Evolutionary perspective on regulation of Nramp1 expression in phagocytes

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The role of the Natural resistance-associated macrophage protein (Nramp1) in host defense against microbial infections, including tuberculosis (TB) is widely accepted. This integral membrane protein limits access of phagocytosed microbes to intracellular nutrients, such iron and manganese. The activated form of Vitamin D3 (VD) is also known to enhance host innate resistance to TB, including treatments in solarium or using food supplements, which involvs increased production of antimicrobial peptides. Intracellular activation of Vitamin D3 precursor in human macrophages results in potent cell-autonomous anti Mycobacterium tuberculosis effect. We showed earlier that NRAMP1 expression is specifically induced by VD in macrophages, implying a possible role in VD antimicrobial effects. We began to delineate promoter regulatory sites and to identify bound factors. Achieving a comprehensive understanding of NRAMP1 regulation by VD will provide knowledge in terms of specific molecular mechanisms and possible avenues to stimulate host innate defenses. We thus propose to scan NRAMP1 gene and surrounding locus to identify all regulatory sites, positive or negative, that are mobilized during the development and maturation of professional phagocytes. We will seek optimal temporal frame for gene activity in time-course studies using inhibitors of chromosomal modeling. Gene activity-related DNA 'marks' and ('marked') bound factors, such as DNA packing histones or specific factors, will be purified using specific antibodies to precipitate fragments of fixed nuclear chromatin; bound DNA will be extracted and labeled before hybridization to a synthetic locus (high resolution tiling oligonucleotide micro-array). Conservation of the sites identified will be analyzed in the human genome to evaluate any significance of sequence polymorphisms, and genomes of other vertebrates to look for elements of a hypothetical ancestral regulatory system. This study will reveal the regulation in vivo of the endogenous NRAMP1 gene in human macrophages, establishing a knowledge basis for future innovative approaches aimed at stimulating host natural immunity and wound antimicrobial defense.