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

基本信息

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
项目类别：
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

项目摘要

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.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以出现在其他 CRISP 条目中列出的机构是。对于中心来说，它不一定是研究者的机构。在人类宿主初次感染期间，1 型单纯疱疹病毒 (HSV-1) 在神经元中建立潜伏期，其中病毒基因组保持与染色质构型中的核小体相关的环状形式，并且大多数病毒基因被沉默。我们进化出新的转录因子控制染色质修饰并在裂解感染和潜伏期调节 HSV-1 基因表达。对 HSV-1 DNA 序列的潜在调控元件的搜索发现了一个。 Roizman 实验室的结果表明，位于 HSV-1 基因 ICP22 和 ICP4 之间的阻遏元件 Element-1 (RE-1) 与 CoREST 具有序列相似性，并且破坏了 REST/CoREST/HDAC 复合物来调节 HSV-1 基因表达。没有解释该复合物如何被招募到 HSV-1 基因组中，我们捕获了阻遏元件沉默转录因子/神经元限制性沉默因子。 (REST/NRSF) 调节 ICP22 和 ICP4 的表达，表明 REST/NRSF 抑制两个启动子的活性，相反，仅编码蛋白质 DNA 结合结构域的 REST/NRSF 突变形式的共转染导致。含有带有染色质结构的游离报告质粒的稳定转化细胞系显示，REST/NRSF 特异性抑制 ICP4 启动子，但不抑制 ICP22。 ICP4 启动子的 REST/NRSF 抑制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) 测定表明辅阻遏物 CoREST 被招募到 ICP4 启动子附近。由于 ICP4 蛋白是 HSV-1 裂解的关键反式激活因子，因此组蛋白 H4 的乙酰化在 REST/NRSF 存在下降低。周期基因，这些结果表明 REST/NRSF 可能在潜伏期 HSV-1 基因沉默的建立和/或维持中发挥重要作用。我们发现 HSV-1 基因 TK 启动子中的甲状腺激素反应元件 (TRE) 是甲状腺激素等核激素受体的结合位点。受体 (TR) 是转录因子，其活性依赖于配体甲状腺激素 (T3)。我们使用神经母细胞瘤细胞研究 TR 和 T3 对 HSV-1 基因表达的作用。组成型表达 TR 亚型的细胞系 ¿ (N2aTR¿)。在本研究中，我们证明，在 T3 存在的情况下，TRs 通过瞬时转染和病毒感染抑制 TK 启动子活性，然而，数据显示，使用 LAT TRE 缺失的质粒，配体 TRs 激活了 LAT 转录。仅当 LAT TRE 存在时，ICP0 转录才会被 TR 和 T3 抑制，这表明 TR 被招募到 TK 和 LAT 中。独立于 T3 和超乙酰化 H4 的 TRE 与转录活性启动子相关。此外，我们的 ChIP 结果表明，在赖氨酸 9 和染色质绝缘子蛋白 CTCF 上修饰的抑制性单甲基化 H3 分别在 TK 和 LAT TRE 中富集。除了组蛋白修饰之外，LAT 的转录还受到染色质重塑因子 BRG1 复合物的过度表达。 BRG1 增强了 LAT 转录，BRG1 K785R 的显性失活突变体消除了 T3 处理的 N2aTR 的激活。细胞在低 moi 下抑制 TK 表达和病毒排出。然而，我们的 RT-PCR 和噬菌斑测定表明，当 T3 被去除时，TK 表达增强并且感染性病毒的释放增加。这些结果表明 T3 可能进行调节。 HSV-1 基因通过其受体通过组蛋白修饰和染色质重塑进行表达，从而控制病毒潜伏期和重新激活。