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.

儿童意味着一个具有高度动态生理的群体，特别是在出生后阶段。我们和 其他研究表明，许多生物转化基因(药物代谢酶和转运蛋白)表现出 出生后激增。我们还表明，激增的同时也伴随着对增长的快速下调 因子独立性-1b基因(GFI-1b)是肝脏发挥血液功能所必需的一种造血基因。 在胎儿和生命早期阶段产生器官。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。这种抑制需要DNA结合域。就在最近，我们有 研究表明，IL-1β(IL-1β)，一种已知下调许多生物转化的促炎细胞因子 基因，诱导GFI1B增加6倍，但使CES1mRNA减少80%。建议中的中心假设 项目是GFI-1b密切参与出生后的激增和许多 生物转化基因。具体目标是：(1)确定GFI-1b在全球范围内参与监管 出生后浪涌中的生物转化基因，以及(2)对反应元件(S)的功能表征 GFI-1b下调CES1的表达。将GFI-1b直接链接到大量被抑制的表达 生物转化基因、GFI1B的过度表达和敲除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(S)，一组 将进行分子分析，包括报告解剖、电泳迁移率分析和位点分析。 定向诱变。这些报告的功能将在GFI-1b过表达和敲打- 往下看牢房。诱导GFI-1b在抑制CES1、GFI-1b表达下调中的作用 细胞将用这种细胞因子处理，并监测是否逆转了被抑制的CES1表达。这个 逆转将通过CES1报告分析进行确认，以获得序列特异性。科学的前提是 这个项目是强大的和原创的。其独创性源于GFI-1b作为一种关键调节器在 生物转化基因的表达，特别是在生命的早期阶段。它是对GFI的解压-- 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控中的作用，表明了一种新的机制联系 在发育调控和炎症调控之间。总体而言，这一项目的完成将填补 知识差距，在这一具有强烈病理生理学意义的研究领域产生了持续的影响， 为基于GFI-1b促进肝细胞分化和分化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。