Mechanism of HTLV-1 Tax-mediated activation of CDK6 transcription.

HTLV-1 Tax 介导的 CDK6 转录激活机制。

• 批准号: 7659886
• 负责人: Isabelle Michele Lemasson
• 金额: $ 17.92万
• 依托单位: EAST CAROLINA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7659886
• 关键词: Accounting; Adult; Affect; Antibodies; Be++ element; Beryllium; Binding; Binding Sites; Biochemical; Biological Assay; Blood Transfusion; Breast Feeding; CREB1 gene; Cell Cycle; Cell Cycle Progression; Cell Line; Cell Proliferation; Cell physiology; Cells; Chronic; Coitus; Complex; DNA; DNA Binding; Disease; Docking; EP300 gene; Elements; Event; Exhibits; Gene Expression; Gene Targeting; Genes; Genetic Enhancer Element; Genetic Transcription; Genome; Human Genome; Human T-lymphotropic virus 1; In Vitro; Individual; Infection; Maintenance; Malignant Neoplasms; Mediating; Modeling; Neurodegenerative Disorders; Paraplegia; Phase Transition; Play; Probability; Proteins; Proviruses; Recruitment Activity; Retroviridae; Role; Series; Site; Spinal Cord Diseases; Stimulation of Cell Proliferation; Surveys; Synapses; T-Cell Leukemia; T-Lymphocyte; Taxes; Testing; Transcription Coactivator; Transcription Regulatory Protein; Transcriptional Activation; Tropical Spastic Paraparesis; Viral; Viral Genome; Viral Load result; Viral Proteins; Virus; base; chromatin immunoprecipitation; cyclin-dependent kinase 6; design; in vivo; insight; intravenous drug use; leukemia; novel; overexpression; promoter; public health relevance; stoichiometry; tax Gene Products; therapeutic development; transcription factor; transmission process

DESCRIPTION (provided by applicant): The complex retrovirus, human T-cell leukemia virus type 1 (HTLV-1), is the etiological agent of a fatal T-cell malignancy termed adult T-cell leukemia and a neurodegenerative disorder defined as tropical spastic paraparesis/HTLV-1-associated myelopathy. The HTLV-1-encoded Tax protein plays an essential role in viral propagation by activating transcription of the chromosomally integrated HTLV-1 genome and by stimulating cell proliferation, which both serve to replicate the viral genome. Stimulation of cell proliferation is also paramount to the persistence of HTLV-1 infection and is believed to underlie the pathological effects caused by the virus. Among several cellular processes affected by Tax, its capacity to deregulate the expression of numerous cellular genes is a key factor leading to cell proliferation. Although the current paradigm suggests that Tax lacks DNA-binding activity, this protein is able to form complexes with the transcription factor CREB that then associate with enhancer elements within the viral promoter. These complexes nucleate the formation of a preinitiation complex that notably includes the coactivator p300, which is recruited directly by Tax. It is believed that Tax utilizes similar mechanisms to activate transcription from certain cellular promoters. To test this hypothesis, we used a ChIP-on-CHIP approach to identify regions within the human genome where Tax is specifically associated. Several sites were found to be potential targets for Tax-recruitment, among which a region of the CDK6 promoter consistently exhibited a high-level of Tax- enrichment. Cdk6 is a positive regulator of the G1- to S-phase transition and maintains cells in a state of proliferation. These functions parallel the effects of Tax in infected cells, and indeed, we have confirmed results from a previous study demonstrating that CDK6 expression is activated by Tax. Unexpectedly, further analysis of the association of Tax upstream of CDK6 revealed that Tax alone binds directly to the DNA, independently of a requirement for an ancillary factor. We also found that p300 is not recruited to this Tax-DNA complex. These findings are unprecedented and challenge some of the current models for Tax-mediated activation of transcription. Our objectives in this proposal are to, first, characterize the novel Tax-DNA complex and, second, define the Tax-dependent events involved in the activation of CDK6 expression. We will utilize a series of biochemical approaches to characterize the DNA element that is recognized by Tax, domains of the viral protein required for DNA-binding, and the stoichiometry of Tax when bound to DNA. We will employ complementary in vitro and in vivo approaches to identify cellular transcriptional regulators that are recruited to the CDK6 promoter by Tax, and define their roles during Tax-mediated transcriptional activation. These studies will yield a more refined understanding of events involved in the chronic proliferation of HTLV-1-infected cells and, ultimately, the pathological effects caused by the virus. In addition, they will provide insight into a novel mechanism through which Tax deregulates cellular gene expression. PUBLIC HEALTH RELEVANCE: Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with an aggressive leukemia and a neurodegenerative disease. This virus encodes a viral protein, Tax, involved in regulating viral replication and in activating cell cycle progression. This proposal investigates a novel mechanism of how Tax activates the transcription of a cellular gene important for cell cycle progression that, when overexpressed, potentially contributes to the proliferation of HTLV-1 infected cells.

