Functional connection between the growth factor independence-1b and post-neonatal regulation of biotransformation genes

生长因子独立1b与生物转化基因新生儿后调控之间的功能联系

• 批准号: 10681617
• 负责人: Bingfang Yan
• 金额: $ 44.55万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-08 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681617
• 关键词: Adult; Age; Age Months; Area; Biological Assay; Birth; Blood; Carboxylesterase 1; Cells; ChIP-seq; Child; Childhood; Cytochrome P450; DNA Binding; DNA Binding Domain; Development; Disease; Dissection; Down-Regulation; Drug Kinetics; Drug toxicity; Elements; Enzymes; Event; Exhibits; Foundations; Future; GFI1B gene; Genes; Genomics; Goals; Health; Hepatic; Human; Human Genome; Inflammation; Inflammatory; Inflammatory Response; Interleukin-1 beta; Knowledge; Laboratories; Life; Link; Liver; Liver Microsomes; Messenger RNA; Metabolic Biotransformation; Metabolism; Molecular; Monitor; Neonatal; Nucleic Acid Regulatory Sequences; Organ; Pharmaceutical Preparations; Pharmacology; Phase; Physiological; Physiology; Population; Production; Publications; Reagent; Recording of previous events; Regulation; Reporter; Repression; Research; Response Elements; Role; Sampling; Site-Directed Mutagenesis; Specific qualifier value; Specificity; Testing; Therapeutic; Toxicology; Xenobiotics; carboxylesterase; chromatin immunoprecipitation; collaborative environment; cytokine; derepression; design; drug efficacy; experimental study; fetal; gene repression; insight; knock-down; medication safety; mutant; novel; originality; overexpression; pharmacologic; postnatal; promoter; stem; transcription factor

Children signify a population with highly dynamic physiology, particularly during the postnatal stage. We and others have shown that many biotransformation genes (drug metabolizing enzymes and transporters) exhibit a postnatal surge. We have also shown that the surge is accompanied by a rapid downregulation of the growth factor independence-1b gene (GFI-1b), a blood-producing gene essential for the liver to function as a blood- producing organ during the fetal and early stage of life. In addition, we have shown that GFI-1b, a sequence- specific transcription factor, represses the promoter of CES1 (carboxylesterase-1), a most abundant drug- metabolizing enzyme in the liver. The repression requires the DNA binding domain. Very recently, we have shown that interleukin-1β (IL-1β), a proinflammatory cytokine known to downregulate many biotransformation genes, induces GFI1b by 6-fold but decreases CES1 mRNA by 80%. The central hypothesis of the proposed project is that GFI-1b is intimately involved in the postnatal surge and inflammatory downregulation of many biotransformation genes. The specific aims are: (1) to ascertain the global engagement of GFI-1b in regulating biotransformation genes in the postnatal surge, and (2) to functionally characterize the response element(s) for GFI-1b to downregulate CES1. To link GFI-1b directly to the repressed expression of a large number of biotransformation genes, overexpression and knockdown of GFI1b as well as chromatin-immunoprecipitation- seq will be performed. To locate the element(s) in the CES1 gene that supports GFI1b repression, a set of molecular assays will be performed including reporter dissection, electrophoretic mobility assay and site- directed mutagenesis. The functionality of these reporters will be tested in GFI-1b overexpression and knock- down cells. To establish the role of induced GFI-1b in suppressed expression of CES1, GFI-1b knockdown cells will be treated with this cytokine and monitored for the reversal of suppressed CES1 expression. The reversal will be confirmed with CES1 reporter assays to gain sequence specificity. The scientific premise of this project is strong and original. The originality stems from the novelty of GFI-1b as a critical regulator in the expression of biotransformation genes, particularly during the early stage of life. It is the de-repression of GFI- 1b that supports the postnatal surge of many biotransformation genes. The project will also investigate a role of GFI-1b in inflammatory regulation of biotransformation genes, pointing to a novel mechanistic connection between developmental and inflammatory regulation. Overall, completion of this project will have filled knowledge gaps, delivered sustained impact in this research area with strong pathophysiological significance, and laid a foundation for a large project on GFI-1b based enhancement of hepatic differentiation and normalization of the expression of biotransformation genes under inflammatory conditions.

儿童是具有高度动态生理机能的人群，特别是在出生后阶段。我们和 其他人已经表明，许多生物转化基因（药物代谢酶和转运蛋白）表现出 产后激增。我们还表明，激增伴随着增长的快速下调 独立因子 1b 基因 (GFI-1b)，一种对肝脏发挥血液功能至关重要的造血基因 胎儿和生命早期阶段的产生器官。此外，我们还证明了 GFI-1b，一个序列- 特异性转录因子，抑制 CES1（羧酸酯酶-1）的启动子，CES1 是一种最丰富的药物 肝脏中的代谢酶。抑制需要 DNA 结合域。最近，我们有 研究表明，白细胞介素-1β (IL-1β) 是一种已知可下调许多生物转化的促炎细胞因子 基因，诱导 GFI1b 6 倍，但 CES1 mRNA 降低 80%。所提出的中心假设 该项目认为，GFI-1b 与许多炎症的产后激增和炎症下调密切相关。 生物转化基因。具体目标是： (1) 确定 GFI-1b 在监管方面的全球参与 出生后激增中的生物转化基因，以及（2）功能性地表征响应元件 GFI-1b 下调 CES1。将 GFI-1b 直接与大量的抑制表达联系起来 生物转化基因、GFI1b 的过表达和敲低以及染色质-免疫沉淀- 将执行 seq。为了定位 CES1 基因中支持 GFI1b 抑制的元件，需要使用一组 将进行分子分析，包括报告基因解剖、电泳迁移率分析和位点分析 定向诱变。这些报告基因的功能将在 GFI-1b 过表达和敲除中进行测试 下调细胞。确定诱导的 GFI-1b 在抑制 CES1 表达、GFI-1b 敲低中的作用 细胞将用这种细胞因子处理并监测受抑制的 CES1 表达的逆转。这 逆转将通过 CES1 报告基因检测来确认，以获得序列特异性。科学前提是 这个项目是强大且原创的。原创性源于 GFI-1b 作为关键调节因子的新颖性 生物转化基因的表达，特别是在生命的早期阶段。这是 GFI 的去抑制- 1b 支持许多生物转化基因的出生后激增。该项目还将调查一个角色 GFI-1b 在生物转化基因炎症调节中的作用，指出一种新的机制联系 发育和炎症调节之间。总体而言，该项目的完成将填补 知识差距，在该研究领域产生了持续的影响，具有很强的病理生理学意义， 并为基于GFI-1b的增强肝脏分化和增强的大型项目奠定了基础 炎症条件下生物转化基因表达的正常化。