Transcriptional Control of Mouse FGF23 Expression in Health and Disease

项目总结/摘要 FGF 23代表最近发现的磷酸尿激素，其主要生理作用是调节 通过肾脏的直接作用改变血液中的磷酸盐含量。FGF 23在成骨细胞中表达， 骨细胞，其输出由磷酸盐、1，25（OH）2D 3和PTH控制。最近的研究表明 炎症和铁缺乏也从多个来源上调FGF 23表达。尽管 然而，尽管人们付出了相当大的努力，但对上述调节因子的调节机制却知之甚少。 FGF 23表达。使用ChIP-seq分析，我们发现了5个位于远端位点的调控区域， 我们假设的小鼠FGF 23基因位点负责体内FGF 23的调节。我们删除 使用CRISPR/Cas9方法在小鼠基因组中的这些潜在调控区域中的每一个， 通过功能丧失研究探讨了它们在介导磷酸盐、1，25（OH）2D 3、PTH和LPS的作用中的作用 通过这些节段。我们的研究结果支持旨在定义分子机制的建议 这是FGF 23在健康和疾病中调节的基础。目标1：细化功能操作和交互 在体内控制组织FGF 23表达的FGF 23基因位点的调节增强子。几 将使用CRISPR/Cas9方法和对CRISPR/Cas9的应答来创建另外的调节突变小鼠品系。 评估磷酸盐、PTH、1，25（OH）2D 3和LPS，以细化和潜在解析基因组作用位点 对于每个监管机构，并确定已经确定的区域是否包括整个监管机构， 在FGF 23基因座的结构域。这些研究将牢固地确立所涉及的功能调节活动的范围 FGF 23的表达。目的2：确定转录因子、染色质调节因子和信号转导 在体内组织中介导FGF 23表达调节的途径及其相互作用。我们 将使用ChIP-seq和ATAC-seq分析和其他技术来解析DNA作用位点， 基序、转录因子和染色质辅助调节因子以及潜在信号传导的无偏鉴定 所涉及的途径。目的3：确定系统性因素在调节 慢性肾脏疾病小鼠模型中FGF 23的表达草酸盐诱导的急性 肾脏疾病影响来自骨和其它组织的FGF 23的表达。我们将检查 草酸诱导组织中FGF 23表达作为我们的FGF 23调控缺失模型的功能。这些 研究还将侧重于评估肾脏、骨骼和肾脏中出现的全身和生物表型。 由于草酸盐治疗后FGF 23上调的改善，血管系统。我们的结果很可能 提供更深入的了解选择性机制，通过该机制，FGF 23在健康和 疾病

PROJECT SUMMARY/ABSTRACT FGF23 represents a recently discovered phosphaturic hormone whose primary physiological role is to regulate phosphate content in the blood through direct actions in the kidney. FGF23 is expressed in osteoblasts and osteocytes where its output is controlled by phosphate, 1,25(OH)2D3, and PTH. More recent studies now suggest that inflammation and iron deficiency also upregulate FGF23 expression from multiple sources. Despite considerable effort, however, little is known of the mechanisms through which the above regulators modulate FGF23 expression. Using ChIP-seq analysis, we discovered 5 regulatory regions located at distal sites across the mouse FGF23 gene locus that we hypothesized were responsible for FGF23 regulation in vivo. We deleted each of these potential regulatory regions in the mouse genome using CRISPR/Cas9 methods, and have explored their role in mediating the actions of phosphate, 1,25(OH)2D3, PTH and LPS via loss of function studies through these segments in vivo. Our results support a proposal aimed at defining the molecular mechanisms which underlie FGF23 regulation in health and disease. Aim 1: Refine the functional actions and interactions of regulatory enhancers at the FGF23 gene locus that control tissue FGF23 expression in vivo. Several additional strains of regulatory mutant mice will be created using a CRISPR/Cas9 approach and response to phosphate, PTH, 1,25(OH)2D3 and LPS assessed to refine and potentially resolve the genomic sites of action for each of these regulators, and to determine whether regions already identified comprise the entire regulatory domain at the FGF23 locus. These studies will firmly establish the span of functional regulatory activities involved in FGF23 expression. Aim 2: Determine the transcription factors, chromatin regulators and signaling pathways and their interactions that mediate the regulation of FGF23 expression in tissues in vivo. We will use ChIP-seq and ATAC-seq analyses and other techniques to resolve DNA sites of action, enabling the unbiased identification of motifs, transcription factor and chromatin coregulators, and potential signaling pathways that are involved. Aim 3: Identify the temporal role(s) of systemic factors in the regulation of FGF23 expression in an oxalate-induced mouse model of chronic kidney disease. Oxalate-induced acute kidney disease impacts the expression of FGF23 from bone and other tissues. We will examine the ability of oxalate to induce FGF23 expression in tissues as a function of our FGF23 regulatory deletion models. These studies will also focus on assessing the systemic and biological phenotypes that emerge in kidney, skeleton and the vascular system due to amelioration of FGF23 upregulation following oxalate treatment. Our results are likely to provide greater insight into the selective mechanisms through which FGF23 is regulated in vivo in health and disease.