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

描述（由申请人提供）：衣原体采用独特的感染周期，其特征是在发育周期中适当时间表达的早、中、晚期基因的时间调控。每一类基因的精确表达时间依赖于不同的转录控制机制。衣原体也可以在细胞培养中被诱导进入一种称为持久性的异常状态，在发育周期的中途停止发育。尽管时间基因调控在衣原体疾病的发展中很重要，但对参与调控的分子机制的了解有限。在这些时间转录控制中，早期衣原体蛋白EUO已被证明与晚期基因启动子结合以抑制转录。拟议研究的目的是确定欧盟施加的监管机制和欧盟本身的监管。该研究的中心假设是，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得到缓解

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.