Neutrophils and differentiating myeloid cells are unusually rich in the expression of non-coding RNA (ncRNA) transcripts, particularly intergenic transcripts from the homeobox-containing (HOX) gene clusters. In contrast to the well-studied functions of HOX genes in embryonic pattern formation, their roles in hematopoiesis are less well understood. We have identified a HOX region ncRNA, HOTAIRM1, that is expressed specifically in myeloid cells and regulates the expression of genes in the proximal HOXA cluster. We propose a detailed study of intergenic transcripts in the HOXA cluster, with the objectives of determining the functions of HOTAIRM1 and other HOXA ncRNAs in the regulation of HOX gene expression, myeloid differentiation, and mature neutrophil function. Central hypothesis: HOTAIRM1 and other non-coding intergenic transcripts in the HOXA gene cluster regulate the expression of developmentally important HOX genes, thereby modulating myeloid differentiation and function. Specifically, we will: 1. Investigate the function of HOTAIRM1 in the regulation of HOX gene expression. Hypothesis: HOTAIRM1 regulates the pattern of HOX gene expression during myeloid differentiation. Each subaim tests a hypothesis regarding a specific regulatory pattern: A. Regulation of individual HOX genes within the HOXA cluster, by either cis or trans actions;B. Differential regulation of sets of 5? versus 3? HOXA cluster genes;C. Preferential repression or activation of HOXB and non-clustered homeobox genes 2. Determine the mechanisms of HOTAIRM1 regulation of HOX gene expression. Each of the following subaims tests a specific, non-exclusive hypothesis that the specific mechanism under examination contributes to the regulatory functions of HOTAIRM1: A. Subcellular localization and molecular neighbors of HOTAIRM1; B. Molecules associated with HOTAIRM1;C. Regulation of transcriptional activity;D. Regulation of chromatin structure;E. Post-transcriptional regulation of mRNA stability and translation. 3. Test the effects of HOXA cluster intergenic ncRNAs on myeloid development and function. A. Effects of HOTAIRM1 knockdown or overexpression on myeloid gene expression, differentiation and function. Hypothesis: HOTAIRM1 regulates myeloid lineage commitment, differentiation, and function through control of myeloid genes and downstream effectors. B. Effects of knockdown or overexpression of additional HOXA intergenic ncRNAs. Hypothesis: Multiple HOX region intergenic transcripts cooperatively regulate myeloid gene expression and function. Characterization of other HOXA intergenic ncRNAs expressed in myeloid cells; knockdown and overexpression of additional HOXA intergenic transcripts The proposed studies will increase our fundamental understanding of myeloid gene regulation, differentiation, and function. The results could reveal potential targets for intervention in disorders of myeloid maturation, such as myelodysplasia and leukemia, as well as neutrophil-mediated inflammation disorders.
The proposed studies should determine the roles of a novel non-coding RNA transcripts in myeloid differentiation and mature neutrophil function. The results will increase our fundamental understanding of these processes and could reveal potential targets for intervention in disorders of myeloid maturation, such as myelodysplasia and leukemia, and in neutrophil-mediated inflammatory disorders, such as arthritis, inflammatory bowel disease, and post-ischemic tissue injury.
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