CTFC Mediated HSV-1 Gene Expression in Latency and Reactivation

单纯疱疹病毒1 （HSV-1）是一种重要的人类病原体，是美国因感染因子导致失明的主要原因。HSV-1在宿主感觉神经元中建立终身潜伏感染，其中裂解基因表达受到抑制。潜伏的HSV-1周期性重新激活，复发通常与HSV-1相关疾病有关。目前人们普遍认识到，HSV-1的LAT启动子和增强子以及即时早期（IE）基因是通过染色质的沉积和维持来调节的。然而，在潜伏期间指导染色质沉积的机制尚未明确，并且在再激活期间调节溶潜开关的机制仍然难以捉摸。我们研究的一个主要重点是克服这些知识上的重大差距，以便将来能够开发出新的1型单纯疱疹病毒治疗干预措施。最近，我们发现了细胞绝缘蛋白CTCF的7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IE区域两侧，这证明CTCF绝缘子可以控制潜在的染色质募集和HSV-1基因表达。我们发现，在潜伏期，该位点被绝缘子蛋白CTCF占据，三个位点作为增强子阻断绝缘子，CTCF在再激活的早期被驱逐。随后，我们开创了一种基因传递方法，证明CTCF消耗会在体内驱动HSV-1再激活。CTCF绝缘子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阶染色质结构。染色质环使增强子和启动子在空间上接近，从而调控基因表达。通过对潜伏感染的小鼠神经元进行深度测序（3C-seq），我们发现了3个不同的染色质环。我们是第一个在甲型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中的三个环通过募集共调节蛋白来控制染色质结构；2)根据环路构象填充能够（或不能）重新激活的神经元亚型。该项目的目的是系统地测试HSV-1中这些CTCF环的三维取向如何控制裂解，潜伏和重新激活基因组。我们开创了新的技术，利用HSV-1感染的体内模型来测试这一假设，使我们的研究不仅在分子水平上相关，而且在生理上相关，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者。总之，我们将通过结合遗传学、分子生物学和体内模型，将疱疹病毒的表观遗传控制领域推向从未探索过的领域，以实现对病毒生命周期各个阶段ctcf -染色质环如何控制病毒转录程序的全面、系统和细致的评估。

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.