基因的表达。对潜伏感染的小鼠神经元(3C-seq)进行深度测序，我们发现了3个明显的染色质环。我们是第一批在阿尔法疱疹病毒中发现染色质环的研究人员。因此，这些环中的每一个都采用了3D构象，将IE基因定位为“关闭”或“准备重新激活”。我们假设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)基于环的构象，填充可以(或不能)重新激活的神经元亚型。这个项目的目的是系统地测试HSV-1中这些CTCF环的3D方向如何控制裂解、潜伏和重新激活基因组。我们开创了使用HSV-1感染体内模型来验证这一假说的新技术，使我们的研究不仅在分子水平上相关，而且在生理上也相关，我们在DNA病毒表观遗传调控方面的专业知识使我们成为开展这些研究的理想研究人员。综上所述，我们将通过将遗传学、分子生物学和体内模型结合起来，将疱疹病毒的表观遗传控制领域推向从未探索的领域，以实现对病毒转录程序如何在病毒生命周期的所有阶段由CTCF-染色质环控制的全面、系统和细致的评估。