Evolutionary perspective on regulation of Nramp1 expression in phagocytes

天然耐药相关巨噬细胞蛋白（Nramp 1）在宿主防御微生物感染（包括结核病（TB））中的作用已被广泛接受。 这种完整的膜蛋白限制了被吞噬的微生物对细胞内营养物质（如铁和锰）的获取。 维生素D3（VD）的活化形式也被称为增强宿主对TB的先天抗性，包括日光浴或使用食物补充剂的治疗，这涉及增加抗菌肽的产生。 人巨噬细胞中维生素D3前体的细胞内活化导致有效的细胞自主抗结核分枝杆菌作用。 我们先前表明，NRAMP 1的表达是由VD在巨噬细胞中特异性诱导的，这意味着可能在VD抗菌作用中发挥作用。 我们开始描绘启动子调控位点，并确定结合因子。 全面了解VD对NRAMP 1的调控将为我们提供特定的分子机制和刺激宿主先天防御的可能途径方面的知识。 因此，我们建议扫描NRAMP 1基因和周围的基因座，以确定所有的监管网站，积极或消极的，动员过程中的发展和成熟的专业吞噬细胞。 我们将寻求最佳的时间框架，基因活性的时程研究使用抑制剂的染色体建模。 基因活性相关的DNA“标记”和（“标记”）结合因子，如DNA包装组蛋白或特异性因子，将使用特异性抗体进行纯化，以沉淀固定的核染色质片段;结合的DNA将在与合成基因座杂交之前进行提取和标记（高分辨率平铺寡核苷酸微阵列）。 将在人类基因组中分析所鉴定的位点的保守性，以评估序列多态性的任何意义，并在其他脊椎动物的基因组中寻找假设的祖先调控系统的元素。这项研究将揭示内源性NRAMP 1基因在人巨噬细胞中的体内调节，为未来旨在刺激宿主天然免疫和伤口抗菌防御的创新方法建立知识基础。

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.