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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 单纯疱疹病毒1型(HSV-1)在其宿主的原发感染过程中，在神经元中建立潜伏期，病毒基因组以与染色质构型的核小体相关的环状形式保持，大多数病毒基因被沉默。导致这一现象的分子机制尚不清楚。我们假设，在裂解感染和潜伏期间，新的转录因子控制染色质的修饰和调节HSV-1基因的表达。对HSV-1 DNA序列的潜在调控元件的搜索发现了位于HSV-1基因ICP22和ICP4之间的抑制元件-1(RE-1)。Roizman实验室的结果表明，ICP0与COREST具有序列相似性，并破坏了REST/COREST/HDAC复合体来调节HSV-1基因的表达。然而，他们没有解释该复合体是如何被招募到HSV-1基因组中的。我们假设抑制因子沉默转录因子/神经元限制性沉默因子(REST/NRSF)调节ICP22和ICP4的表达。瞬时共转染表明，REST/NRSF抑制了两个启动子的活性。相反，共转染突变形式的REST/NRSF只编码蛋白质的DNA结合区域，导致明显较少的抑制。稳定转化的含有染色质结构的上体报告质粒的细胞系表明，REST/NRSF特异性地抑制ICP4启动子，但不抑制ICP22启动子。组蛋白脱乙酰酶(HDAC)抑制剂曲古抑素A(TSA)可逆转对ICP4启动子的REST/NRSF抑制作用。此外，染色质免疫沉淀(ChIP)分析表明，辅阻遏子核心位于ICP4启动子附近，在REST/NRSF存在下，组蛋白H4的乙酰化程度降低。由于ICP4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HSV-1潜伏期基因沉默的建立和/或维持中起重要作用。 目前已提出多种因素/途径来控制HSV-1潜伏和重新激活。我们鉴定了HSV-1基因TK和LAT启动子中的甲状腺激素反应元件(Tres)。TRES是甲状腺激素受体(TRs)等核激素受体的结合部位。TRs是一种转录因子，其活性依赖于配体甲状腺激素(T3)。我们利用一个组成性表达tr异构体的神经母细胞瘤细胞系(N2atr)，研究了tr和t3对HSV-1基因表达的影响。在目前的研究中，我们证明了在存在T3的情况下，TRS通过瞬时转染和病毒感染抑制了TK启动子的活性。然而，连接的TRs激活了LAT转录。利用LAT TRES中缺失的质粒，数据表明，只有当LAT TRES存在时，ICP0的转录才被TR和T3抑制。染色质免疫沉淀法(CHIP)表明，TK和LAT的TRES与T3无关，H4的高乙酰化与转录活性的启动子有关。此外，我们的芯片结果显示，在TR和T3存在下，在TK和LAT TRES中，赖氨酸9位修饰的抑制性单甲基化H3和染色质绝缘蛋白CTCF分别富含。除了组蛋白修饰外，染色质重塑因子BRG1复合体还进一步调节LAT的转录，因为BRG1的过表达促进了LAT的转录，而BRG1K785R的显性阴性突变体则取消了这种激活。低MOI时，T3处理的N2aTR细胞对TK的表达和病毒的排出均有抑制作用。然而，我们的RT-PCR和空斑分析表明，当去除T3时，TK的表达增强，感染性病毒的释放增加。这些结果提示，T3可能通过组蛋白修饰和染色质重塑，通过其受体调控HSV-1基因的表达，从而控制病毒潜伏期和再激活。

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.