Investigation of histone exchange mechanisms and their roles in viral gene expression

组蛋白交换机制及其在病毒基因表达中的作用的研究

• 批准号: RGPIN-2019-05452
• 负责人: Conn, Kristen
• 金额: $ 2.19万
• 依托单位: University of Saskatchewan
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=766040
• 关键词: Investigation; histone; exchange; mechanisms; their

The regulated expression of genes is fundamental to growth and development. Although much progress has been made towards a comprehensive understanding of the mechanisms that regulate gene expression, many areas remain unknown. Transcription and other processes that require access to the DNA are physically impeded by the assembly of DNA into chromatin with histones. DNA assembled in accessible chromatin is competent for transcription whereas that assembled in inaccessible chromatin is not. The mechanisms that regulate chromatin accessibility are not fully understood. It is also not clear how chromatin accessibility is modulated to enable transcription activation of DNA that is assembled into inaccessible chromatin. This proposed research program will use herpes simplex virus 1 (HSV) as a model system to investigate the processes that regulate chromatin accessibility and how they relate to transcription activation. Infecting HSV genomes are assembled into chromatin with cellular histones shortly after nuclear entry. Some HSV genomes are assembled into highly accessible, transcriptionally competent, chromatin, whereas the rest are assembled into highly inaccessible, transcriptionally incompetent, chromatin. The two populations of HSV chromatin within the same nucleus support a model whereby cellular antiviral defences attempt to silence HSV genomes through their assembly into inaccessible chromatin. HSV must counteract and disrupt silencing for viral genes to be expressed. Consistent with this model, the only essential HSV transcription activator, ICP4, enhances histone dynamics. Enhancing the dynamics of histones within the HSV chromatin would increase its accessibility and allow transcription. The enhancement of histone dynamics appears to be a novel mechanism of gene activation acquired by HSV to counteract cellular silencing attempts, although the mechanisms whereby ICP4 enhances histone dynamics are not known.  Our hypothesis is that histone dynamics regulate HSV chromatin accessibility. Graduate (4 MSc, 2 PhD) and Undergraduate students (5) will be recruited and trained in virology, cell culture, DNA- and protein-centered techniques, and advanced cell imaging to: (1) Identify the minimal ICP4 domains necessary and sufficient to enhance histone dynamics, (2) Identify ICP4-interacting proteins functionally relevant to the enhancement of histone dynamics, and (3) Test the conservation of ICP4 enhancement of histone dynamics and chromatin accessibility among alphaherpesviruses. This research will support my long-term objective to characterize the mechanisms that regulate chromatin accessibility in order to increase our fundamental understanding of the processes that activate transcription of DNA assembled into inaccessible chromatin. Knowledge gained from this research program will be of importance to researchers in the fields of virology, epigenetics and chromatin biology, developmental biology, and oncology, among others.

基因的调控表达是生长发育的基础。尽管在全面了解调控基因表达的机制方面取得了很大进展，但许多领域仍然未知。转录和其他需要进入DNA的过程在物理上受到DNA与组蛋白组装成染色质的阻碍。在可接近的染色质中组装的DNA具有转录能力，而在不可接近的染色质中组装的DNA则没有转录能力。调控染色质可及性的机制尚不完全清楚。也不清楚如何调节染色质的可接近性以使组装成不可接近的染色质的DNA的转录激活。这个拟议的研究计划将使用单纯疱疹病毒1 （HSV）作为模型系统来研究调节染色质可及性的过程及其与转录激活的关系。感染HSV的基因组在细胞核进入后不久与细胞组蛋白一起组装成染色质。一些HSV基因组组装成高度可接近的、转录能力强的染色质，而其余的则组装成高度不可接近的、转录能力弱的染色质。在同一细胞核内的两个HSV染色质群体支持一种模型，即细胞抗病毒防御试图通过将HSV基因组组装到不可接近的染色质中来沉默HSV基因组。单纯疱疹病毒必须抵消和破坏沉默，才能表达病毒基因。与该模型一致的是，唯一必需的HSV转录激活子ICP4增强了组蛋白动力学。增强HSV染色质内组蛋白的动态将增加其可及性并允许转录。虽然ICP4增强组蛋白动力学的机制尚不清楚，但组蛋白动力学的增强似乎是HSV获得基因激活的新机制，以抵消细胞沉默的企图。我们的假设是组蛋白动力学调节HSV染色质的可及性。研究生（4名硕士，2名博士）和本科生（5名）将被招募并接受病毒学，细胞培养，DNA和蛋白质中心技术以及高级细胞成像方面的培训，以：(1)确定增强组蛋白动力学所需和足够的最小ICP4结构域，(2)确定与增强组蛋白动力学功能相关的ICP4相互作用蛋白，以及(3)测试ICP4增强组蛋白动力学和α疱疹病毒染色质可及性的保护。这项研究将支持我的长期目标，即表征调节染色质可及性的机制，以增加我们对激活DNA转录组装成不可及的染色质的过程的基本理解。从这个研究项目中获得的知识将对病毒学、表观遗传学和染色质生物学、发育生物学和肿瘤学等领域的研究人员具有重要意义。