Cytokine Regulation of Human Eosinophil Genes

描述(申请人提供)：嗜酸性粒细胞在哮喘、过敏性疾病、寄生虫病和某些特发性疾病的发病机制中是促炎效应细胞。它们在骨髓中的分化，在组织中的启动和功能激活，以及延长组织的存活都是对包括GM-CSF、IL-3和IL-5在内的细胞因子的反应。IL-5对嗜酸性粒细胞有选择性，在感染和过敏过程中调节血液和组织中的嗜酸性粒细胞起关键作用。IL-5通过其高亲和力受体(IL-5R)传递信号，该受体由嗜酸性粒细胞特异性的IL-5结合的α亚基和IL-3和GM-CSF受体共有的Betac亚基组成。IL-5R的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。在发育中的人类祖细胞中，调节IL-5R表达、嗜酸性粒细胞终末分化和嗜酸性粒细胞系特异性基因表达的转录网络和机制尚未被描述。这笔拨款的重点是定义关键的转录因子及其组合网络，以调节人类嗜酸性粒细胞系特异性基因的表达。这次更新建议扩大这些研究，将它们带入体内染色质重塑的水平，以便评估到目前为止我们和其他人在体外发现的在谱系指定、末端分化和功能成熟方面对嗜酸性粒细胞基因表达至关重要的转录网络的相关性。我们的目标是在体内继续这些研究，使用IL-5Rpha亚基和颗粒主要碱性蛋白(MBP)基因作为模型，在一般的骨髓生成的背景下定义嗜酸性粒细胞的发育程序。提出了三个具体的问题：(1)在嗜酸性粒细胞发育过程中调节嗜酸性粒细胞IL-5Rpha基因表达的转录因子及其相互作用和转录机制是什么？染色质免疫沉淀(ChIP)和染色质重塑的分析，包括DNase I超敏图谱和DNaseI在体内的LM-PCR足迹分析，将被用来确认和进一步表征我们迄今已确定的转录因子(C/EBPs，GATA结合蛋白，AP-1，RFX，RFX相关蛋白)的作用，以及重要的是，确定IL-5Rpha基因座上新的调节区；(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？芯片、DNase I超敏定位和体内DNase I足迹的方法，将被用来研究体外发现的MBP基因表达的关键转录调控因子(C/EBPs、GATA-1及其辅助激活或抑制因子，以及ETS因子PU.1)在协同或拮抗嗜酸性粒细胞基因表达方面的比较作用、物理和功能相互作用，并寻找新的调控区域和转录因子靶点；(3)各种C/EBP家族成员(尤其是C/EBPepsilon亚型)、GATA结合蛋白(GATA-1)及其辅活化子/辅阻遏物(FOGs、CtBPs)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？采用HIVTAT-转录因子融合蛋白的高效细胞转导方法，转导CD34+髓系、CD34+/L-5R+嗜酸性粒细胞祖细胞、嗜酸性粒细胞株和成熟嗜酸性粒细胞，研究其对嗜酸性粒细胞分化和内源性基因表达的影响。我们的长期目标是更好地了解嗜酸性粒细胞异常产生、激活和功能的过程，以确定调节嗜酸性粒细胞发育和嗜酸性粒细胞疾病病理性炎症活动的新靶点。

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.