Tat and CDK9 on gene expression alterations coupled to HIV-1 Associated Dementia

Tat 和 CDK9 与 HIV-1 相关痴呆相关的基因表达改变

• 批准号: 7685229
• 负责人: Xavier Grana
• 金额: $ 22.5万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-15 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7685229
• 关键词: AIDS Dementia Complex; Affect; Astrocytes; Astrocytoma; B-Lymphocytes; Binding; Boxing; Brain; Catalytic Domain; Cell Line; Cell physiology; Cells; Cognition Disorders; Complex; Coupled; Cyclins; Data; Dementia; Dendritic Cells; Elements; Elongation Factor; Equilibrium; FGF2 gene; GADD45A gene; GADD45B; Gene Expression; Gene Expression Alteration; Genes; Genetic Transcription; Goals; HIV-1; Hela Cells; Highly Active Antiretroviral Therapy; Homeostasis; Human; IL8 gene; Immune; Incidence; Indium; Infection; Interleukin-12; Mediating; Molecular; NF-kappa B; Names; Nerve Degeneration; Neurons; Pathogenesis; Patients; Phosphotransferases; Physiological; Play; Positioning Attribute; Positive Transcriptional Elongation Factor B; Prevalence; Property; Proteins; RNA; RNA Polymerase II; Recruitment Activity; Reporter; Response to stimulus physiology; Role; SOD2 gene; Stress; Survival Rate; System; Tars; Testing; Trans-Activators; Transactivation; Transcript; Transcription Coactivator; Transcription Elongation; Up-Regulation; Viral; Viral Proteins; Virus; cell type; chromatin immunoprecipitation; cyclin T1; factor EF-P; insight; macrophage; mutant; negative elongation factor; nervous system disorder; neurogenesis; neurotoxic; promoter; public health relevance; restoration; transcription factor; uptake

DESCRIPTION (provided by applicant): Dementia and other milder forms of cognitive diseases of the CNS affect HIV-1 infected patients. While HAART has diminished the incidence of HIV-1-associated dementia (HAD), milder forms of neurological disease persist. Also, increased survival rates resulting from HAART have led to an increase in the prevalence of neurological disorders. HIV-induced neurodegeneration may result at least in part from changes in cellular gene expression induced by HIV-1 Tat in a variety of cell types including astrocytes and neurons. Tat is an essential viral transactivator that requires association with a cellular kinase composed of a regulatory subunit designated cyclin T1 and a catalytic subunit named CDK9. This host complex is recruited by Tat to TAR, an RNA element in the nascent HIV-1 transcript, where CDK9 is positioned to phosphorylate RNA polymerase II (RNAPII) and negative elongation factors, strongly activating HIV-1 transcription. CDK9 in association with T-type cyclins forms Positive Transcription Elongation Factors (P-TEFb) thought to be required for the expression of cellular genes. We have investigated the effect of limiting CDK9 activity in a human astrocytoma cell line. Our gene profiling analysis shows that CDK9 activity is not homogeneously required for the expression of cellular genes, but rather that inhibition of CDK9 leads to selective changes in gene expression. Among the genes downregulated we have identified various transcription factors. Our data also show that inhibition of CDK9 results in the upregulation of a subset of genes including stress and stimuli response genes (Gadd45A, Gadd45B, IL-8 and IL-12), as well as deregulation of a number of genes that have been associated with dementia (GLS, SOD2, FGF2 and CHMP2B). Cellular CDK9 is sequestered in catalytically inactive and active complexes in an equilibrium that is in place to restrict gene expression. Importantly, Tat inhibits cyclin T1/CDK9-dependent gene expression of certain genes in macrophages, dendritic cells and B cells via a competition mechanism with specific transcriptional activators. Since Tat reconfigures pools of CDK9 complexes in HIV-1 infected HeLa cells and Tat expressing primary human astrocytes and astrocytoma cells, it is likely that similar competition mechanisms operate in astrocytes and neurons with other transcription factors. Thus, the goal of this exploratory R21 proposal is to test the hypothesis that the effects of Tat in gene expression that may contribute to HAD are at least in part mediated through interference with the host CDK9 function and that restoration of normal CDK9 function should ameliorate the detrimental effects of Tat expression and HIV-1 infection in the CNS. To test this hypothesis we propose: (Aim 1) to determine the role of cellular CDK9 in the alterations on gene expression triggered by HIV-1 Tat in astrocytes and neurons; and (Aim 2) to determine whether the effects of Tat in gene expression can be reverted by restoring normal cellular CDK9 function in Tat expressing astrocytes and neurons. The short-term benefit of these studies is gaining insight into the molecular mechanisms by which Tat remodels cellular gene expression in astrocytes and neurons contributing to neurodegeneration. The results of these studies could set the path to subsequent structural studies to search for small compounds with the ability to bind cyclin T1 blocking Tat without altering CDK9 dependent cellular gene expression. in astrocytes and neurons PUBLIC HEALTH RELEVANCE: Astrocytes are the most abundant cell type in the brain and are critical for neuronal function, survival and neurogenesis. Astrocytes are infected by HIV-1, and viral proteins including Tat are made, but the virus does not productively replicate. Given their abundance and importance, astrocytes represent a very significant reservoir of infected cells that express HIV-1 proteins with neurotoxic properties. These proteins may affect astrocyte homeostasis, which in turn will influence neuronal function or might be secreted and taken up by neurons to directly affect them contributing to HAD. HIV-1 Tat is an essential viral transactivator that hijacks the cellular cyclin T1/CDK9 transcription elongation factor to promote HIV-1 replication. Tat also alters cellular gene expression, which contributes to HIV-1-associated pathogenesis by altering immune cell function. As Tat expression rearranges cellular P-TEFb complexes in human astrocytes, and these complexes are thought to play rate-limiting roles in transcription, it is hypothesized that P-TEFb deregulation by Tat induces selective changes in gene expression in astrocytes and neurons that contribute to HIV-1-associated dementia. We propose to test the hypothesis that the effects of Tat in gene expression are at least in part mediated through interference with the host CDK9 function and that restoration of normal CDK9 function should ameliorate the detrimental effects of Tat expression and HIV-1 infection in the CNS.

