ROLE OF CHROMATIN IN HERPES SIMPLEX VIRUS TYPE 1 (HSV-1) GENE REGULATION

染色质在 1 型单纯疱疹病毒 (HSV-1) 基因调控中的作用

• 批准号: 7720007
• 负责人: Shaochung Victor Hsia
• 金额: $ 12.41万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720007
• 关键词: Acetylation; Binding Sites; Biological Assay; CCCTC-binding factor; Cell Line; Cells; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA Binding Domain; DNA Sequence; Data; Dominant-Negative Mutation; Elements; Exhibits; Funding; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic Transcription; Genome; Grant; Herpesvirus 1; Histone Deacetylase; Histone Deacetylase Inhibitor; Histone H4; Histones; Human; Infection; Institution; Ligands; Lysine; Lytic Phase; Maintenance; Modification; Molecular; Mono-S; Neurons; Nuclear Hormone Receptors; Nucleosomes; Pathway interactions; Plaque Assay; Plasmids; Precipitation; Protein Isoforms; Protein Overexpression; Proteins; Recruitment Activity; Regulatory Element; Reporter; Research; Research Personnel; Resources; Reverse Transcriptase Polymerase Chain Reaction; Role; SMARCA4 gene; Source; TK Gene; Thyroid Hormone Receptor; Thyroid Hormones; Trans-Activators; Transcriptional Silencer Elements; Transfection; Transformed Cell Line; Trichostatin A; Triiodothyronine; United States National Institutes of Health; Viral; Viral Genome; Virus; Virus Diseases; Virus Latency; chromatin immunoprecipitation; chromatin remodeling; hormone response element; mutant; novel; promoter; reactivation from latency; receptor; transcription factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During primary infection of its human host, Herpes Simplex Virus Type-1 (HSV-1) establishes latency in neurons where the viral genome is maintained in a circular form associated with nucleosomes in a chromatin configuration and most viral genes are silenced. The molecular mechanisms responsible for this are unclear. We hypothesized that novel transcription factors control chromatin modifications and regulate HSV-1 gene expression during lytic infection and latency. A search of the HSV-1 DNA sequence for potential regulatory elements identified a Repressor Element-1 (RE-1) located between HSV-1 genes ICP22 and ICP4. Results from Roizman's lab suggested that ICP0 exhibited sequence similarity to CoREST and disrupted REST/CoREST/HDAC complex to regulate HSV-1 gene expression. However, they did not explain how the complex was recruited to HSV-1 genome. We hypothesize that the Repressor Element Silencing Transcription Factor/Neuronal Restrictive Silencer Factor (REST/NRSF) regulates expression of ICP22 and ICP4. Transient cotransfection indicated that REST/NRSF inhibited the activity of both promoters. In contrast, cotransfection of a mutant form of REST/NRSF encoding only the DNA-binding domain of the protein resulted in significantly less inhibition. Stably transformed cell lines containing episomal reporter plasmids with a chromatin structure showed that REST/NRSF specifically inhibited the ICP4 promoter, but not the ICP22 promoter. REST/NRSF inhibition of the ICP4 promoter was reversed by histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). Additionally, chromatin immuno-precipitation (ChIP) assays indicated that the corepressor CoREST was recruited to the proximity of ICP4 promoter and that acetylation of histone H4 was reduced in the presence of REST/NRSF. Since the ICP4 protein is a key transactivator of HSV-1 lytic cycle genes, these results suggest that