Regulation of HSV-1 gene expression and reactivation by insulator protein CTCF

描述（由申请人提供）：单纯疱疹病毒1 （HSV-1）影响数百万人，是美国感染性失明的最大单一原因，HSV-1在感觉神经元中建立终身潜伏期，其中丰富的病毒基因表达仅限于潜伏期相关转录物（LAT）。HSV-1仍然作为与感觉神经元组蛋白相关的循环发作潜伏，但有能力在应激源下重新激活。这种重新激活导致反复暴发，这是造成大多数1型单纯疱疹病毒相关疾病和相关卫生保健费用的原因。不幸的是，调控潜伏期和HSV-1再激活的分子机制尚不明确。我们研究的一个主要重点是确定控制HSV-1潜伏期和促进复发性HSV-1感染的分子机制。最近，我们发现，当病毒从潜伏期重新激活时，存在于HSV-1基因组上的表观遗传标记发生了变化。我们还在HSV-1的基因组区域周围发现了细胞绝缘蛋白CTCF的DNA结合基序簇，这些基序对潜伏期的建立和再激活都至关重要。这些结合基序在潜伏期时在CTCF中富集，我们已经将三个CTCF位点描述为染色质绝缘体。本研究要验证的总体假设是，细胞绝缘蛋白CTCF通过远程染色质相互作用调节HSV-1基因在潜伏期的表达，这种相互作用可以被破坏以促进HSV-1的再激活。在Aim 1中，我们会

DESCRIPTION (provided by applicant): Herpes Simplex Virus 1 (HSV-1) affects millions of individuals, and is the single largest cause of infectious blindness in the U.S. HSV-1 establishes life-long latency in sensory neurons, where abundant viral gene expression is limited to the latency associated transcript (LAT). HSV-1 remains latent as a circular episome associated with histones in sensory neurons, but has the ability to reactivate in response to stressors. This reactivation causes recurrent outbreaks, which is responsible for the majority of HSV-1-related disease and the associated health care costs. Unfortunately, the molecular mechanisms that regulate latency and HSV-1 reactivation are not well defined. A major focus of our research is to determine the molecular mechanisms that control HSV-1 latency and contribute to recurrent HSV-1 infections. Recently, we found that epigenetic marks present on the HSV-1 genome change as the virus reactivates from latency. We have also identified clusters of DNA binding motifs for the cellular insulating protein, CTCF around genomic regions of HSV-1 that are critical for both the establishment of latency as well as in reactivation. These binding motifs are enriched in CTCF during latency and we have already characterized three CTCF sites as chromatin insulators. The overall hypothesis to be tested in this proposal is that the cellular insulating protein, CTCF, regulates HSV-1 gene expression during latency through long-range chromatin interactions that can be destabilized to facilitate HSV-1 reactivation. In Aim 1, we will characterize the insulator function of the CTCF binding motifs in HSV-1 using chromatin immunoprecipitation assays, and map the histone profiles around these CTCF binding motifs. This will allow us to characterize additional elements within the HSV-1 genome that might be responsible for facilitating HSV-1 gene expression during latency. We will also use chromatin conformation capture (3-C) assays to determine whether CTCF regulates HSV-1 gene expression through classic insulator function (in a linear manner) or indirectly through the formation of chromatin loop domains. Finally, we will determine whether mutations to the CTCF binding domains in HSV-1 affect HSV-1 gene expression during latency. In Aim 2 of this proposal, we will be investigating the role of CTCF-mediated gene expression during in vivo reactivation using the highly efficient rabbit ocular model to characterize ocular pathogenesis and CTCF-mediated HSV-1 gene expression during reactivation in vivo. The proposed studies will provide key insight into how the HSV-1 genome is regulated epigenetically, as well as define the role of CTCF and chromatin insulators in HSV-1 gene regulation during latency and in reactivation. In addition, this work will likely increase our understanding of how CTCF regulates cellular transcription from chromosomal elements. .