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人口，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 产后高潮我们还表明，激增伴随着快速下调的增长， 因子独立性-1b基因（GFI-1b）是肝脏发挥造血功能所必需的造血基因， 在胎儿和生命早期阶段产生器官。此外，我们已经表明，GFI-1b，一个序列- 特异性转录因子，抑制CES 1（羧酸酯酶-1）的启动子，CES 1是一种最丰富的药物， 肝脏中的代谢酶。抑制需要DNA结合结构域。最近，我们有 显示白细胞介素-1 β（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GFI 1b的6倍，但减少CES 1 mRNA的80%。提出的中心假设 GFI-1b密切参与了出生后的激增和炎症下调， 生物转化基因具体目标是：（1）确定GFI-1b在全球参与监管 生物转化基因在出生后激增，和（2）功能上表征的反应元件， GFI-1b下调CES 1。为了将GFI-1b直接与大量受抑制的 生物转化基因、GFI 1b的过表达和敲除以及染色质免疫沉淀- 将执行seq。为了定位CES 1基因中支持GFI 1b抑制的元件，使用了一组 将进行分子测定，包括报告分子解剖、电泳迁移率测定和位点- 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。 向下的细胞。为了确定诱导的GFI-1b在抑制CES 1表达中的作用，GFI-1b敲低 用该细胞因子处理细胞并监测受抑制的CES 1表达的逆转。的 逆转将用CES 1报告基因测定法确认，以获得序列特异性。科学的前提是 这个项目是强大的和原始的。独创性源于GFI-1b作为一种关键调节剂的新奇， 生物转化基因的表达，特别是在生命的早期阶段。它是GFI的去压抑- 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在生物转化基因的炎症调节中的作用，指出了一种新的机制联系 发育和炎症调节之间的联系总的来说，该项目的完成将填补 知识差距，在这一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。