REST/NRSF may have an important role in the establishment and/or maintenance of HSV-1 gene silencing during latency. Multiple factors/pathways have been suggested to control HSV-1 latency and reactivation. We identified thyroid hormone response elements (TREs) in the promoters of HSV-1 genes TK and LAT. TREs are the binding sites of nuclear hormone receptors such as thyroid hormone receptors (TRs). TRs are transcription factors whose activity is dependent on the ligand thyroid hormone (T3). We investigate the roles of TR and T3 on HSV-1 gene expression using a neuoroblastoma cell line constitutively expressing TR isoform ¿ (N2aTR¿). In the present study, we demonstrated that TRs repressed TK promoter activity in the presence of T3 by transient transfection and viral infection. However, the liganded TRs activated LAT transcription. Using plasmids with deletion in LAT TREs, the data showed that ICP0 transcription was repressed by TR and T3 only when the LAT TREs were present. Chromatin immunoprecipitation (ChIP) illustrated that TRs were recruited to TK and LAT TREs independently of T3 and hyperacetylated H4 was associated with promoters that were transcriptionally active. In addition, our ChIP results showed that a repressive mono methylated H3 modified at lysine 9 and chromatin insulator protein CTCF was enriched in the TK and LAT TREs, respectively, in the presence of TR and T3. Besides histone modification, transcription of LAT was further regulated by chromatin remodeling factor BRG1 complex since overexpression of BRG1 enhanced the LAT transcription and dominant negative mutant of BRG1 K785R abolished the activation. T3-treated N2aTR¿ cells were suppressive to TK expression and virus egress at low moi. However, our RT-PCR and plaque assays showed that the TK expression was enhanced and the release of infectious viruses was increased when the T3 was removed. These results suggested that T3 may regulate the HSV-1 gene expression through its receptor via histone modification and chromatin remodeling and therefore control viral latency and reactivation.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在其人类宿主的初次感染期间，单纯疱疹病毒1型（HSV-1）在神经元中建立潜伏期，其中病毒基因组以与核小体相关的环状形式维持在染色质构型中，并且大多数病毒基因被沉默。对此负责的分子机制尚不清楚。我们假设，新的转录因子控制染色质修饰和调节HSV-1基因表达在裂解感染和潜伏期。对HSV-1 DNA序列进行搜索以寻找潜在的调控元件，鉴定出位于HSV-1基因ICP 22和ICP 4之间的阻遏元件-1（RE-1）。Roizman实验室的结果表明，ICP 0与CoREST具有序列相似性，并破坏REST/CoREST/HDAC复合物以调节HSV-1基因表达。然而，他们没有解释复合物是如何被招募到HSV-1基因组中的。我们假设阻遏元件沉默转录因子/神经元限制性沉默因子（REST/NRSF）调节ICP 22和ICP 4的表达。瞬时共转染表明REST/NRSF抑制两种启动子的活性。与此相反，共转染的REST/NRSF的突变体形式编码的DNA结合结构域的蛋白质导致显着减少抑制。含有具有染色质结构的附加型报告质粒的稳定转化的细胞系表明，REST/NRSF特异性抑制ICP 4启动子，但不抑制ICP 22启动子。REST/NRSF对ICP 4启动子的抑制作用可被组蛋白脱乙酰酶（HDAC）抑制剂曲古抑菌素A（TSA）逆转。此外，染色质免疫沉淀（ChIP）分析表明，辅阻遏物CoREST被募集到ICP 4启动子附近，组蛋白H4的乙酰化在REST/NRSF的存在下减少。由于ICP 4蛋白是HSV-1裂解周期基因的关键反式激活因子，这些结果表明REST/NRSF可能在潜伏期HSV-1基因沉默的建立和/或维持中起重要作用。 已提出多种因素/途径来控制HSV-1潜伏期和再激活。我们在HSV-1基因TK和LAT的启动子中鉴定了甲状腺激素反应元件（TRE）。TREs是核激素受体如甲状腺激素受体（TR）的结合位点。TR是转录因子，其活性取决于配体甲状腺激素（T3）。我们使用组成型表达TR亚型（N2 aTR）的神经母细胞瘤细胞系研究TR和T3对HSV-1基因表达的作用。在本研究中，我们通过瞬时转染和病毒感染证明，在T3存在的情况下，TR抑制TK启动子活性。然而，配体TR激活LAT转录。使用在LAT TREs中具有缺失的质粒，数据显示，只有当LAT TREs存在时，ICP 0转录才被TR和T3抑制。染色质免疫沉淀（ChIP）表明，TRs募集到TK和LAT TRs独立的T3和超乙酰化的H4是与启动子的转录活性。此外，我们的ChIP结果表明，在TR和T3的存在下，在赖氨酸9和染色质绝缘子蛋白CTCF处修饰的抑制性单甲基化H3分别富集在TK和LAT TREs中。除了组蛋白修饰外，染色质重塑因子BRG 1复合物还进一步调节LAT的转录，因为BRG 1的过表达增强了LAT的转录，而BRG 1的显性负突变体K785 R取消了激活。T3处理的N2 aTR？细胞在低moi下抑制TK表达和病毒排出。然而，我们的RT-PCR和空斑试验表明，TK表达增强，感染性病毒的释放增加时，T3被删除。提示T3可能通过其受体介导的组蛋白修饰和染色质重塑来调控HSV-1基因的表达，从而控制病毒的潜伏期和再激活。