描述（由申请人提供）：HIV-1感染患者会出现痴呆和其他较轻形式的中枢神经系统认知疾病。虽然HAART降低了hiv -1相关痴呆（HAD）的发病率，但较轻形式的神经系统疾病仍然存在。此外，高效抗逆转录病毒疗法提高的存活率导致神经系统疾病的患病率增加。hiv诱导的神经退行性变可能至少部分是由HIV-1 Tat在包括星形胶质细胞和神经元在内的多种细胞类型中诱导的细胞基因表达变化引起的。Tat是一种必需的病毒反激活因子，它需要与细胞激酶结合，细胞激酶由一个被称为周期蛋白T1的调节亚基和一个被称为CDK9的催化亚基组成。这个宿主复合体被Tat招募到TAR， TAR是新生HIV-1转录物中的一种RNA元件，CDK9定位于磷酸化RNA聚合酶II （RNAPII）和负伸长因子，强烈激活HIV-1转录。CDK9与t型细胞周期蛋白相关，形成被认为是细胞基因表达所必需的正转录延伸因子（P-TEFb）。我们研究了限制CDK9活性对人类星形细胞瘤细胞系的影响。我们的基因谱分析表明，CDK9活性不是细胞基因表达所必需的，而是抑制CDK9导致基因表达的选择性改变。在下调的基因中，我们已经确定了各种转录因子。我们的数据还显示，抑制CDK9导致包括应激和刺激反应基因（Gadd45A、Gadd45B、IL-8和IL-12）在内的一组基因上调，以及与痴呆相关的一些基因（GLS、SOD2、FGF2和CHMP2B）的失调。细胞CDK9在限制基因表达的平衡中被隔离在催化无活性和活性复合物中。重要的是，Tat通过与特定转录激活因子的竞争机制抑制巨噬细胞、树突状细胞和B细胞中cyclin T1/ cdk9依赖基因的某些基因的表达。由于Tat在HIV-1感染的HeLa细胞和表达原代人星形胶质细胞和星形细胞瘤细胞的Tat中重新配置了CDK9复合物池，因此星形胶质细胞和神经元中可能存在与其他转录因子相似的竞争机制。因此，本探索性R21提案的目的是验证Tat对HAD基因表达的影响至少部分是通过干扰宿主CDK9功能介导的，并且恢复正常的CDK9功能应该可以改善Tat表达和HIV-1感染在中枢神经系统中的有害影响。为了验证这一假设，我们提出：（目的1）确定细胞CDK9在星形胶质细胞和神经元中由HIV-1 Tat引发的基因表达改变中的作用；以及（目的2）确定Tat对基因表达的影响是否可以通过恢复表达Tat的星形胶质细胞和神经元的正常细胞CDK9功能来逆转。这些研究的短期利益是深入了解Tat在星形胶质细胞和神经元中重塑细胞基因表达的分子机制，从而促进神经退行性变。这些研究的结果可以为后续的结构研究开辟道路，以寻找能够结合cyclin T1阻断Tat而不改变CDK9依赖性细胞基因表达的小化合物。公共卫生相关性：星形胶质细胞是大脑中最丰富的细胞类型，对神经元功能、存活和神经发生至关重要。星形胶质细胞被HIV-1感染后，会产生包括Tat在内的病毒蛋白，但病毒不会进行有效复制。鉴于星形胶质细胞的丰度和重要性，它们代表了表达具有神经毒性的HIV-1蛋白的感染细胞的一个非常重要的储存库。这些蛋白可能影响星形胶质细胞的稳态，进而影响神经元的功能，或者可能被神经元分泌和吸收，直接影响它们，从而导致HAD。HIV-1 Tat是一种重要的病毒反激活因子，它劫持细胞周期蛋白T1/CDK9转录延伸因子来促进HIV-1复制。这也会改变细胞基因表达，从而通过改变免疫细胞功能导致hiv -1相关发病。由于Tat的表达重排了人类星形胶质细胞中的P-TEFb复合物，并且这些复合物被认为在转录中起限速作用，因此假设Tat对P-TEFb的失调会诱导星形胶质细胞和神经元中基因表达的选择性改变，从而导致hiv -1相关痴呆。我们建议验证Tat对基因表达的影响至少部分是通过干扰宿主CDK9功能介导的，并且恢复正常的CDK9功能应该可以改善Tat表达和HIV-1感染在中枢神经系统中的有害影响。