描述（由申请方提供）：复杂逆转录病毒，人T细胞白血病病毒1型（HTLV-1），是一种称为成人T细胞白血病的致死性T细胞恶性肿瘤和一种定义为热带痉挛性下肢轻瘫/HTLV-1相关脊髓病的神经退行性疾病的病原体。HTLV-1编码的Tax蛋白通过激活染色体整合的HTLV-1基因组的转录和刺激细胞增殖在病毒繁殖中起重要作用，这两者都用于复制病毒基因组。刺激细胞增殖对于HTLV-1感染的持续性也至关重要，并且被认为是该病毒引起的病理效应的基础。在Tax影响的几个细胞过程中，其解除许多细胞基因表达的能力是导致细胞增殖的关键因素。尽管目前的研究表明Tax缺乏DNA结合活性，但这种蛋白能够与转录因子CREB形成复合物，然后与病毒启动子内的增强子元件结合。这些复合物使预起始复合物的形成成核，该预起始复合物特别包括由Tax直接募集的共激活因子p300。据信Tax利用类似的机制来激活某些细胞启动子的转录。为了验证这一假设，我们使用ChIP-on-CHIP方法来识别人类基因组中与Tax特异相关的区域。发现几个位点是Tax募集的潜在靶点，其中CDK 6启动子的一个区域始终表现出高水平的Tax富集。Cdk 6是G1-到S-期转变的正调节剂，并维持细胞处于增殖状态。这些功能与Tax在感染细胞中的作用相似，事实上，我们已经证实了先前研究的结果，表明Tax激活了CDK 6表达。出乎意料的是，对CDK 6上游的Tax关联的进一步分析显示，Tax单独直接与DNA结合，而不需要辅助因子。我们还发现，p300不招募到这个Tax-DNA复合物。这些发现是前所未有的，并挑战了目前的一些Tax介导的转录激活模型。我们的目标是，首先，描述新的Tax-DNA复合物，其次，定义参与CDK 6表达激活的Tax依赖性事件。我们将利用一系列生化方法来表征Tax识别的DNA元件、DNA结合所需的病毒蛋白结构域以及Tax与DNA结合时的化学计量。我们将采用互补的体外和体内方法来鉴定被Tax招募到CDK 6启动子的细胞转录调节因子，并定义它们在Tax介导的转录激活过程中的作用。这些研究将对HTLV-1感染细胞的慢性增殖所涉及的事件以及最终由病毒引起的病理效应产生更精确的理解。此外，他们还将深入了解Tax解除细胞基因表达调控的新机制。公共卫生相关性：人类T细胞白血病病毒1型（HTLV-1）是一种与侵袭性白血病和神经退行性疾病相关的逆转录病毒。该病毒编码一种病毒蛋白Tax，参与调节病毒复制和激活细胞周期进程。该提案研究了Tax如何激活对细胞周期进展重要的细胞基因的转录的新机制，该细胞基因在过度表达时可能有助于HTLV-1感染细胞的增殖。