Cytokine Regulation of Human Eosinophil Genes

描述（申请人提供）：嗜酸性粒细胞是哮喘、过敏性疾病、寄生虫病和某些特发性疾病发病机制中的促炎效应物。它们在骨髓中的分化、组织中的引发和功能活化以及延长的组织存活响应于包括GM-CSF、IL-3和IL-5的细胞因子而发生。IL-5对嗜酸性粒细胞谱系具有选择性，在感染和过敏过程中调节血液和组织嗜酸性粒细胞中起关键作用。IL-5通过其高亲和力受体（IL-5 R）进行信号传导，所述高亲和力受体由嗜酸性粒细胞特异性IL-5结合α亚基和IL-3和GM-CSF受体共享的β亚基组成。IL-5 R表达是启动和完成嗜酸性粒细胞发育和功能程序的先决条件。调控IL-5 R表达、嗜酸性粒细胞终末分化和发育中的人祖细胞中嗜酸性粒细胞谱系特异性基因表达的转录网络和机制尚未阐明。该基金的重点是确定调节人类嗜酸性粒细胞谱系特异性基因表达的关键转录因子及其组合网络。这次更新建议扩大这些研究，把它们在体内的染色质重塑的水平，以评估相关的转录网络，我们和其他人已经确定，迄今为止在体外作为关键的嗜酸性粒细胞基因表达的谱系规范，终端分化和功能成熟。我们的目标是继续这些研究在体内使用IL-5 R α亚基和颗粒主要碱性蛋白（MBP）基因作为模型，用于定义嗜酸性粒细胞的发育程序在骨髓生成的一般情况下。本文提出了三个具体问题：（1）在嗜酸性粒细胞发育过程中，调控IL-5 R α基因表达的转录因子有哪些，它们之间的相互作用和转录机制是什么？染色质免疫沉淀（CHIP）和染色质重塑分析（包括DNA酶I超敏反应图谱和DNA酶I体内足迹与LM-PCR）将用于确认和进一步表征转录因子的作用（C/EBP、GATA结合蛋白、AP-1、RFX、RFX相关蛋白），重要的是，鉴定了IL-5 R α基因座中的新调控区;（2）在嗜酸性粒细胞发育过程中，体内调节嗜酸性粒细胞MBP基因表达的转录机制是什么？CHIP、DNase I超敏反应图谱和DNase I体内足迹法将用于表征迄今为止在体外鉴定的MBP基因表达的关键转录调控因子的比较作用以及物理和功能相互作用。（C/EBP、加塔-1和它们的共激活物或阻遏物，以及ets因子PU. 1）在它们对嗜酸性粒细胞基因表达的协同作用或拮抗作用方面，（3）各种C/EBP家族成员（特别是C/EB肽亚型）、GATA结合蛋白（加塔-1）及其辅激活子/辅抑制子（FOG、CtBP）和ets因子PU的“体内”活性是什么。1在嗜酸性粒细胞基因转录和发育中的个体和组合作用？将使用HIV Tat-转录因子融合蛋白的高效细胞转导方法来鉴定真实的CD 34+髓样和CD 34 +/L-5 R+嗜酸性粒细胞祖细胞、嗜酸性粒细胞系和成熟嗜酸性粒细胞，以确定它们对嗜酸性粒细胞分化和内源性基因表达的影响。我们的长期目标是提供更多的了解异常生产，激活和嗜酸性粒细胞的功能，以确定新的目标，调节嗜酸性粒细胞的发展和嗜酸性粒细胞疾病的病理炎症活动的过程。

DESCRIPTION (provided by applicant): Eosinophils are pro-inflammatory effectors in the pathogenesis of asthma and allergic, parasitic and certain idiopathic diseases. Their differentiation in the bone marrow, priming and functional activation in tissues, and prolonged tissue survival occur in response to cytokines including GM-CSF, IL-3 and IL-5. IL-5 is selective for the eosinophil lineage, playing a key role in regulating blood and tissue eosinophilia in infectious and allergic processes. IL-5 signals through its high affinity receptor (IL-5R) comprised of an eosinophil-specific, IL-5-binding alpha subunit, and a betac subunit shared by the IL-3 and GM-CSF receptors. IL-5R expression is a prerequisite for initiating and fulfilling the eosinophil's developmental and functional programs. The transcriptional networks and mechanisms that regulate expression of the IL-5R, eosinophil terminal differentiation, and expression of eosinophil lineage-specific genes in developing human progenitors has not been delineated. This grant has been focused on defining key transcription factors and their combinatorial networks that regulate human eosinophil lineage-specific gene expression. This renewal proposes to expand these studies, taking them in vivo to the level of chromatin remodeling in order to evaluate the relevance of the transcriptional networks that we and others have identified thus far in vitro as critical to eosinophil gene expression in terms of lineage specification, terminal differentiation and functional maturation. Our objective is to continue these studies in vivo using the IL-5Ralpha subunit and granule major basic protein (MBP) genes as models for defining the eosinophil developmental program in the general context of myelopoiesis. Three specific questions (aims) are proposed: (1) What are the transcription factors, their interactions and transcriptional mechanisms that regulate eosinophil IL-5Ralpha gene expression during eosinophil development? Chromatin immunoprecipitation (CHIP) and analyses of chromatin remodeling including DNAse I hypersensitivity mapping and DNase I in vivo footprinting with LM-PCR will be used to confirm and further characterize the roles of transcription factors (C/EBPs, GATA-binding proteins, AP-1, RFX, RFX-associated proteins) we have identified thus far, and importantly, to identify novel regulatory regions in the IL-5Ralpha gene locus; (2) What are the transcriptional mechanisms that regulate eosinophil MBP gene expression in vivo during eosinophil development? The methods of CHIP, DNase I hypersensitivity mapping and DNase I in vivo footprinting and will be used to characterize the comparative roles, and physical and functional interactions of key transcriptional regulators of MBP gene expression identified thus far in vitro (C/EBPs, GATA-1 and their co-activators or repressors, and the ets factor PU.1) in terms of their synergy or antagonism of eosinophil gene expression, and to identify new regulatory regions and transcription factor targets; (3) What are the "in vivo" activities of the various C/EBP family members (especially C/EBPepsilon isoforms), GATA-binding proteins (GATA-1) and their coactivators/corepressors (FOGs, CtBPs), and the ets factor PU. 1 in terms of individual and combinatorial roles in eosinophil gene transcription and development? A highly efficient cell transduction method with HIV Tat-transcription factor fusion proteins will be used to transduce authentic CD34 + myeloid and CD34+/L-5R+ eosinophil progenitors, eosinophil lines and mature eosinophils to determine their effects on eosinophil differentiation and endogenous gene expression. Our long-term goal is to provide greater understanding of the processes involved in the abnormal production, activation and functions of eosinophils in order to identify novel targets for modulating eosinophil development and pathologic inflammatory activities in eosinophilic disease.