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

基本信息

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
项目类别：
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

项目摘要

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）是一种显着的人类病原体，由于天空，在美国失明的主要原因。 T启动子和增强子和HSV-1的立即（IE）基因是定期的染色质的沉积和维持，涉及的机制尚未定义我们的知识使新的HSV-1治疗疗法在未来进行了蛋白质CTCF。 -1基因表明，在潜伏期期间，绝缘子蛋白CTCF占据，三个位点起到增强子阻滞剂的作用，而CTCF则在早期被驱逐出来。 CTCF耗尽在体内驱动HSV-1的重新激活。。共同调节的蛋白质；，我们的研究不是在分子水平上进行的，但生理学也相关，而我们在表观遗传调节的DNA病毒方面的专业知识使我们在总结上进行了研究。从未通过梳理遗传学，分子生物学和体内模型梳理的领域，以在病毒生命周期的所有阶段受到CTCF-染色质素环的控制，以实现病毒转录程序如何控制病毒转录程序。