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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 在其人类宿主的初次感染期间，单纯疱疹病毒类型-1（HSV-1）在神经元中建立了潜伏期，在神经元中，病毒基因组以与核形式相关的圆形形式保持在染色质构型中，并且大多数病毒基因被沉默。负责此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并调节裂解感染和潜伏期期间的HSV-1基因表达。对潜在调节元件的HSV-1 DNA序列的搜索确定了位于HSV-1基因ICP22和ICP4之间的阻遏元件-1（RE-1）。罗伊曼（Roizman）实验室的结果表明，ICP0暴露了与Corest的序列相似性，并破坏了REST/COREST/HDAC复合物以调节HSV-1基因表达。但是，他们没有解释如何将复合物招募到HSV-1基因组。我们假设阻遏物元件沉默转录因子/神经元素限制性沉默因子（REST/NRSF）调节ICP22和ICP4的表达。瞬时共转染表明，静止/NRSF抑制了两个启动子的活性。相比之下，仅编码蛋白质的DNA结合结构域的静止/NRSF突变形式的共转染导致抑制作用明显较小。含有染色质结构的含有伴有染色质结构的杂物记者质粒的细胞系稳定转化，表明REST/NRSF专门抑制ICP4启动子，但没有抑制ICP22启动子。组蛋白脱乙酰基酶（HDAC）抑制剂trichostatin A（TSA）逆转了ICP4启动子的静息/NRSF抑制。此外，染色质免疫沉淀（CHIP）测定表明，将CoreST募集到ICP4启动子的接近度，并且在REST/NRSF存在下，组蛋白H4的乙酰化H4降低。由于ICP4蛋白是HSV-1裂解周期基因的关键反式激活剂，因此这些结果表明REST/NRSF在延迟期间HSV-1基因沉默的建立和/或维持中可能具有重要作用。 已经提出了多种因素/途径来控制HSV-1潜伏期和重新激活。我们确定了HSV-1基因TK和LAT启动子中的甲状腺激素反应元件（TRE）。 TRE是核马酮受体的结合位点，例如甲状腺激素受体（TRS）。 TR是转录因子，其活性取决于配体甲状腺激素（T3）。我们使用神经细胞瘤细胞系组成型表达TR tr同工型（N2ATR？），研究TR和T3在HSV-1基因表达上的作用。在本研究中，我们证明了通过瞬时转染和病毒感染在T3存在下将TRS复制的。但是，配体TRS激活了LAT转录。使用LAT TRES中缺失的质粒，数据表明，仅当存在LAT TRE时，ICP0转录才由TR和T3复制。染色质免疫沉淀（CHIP）说明，将TRS独立于T3募集到TK和LAT TRE，而高乙酰化H4与转录活性的启动子有关。此外，我们的芯片结果表明，在TR和T3的存在下，分别将赖氨酸9和染色质蛋白CTCF的反射单甲基化H3分别富含TK和LAT TRE。除组蛋白的修饰外，LAT的转录还受染色质重塑因子BRG1复合物的进一步调节，因为BRG1的过表达增强了BRG1 K785R的LAT转录和显性阴性突变体废除了激活。 T3处理的N2ATR？细胞在低MOI处抑制了TK表达和病毒出口。但是，我们的RT-PCR和斑块测定法表明，去除T3时，TK表达得到了增强，并释放了传染性病毒。这些结果表明，T3可以通过组蛋白修饰和染色质重塑来调节HSV-1基因的表达，从而控制病毒潜伏期和重新激活。