Mechanism of Bach1-Mediated Transcriptional Regulation and Immune Function

描述（由申请人提供）：需要免疫系统的精确调节，以保护免受外来入侵者和自我容忍。功能失调的免疫调节可导致对感染的免疫反应受损以及自身免疫性疾病的发展。转录因子Bach1在调节免疫过程中的作用已被间接牵连，但尚未直接探索；因此，本项目的广泛和长期目标是确定Bach1的功能及其在细胞和分子水平上介导保护性免疫和自身免疫性疾病的机制。为了实现这一目标，我将采用以下的研究设计和方法。1)首先，阐明Bach1调控转录的机制。具体来说，染色质免疫沉淀和微阵列（ChIP-chip）将用于确定Bach1是否结合启动子近端和/或远端调控元件直接引发转录激活和抑制。计算基序分析和荧光素酶报告基因分析将用于确定Bach1对转录调控重要的功能域。2)第二，确定Bach1在病毒和细菌感染中的作用。在感染期间，巨噬细胞和小鼠体内的Bach1将被清除，以表征Bach1调节保护性免疫反应的分子途径。3)第三，探讨Bach1在自身免疫中的作用。初步数据表明，Bach1促进小鼠实验性自身免疫性脑脊髓炎（EAE）的发展，这是一种模拟人类多发性硬化症的疾病。因此，骨髓重建和过继移植实验将用于鉴定那些具有Bach1内在功能调节EAE的免疫细胞。微阵列实验将在这些细胞中进行，以确定Bach1调控的潜在靶点和下游信号通路。4)第四，确定介导其在EAE发展过程中的Bach1靶点，并对其进行表征。巴赫1介导的EAE对这些靶标的消融作用将通过产生巴赫1和感兴趣靶标的双敲除动物来检验。此外，Bach1在体内调节功能所必需的功能域将被确定。总的来说，本项目将确定一个新的免疫系统调节因子（Bach1），并揭示其在感染和功能障碍期间调节保护性免疫和自身免疫的信号级联。因此，这一项目的成功完成将通过确定治疗免疫相关疾病和自身免疫性疾病的新靶点，使公共卫生受益。

DESCRIPTION (provided by applicant): Precise regulation of the immune system is required for protection from foreign invaders and tolerance of self. Dysfunctional immune regulation can lead to impaired immune responses to infections as well as development of autoimmune diseases. The role of the transcription factor Bach1 in regulating immune processes has been indirectly implicated but has not been directly explored; thus, the broad and long-term objective of this project is to determine the function of Bach1 and the mechanism by which it mediates protective immunity and autoimmune diseases at a cellular and molecular level. The following research design and methods will be employed to achieve this goal. 1) First, the mechanism by which Bach1 regulates transcription will be elucidated. Specifically, chromatin immunoprecipitation followed by microarray (ChIP-chip) will be used to determine whether Bach1 binds promoter proximal and/or distal regulatory elements to directly elicit transcriptional activation and repression. Computational motif analyses and luciferase reporter assays will be used to define those functional domains of Bach1 important for transcriptional regulation. 2) Second, the role of Bach1 during viral and bacterial infections will be determined. Bach1 will be ablated in macrophages and in mice during infection in order to characterize the molecular pathways by which Bach1 regulates protective immune responses. 3) Third, the role of Bach1 in autoimmunity will be investigated. Preliminary data demonstrated that Bach1 promotes the development of murine experimental autoimmune encephalomyelitis (EAE), which mimics human multiple sclerosis. Accordingly, bone marrow reconstitution and adoptive transfer experiments will be used to identify those immune cell(s) in which the intrinsic function of Bach1 modulates EAE. Microarray experiments will be performed in those cells to identify potential targets and downstream signaling pathways regulated by Bach1. 4) Fourth, Bach1 targets responsible for mediating its role in the development of EAE will be identified and characterized. The effect of Bach1-mediated EAE upon ablating these targets will be examined by generating double knockout animals of Bach1 and the target(s) of interest. Furthermore, functional domains of Bach1 necessary for its regulatory function in vivo will be identified. Overall, this project will identify a new regulator of the immune system (Bach1) and reveal those signaling cascades by which it modulates protective immunity during infection and autoimmunity during dysfunction. The successful completion of this project will therefore benefit public health by identifying new targets for therapy against immune-related and autoimmune diseases.