Functional analysis of downstream open chromatin induced in HSV-1 infection

单纯疱疹病毒1型(HSV-1)是常见的唇疱疹的病原体，但也是严重威胁生命的疾病，包括脑炎、肺炎、肝炎和全身皮肤感染。此外，HSV-1是一种在RNA水平上实现病毒诱导的宿主关闭的范例。最近，我们发现HSV-1触发了细胞内广泛的转录终止中断(DOTT)，而不是病毒基因，导致了Poly(A)位点以外的广泛的直读转录。随后，在细胞应激反应和癌症中也报道了类似的观察结果。我们现在发现，HSV-1诱导的DOTT类似于细胞应激反应，导致通读转录本的核滞留，从而导致宿主关闭。目前应用的焦点是引人注目的观察到，HSV-1诱导的Dott，而不是压力诱导的通读转录，伴随着受影响的Poly(A)位点下游染色质可及性的广泛增加。这些下游的开放染色质区域(DOCR)延伸到数万个核苷酸，基本上与转录通读区域匹配。在这项建议中，我们现在试图确定诱导dOCRs的分子机制，并评估其与产生性HSV-1感染的功能相关性。拟议的项目结合了湿实验室工作和生物信息学分析来检验我们的假设：(1)在HSV-1感染中，组蛋白的流动性增加是由于组蛋白在PolII转录时重新定位到基因外的基因组区域而造成的；(2)组蛋白在DOCR形成过程中的释放解释了先前报道的游离核质组蛋白池的增加；(3)组蛋白流动性的相关增加促进了复制病毒DNA的染色质化。从这项工作中获得的结果将详细说明一种重要的人类病原体如何操纵转录机制以促进有效的病毒复制。此外，它还将开创一个迷人的新模型，用于研究人类细胞中协调基因表达和染色质结构的分子机制。

Herpes simplex virus 1 (HSV-1) is the causative agent of the common cold sores but also responsible for severe life-threatening diseases including encephalitis, pneumonia, hepatitis and generalized skin infections. Furthermore, HSV-1 is a paradigm for virus-induced host shut-off exerted at RNA level. Recently, we showed that HSV-1 triggers a widespread disruption of transcription termination (DoTT) of cellular but not viral genes, resulting in extensive read-through transcription beyond poly(A) sites. Subsequently, similar observations were also reported in cellular stress responses and cancer. We now found that HSV-1 induced DoTT resembles a cellular stress response and leads to nuclear retention of read-through transcripts, thereby contributing to host shut-off. The focus of the current application is the striking observation that HSV-1-induced DoTT, but not stress-induced read-through transcription, was accompanied by an extensive increase in chromatin accessibility downstream of the affected poly(A) sites. These downstream open chromatin regions (dOCRs) extend for tens-of-thousands of nucleotides and essentially match the region of transcription read-through. In this proposal, we now seek to identify the molecular mechanism responsible for the induction of dOCRs and assess its functional relevance for productive HSV-1 infection. The proposed project combines wet-lab work and bioinformatics analyses to test our hypothesis that: (i) dOCRs in HSV-1 infection arise from an impairment in histone repositioning upon Pol II transcription into genomic regions outside of genes; (ii) histone release during dOCR formation explains the previously reported increase in the pool of free nucleoplasmic histones; (iii) the associated increase in histone mobility facilitates chromatinization of the replicating viral DNA. Results obtained from this work will detail how an important human pathogen manipulates the transcriptional machinery to promote productive viral replication. In addition, it will pioneer a fascinating new model for studying the molecular mechanisms orchestrating gene expression and chromatin architecture in human cells.