CTFC Mediated HSV-1 Gene Expression in Latency and Reactivation

单纯疱疹病毒1型（HSV-1）是一种重要的人类病原体，是美国因感染性病原体而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，其中裂解基因表达被抑制。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。目前普遍认为，HSV-1的LAT启动子和增强子以及立即早期（IE）基因通过染色质的沉积和维持来调节。然而，参与指导染色质沉积在潜伏期的机制尚未被定义，并在再激活过程中调节潜伏裂解开关的机制的识别仍然难以捉摸。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，以便将来可以开发新的HSV-1治疗干预措施。最近，我们确定了七个结合基序的细胞绝缘蛋白CTCF。HSV-1中每个基序的基因组位置显示CTCF结合结构域分别位于基因组的LAT和每个IE区域的侧翼，这提供了CTCF绝缘子可以控制潜在染色质募集和HSV-1基因表达的证据。我们发现，该网站被占领的绝缘子蛋白CTCF在潜伏期，三个网站的功能作为增强子阻断绝缘子，CTCF被驱逐在早期的时间在重新激活。随后，我们开创了一种基因递送方法，以证明CTCF耗竭驱动体内HSV-1再活化。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级染色质结构。染色质环使增强子和启动子在空间上接近以调节基因表达。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个不同的染色质环。我们是第一个发现α-疱疹病毒染色质环的研究者。因此，这些环中的每一个都采用3D构象，将IE基因定向为“关闭”或“准备重新激活”。我们假设HSV-1中的三个环1）通过募集共调节蛋白来控制染色质结构，和; 2）基于环构象，填充能够（或不能）再活化的神经元亚型。该项目的目的是系统地测试HSV-1中这些CTCF环的3D方向如何控制裂解，潜伏和再活化基因组。我们开创了新的技术来测试这一假设使用HSV-1感染的体内模型，使我们的研究不仅在分子水平上相关，但生理相关，以及我们在DNA病毒的表观遗传调控的专业知识，使我们的理想调查员进行这些研究。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，有条不紊和细致的评估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。

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.