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，由于感染剂而导致美国失明的主要原因。 HSV-1在宿主感觉神经元中建立了终生的潜在感染，其中反映了裂解基因表达。潜在的HSV-1会定期重新激活，并且回报通常与HSV-1相关疾病有关。现在已广泛认识到，HSV-1的LAT启动子和增强子以及直接的早期基因通过染色质的沉积和维持来调节。然而，尚未定义引导染色质沉积物涉及的机制，并且鉴定机制调节重新激活过程中潜在的宽差开关仍然难以捉摸。我们研究的重点是克服我们知识上的这些显着差距，以便将来可以开发出新的HSV-1治疗干预措施。最近，我们确定了七个用于细胞绝缘蛋白CTCF的结合基序。 HSV-1中每个基序的基因组位置表明，CTCF结合结构域分别侧翼和基因组的每个IE区域，提供了证据表明CTCF绝缘子可以控制潜在的染色质募集和HSV-1基因表达。我们表明，在潜伏期期间，绝缘子蛋白CTCF占据了位点，三个位点起到增强剂阻滞剂的绝缘子的作用，并且CTCF随后我们率先开发了一种基因递送方法，以表明CTCF的部署驱动了HSV-1在体内驱动HSV-1。 CTCF绝缘子通过多种机制调节基因表达，其中之一是形成高阶染色质结构，称为染色质环。染色质环将增强子和启动子带入密切的空间接近度以调节基因表达。使用潜在感染的小鼠神经元（3C-Seq）上的深层测序，我们发现了3个不同的染色质环。我们是第一个发现α-疱疹病毒中染色质环的研究者。因此，这些循环中的每一个都采用了3D会议，将IE基因定向为“关闭”或“准备重新激活”。我们假设HSV-1中的三个回路1）通过募集共同调节蛋白质来控制染色质结构，并且； 2）基于循环构象可以（或不能）重新激活的神经元亚型。该项目的目的有条不紊地测试了HSV-1中这些CTCF循环的3D方向如何控制裂解，潜在和重新激活的基因组。我们采用了新型技术来使用HSV-1感染的体内模型检验这一假设，这不仅使我们的研究在分子水平上相关，而且在物理上相关，并且我们在DNA病毒表观遗传调节方面的专业知识使我们成为理想的研究者进行这些研究。总而言之，我们将将疱疹病毒的表观遗传控制领域推向从未通过将遗传学，分子生物学和体内模型组合在一起来探索的领域，以实现对病毒转录程序如何通过CTCF-染料素环在整个阶段的病毒生活周期中控制病毒转录程序的全面，有方法和细致的评估。