Neutrophils provide the first line of host defense against microbial infections and play a major role in inflammation and tissue damage. Previous studies of RNA expression in neutrophils have revealed a remarkably vigorous transcriptional response to activation by various stimuli. Our studies in a myeloid cell line model of terminal differentiation have also indicated that mRNA levels for a large number of transcription factors change during cell maturation. We now propose a coordinated and comprehensive investigation of the transcribed regions and the regulators of transcriptional activity in developing and mature neutrophils. We will investigate: 1. """"""""Novel"""""""" transcripts. We will use tiling arrays to determine the cell specificity and strandedness of novel transcripts (i.e. not corresponding to known genes) expressed in resting and activated peripheral blood neutrophils and in NB4 promyelocytic leukemia cells during retinoid-induced terminal differentiation. 2. Transcription factors. We will use chromatin immunoprecipitation and both promoter and genomic arrays to identify promoters bound by sequence-specific transcription factors expressed in resting and activated neutrophils. We will also investigate which gene promoters are bound by transcription factors during NB4 cell differentiation to determine whether the novel factors affect important downstream targets. 3. Chromatin structure and remodeling proteins: We will use oligonucleotide tiling arrays to test whether neutrophils regulate activation responses by alterations in the association of DNA with chromatin remodeling proteins or specifically modified histones. We will also determine the association of specific promoter sequences with chromatin modifying proteins and modified histones during terminal differentiation of NB4 cells. 4. DNA methylation: Using complementary systems to determine the methylation status and location of cytosine residues, both within and outside CpG islands, we will investigate changes in the sites of cytosine methylation in activated neutrophils and during myeloid maturation in NB4 cells. Identification of novel neutrophil-specific genes and regulatory networks could provide new targets for augmentation of host defense, attenuation of inflammation, and treatments of disorders of myelopoiesis.
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