Regulation of late genes and the transcriptional repressor protein EUO in Chlamyd

衣原体晚期基因和转录抑制蛋白 EUO 的调控

• 批准号: 8715506
• 负责人: Brett Hanson
• 金额: $ 5.15万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-09 至 2016-05-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8715506
• 关键词: 5' Untranslated Regions; Adopted; Affinity; Bacteria; Binding; Binding Sites; Cell Culture Techniques; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; DNA; DNA Sequence; Data; Development; Down-Regulation; Escherichia coli; Expression Library; Functional RNA; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Glean; Goals; Health; In Vitro; Infection; Knowledge; Late Promoters; Lead; Libraries; Light; Massive Parallel Sequencing; Mediating; Methods; Mission; Modeling; Molecular; Morbidity - disease rate; National Institute of Allergy and Infectious Disease; Nature; Proteins; Public Health; Recombinants; Regulation; Regulator Genes; Reporter; Reporter Genes; Repression; Repressor Proteins; Research; Role; Site; Staging; System; Time; Transcript; Transcription Repressor/Corepressor; Transcriptional Regulation; Translational Repression; Translations; Validation; chromatin immunoprecipitation; derepression; design; improved; in vivo; innovation; insight; novel therapeutic intervention; pathogen; premature; prevent; promoter; public health relevance

DESCRIPTION (provided by applicant): The unique infectious cycle adopted by Chlamydia is characterized by temporal regulation of early, middle, and late genes expressed at appropriate times during the developmental cycle. The precise timing of expression for each of these temporal classes of genes relies on distinct mechanisms of transcriptional control. Chlamydia can also be induced to enter an aberrant state in cell culture called persistence, with development halted midway through the developmental cycle. Despite the importance of temporal gene regulation in development of chlamydial disease, knowledge of the molecular mechanisms involved in regulation is limited. Among these temporal transcriptional controls is an early chlamydial protein, EUO, which has been shown to bind to late gene promoters to inhibit transcription. The objective of the proposed research is to identify the regulatory mechanisms imposed by EUO and regulation of EUO itself. The central hypothesis of the proposed study is that EUO binds and represses late gene promoters during early and middle stages of the developmental cycle, and that this repression is alleviated prior to progression into the late developmental stage. Furthermore, during persistence EUO may act to maintain a persistent state of infection by blocking transition into the late developmental stage. The specifi aims of the proposed study are; 1) Determine promoters bound in vivo by EUO ; 2) Identify the mechanism used to alleviate EUO mediated repression; and 3) Determine the role of EUO in persistence and reactivation. In the first aim, analysis of in vivo binding of EUO to late gene promoters during infection will use Chromatin Immunoprecipitation (ChIP) with subsequent sequencing of the DNA to identify EUO binding sites in vivo. In the second aim, there is evidence that EUO protein levels are downregulated to alleviate repression, and this is likely due to inhibition of translation through a small non-coding RNA (sRNA). A regulatory sRNA will be investigated using a sRNA expression library in conjunction with a reporter system fused to euo to assess translational inhibition. In the third aim, persistence is characterized by a block i late gene expression and we propose that regulation of EUO levels is altered, leading to continuous repression of late genes. We will use the methods from Aims 1 and 2 to investigate this. The proposed research is innovative because it will shed light on an important regulator of the chlamydial developmental cycle. This contribution is significant to the mission of NIAID because insight gleaned from these studies may lead to successful new strategies for treating chlamydial infections by targeting master regulators of the developmental cycle, such as EUO.

描述（由申请方提供）：衣原体采用的独特感染周期的特征是在发育周期的适当时间表达的早期、中期和晚期基因的时间调节。这些基因的时间类别中的每一个的精确表达时间依赖于转录控制的不同机制。衣原体也可以被诱导进入一个异常的状态，在细胞培养称为持久性，与发展停止中途通过发展周期。尽管时间基因调控在衣原体疾病发展中的重要性，但对调控中涉及的分子机制的认识是有限的。在这些时间转录控制是早期衣原体蛋白，EUO，它已被证明可以结合到晚期基因启动子，以抑制转录。拟议的研究的目的是确定EUO和EUO本身的监管规定的监管机制。这项研究的中心假设是，EUO在发育周期的早期和中期结合并抑制晚期基因启动子，并且这种抑制在进入发育周期之前减轻。 发育后期。此外，在持久性EUO可能会采取行动，以保持一个持久的状态，通过阻止过渡到后期发展阶段的感染。所提出的研究的具体目的是：1）确定EUO在体内结合的启动子; 2）确定用于减轻EUO介导的抑制的机制;和3）确定EUO在持久性和再激活中的作用。在第一个目标中，在感染期间EUO与晚期基因启动子的体内结合的分析将使用染色质免疫沉淀（ChIP），随后对DNA进行测序以鉴定体内EUO结合位点。在第二个目标中，有证据表明EUO蛋白水平下调以减轻抑制，这可能是由于通过小的非编码RNA（sRNA）抑制翻译。将使用sRNA表达文库结合与euo融合的报告系统研究调节sRNA，以评估翻译抑制。在第三个目标中，持久性的特点是一个块I晚期基因表达，我们建议，EUO水平的调节被改变，导致持续的晚期基因的抑制。我们将使用目标1和目标2中的方法对此进行研究。这项拟议的研究具有创新性，因为它将揭示衣原体发育周期的重要调节因子。这一贡献对于NIAID的使命具有重要意义，因为从这些研究中获得的见解可能会导致通过靶向发育周期的主要调节因子（如EUO）来治疗衣原体感染的成功新策略。