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.
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