Cytokine Regulation of Human Eosinophil Genes

描述（由申请人提供）：嗜酸性粒细胞是哮喘、过敏性、寄生虫和某些特发性疾病发病机制中的促炎效应物。GM-CSF、IL-3和IL-5等细胞因子的作用使它们在骨髓中分化，在组织中启动和功能激活，延长组织存活。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发挥关键作用。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α亚基和IL-3和GM-CSF受体共享的β亚基组成。IL-5R的表达是启动和实现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、嗜酸性粒细胞末端分化和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明确。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。这项更新提议扩大这些研究，将它们在体内的染色质重塑水平，以评估我们和其他人迄今为止在体外鉴定的转录网络的相关性，这些转录网络在谱系规范，终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。我们的目标是在体内继续这些研究，使用il - 5rα亚基和颗粒主要碱性蛋白（MBP）基因作为模型，在骨髓形成的一般背景下定义嗜酸性粒细胞发育程序。提出三个具体问题（目标）：(1)在嗜酸性粒细胞发育过程中调控嗜酸性粒细胞il - 5rα基因表达的转录因子、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DNAse I超敏定位和DNAse I在LM-PCR中的体内足迹，将用于确认和进一步表征我们迄今已确定的转录因子（C/ ebp， gta结合蛋白，AP-1， RFX， RFX相关蛋白）的作用，重要的是，鉴定il - 5rα基因位点的新调控区域；(2)在体内嗜酸性粒细胞发育过程中，调控嗜酸性粒细胞MBP基因表达的转录机制是什么？CHIP， DNase I超敏定位和DNase I体内足迹的方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节因子（C/ ebp， GATA-1及其共激活或抑制因子，以及ets因子PU.1）在其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，并确定新的调控区域和转录因子靶点；(3)各种C/EBP家族成员（特别是C/EBPepsilon亚型）、gata结合蛋白（GATA-1）及其共激活因子/共抑制因子（FOGs、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？我们将采用HIV转录因子融合蛋白的高效细胞转导方法转导真实的CD34+髓系和CD34+/L-5R+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。我们的长期目标是进一步了解嗜酸性粒细胞的异常产生、激活和功能的过程，以确定嗜酸性粒细胞发育和病理性炎症活动调节的新靶点。

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.