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限于潜伏期相关转录本（LAT）。HSV-1作为与感觉神经元中的组蛋白相关的环状附加体保持潜伏，但具有响应于应激物而重新激活的能力。这种重新激活导致反复爆发，这是大多数HSV-1相关疾病和相关医疗保健费用的原因。不幸的是，调节潜伏期和HSV-1再激活的分子机制还没有很好的定义。我们研究的一个主要重点是确定控制HSV-1潜伏期和导致复发性HSV-1感染的分子机制。最近，我们发现HSV-1基因组上的表观遗传标记随着病毒从潜伏期重新激活而发生变化。我们还确定了细胞绝缘蛋白CTCF的DNA结合基序簇，这些基序围绕HSV-1的基因组区域，对于潜伏期的建立以及再激活都至关重要。这些结合基序在潜伏期期间在CTCF中富集，并且我们已经将三个CTCF位点表征为染色质绝缘体。在这个提议中要测试的总体假设是，细胞绝缘蛋白CTCF通过长距离染色质相互作用在潜伏期期间调节HSV-1基因表达，所述长距离染色质相互作用可以不稳定以促进HSV-1再激活。在目标1中，我们 使用染色质免疫沉淀测定表征HSV-1中CTCF结合基序的绝缘子功能，并绘制这些CTCF结合基序周围的组蛋白谱。这将使我们能够表征HSV-1基因组中可能负责促进HSV-1基因在潜伏期表达的其他元件。我们还将使用染色质构象捕获（3-C）测定来确定CTCF是否通过经典绝缘子功能（以线性方式）或间接通过形成染色质环结构域来调节HSV-1基因表达。最后，我们将确定HSV-1中CTCF结合结构域的突变是否会影响HSV-1潜伏期的基因表达。在本提案的目标2中，我们将使用高效兔眼模型研究CTCF介导的基因表达在体内再活化过程中的作用，以表征眼部发病机制和CTCF介导的HSV-1基因表达在体内再活化过程中的作用。拟议的研究将提供有关HSV-1基因组如何表观遗传学调节的关键见解，并定义CTCF和染色质绝缘子在潜伏期和重新激活期间在HSV-1基因调节中的作用。此外，这项工作可能会增加我们对CTCF如何从染色体元件调节细胞转录的理解。.

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. .