RNA m6A methylation contributes to hepatic inflammation in obesity-associated NAFLD

RNA m6A 甲基化导致肥胖相关 NAFLD 的肝脏炎症

• 批准号: 10566443
• 负责人: Xinshou Ouyang
• 金额: $ 39.63万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10566443
• 关键词: Binding; Biogenesis; Biological; Cells; Characteristics; Collaborations; DNA Damage; Data; Diet; Disease Progression; Enzymes; Epigenetic Process; FRAP1 gene; Fibrosis; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Grant; Hepatic; Homeostasis; Inflammation; Inflammatory; Kupffer Cells; Liver; Liver diseases; Macrophage; Maps; Messenger RNA; Metabolic; Methylation; Methyltransferase; Modification; Molecular; Mus; Myelogenous; Myeloid Cells; NF-kappa B; Nucleotides; Obesity; Organism; Pathway interactions; Phenocopy; Phenotype; Play; Population; Post-Transcriptional Regulation; Prevention; Protein Methyltransferases; Proteins; RNA; RNA Editing; RNA Helicase; RNA Transport; Reaction; Reader; Reporting; Research; Resolution; Ribosomes; Risk Factors; Role; Site; Specificity; Steatohepatitis; Testing; Therapeutic; Transcript; biological adaptation to stress; diet-induced obesity; differential expression; efficacy testing; endoplasmic reticulum stress; epigenome; immune cell infiltrate; improved; inflammatory marker; liver inflammation; mRNA Stability; mRNA Transcript Degradation; monocyte; non-alcoholic fatty liver disease; novel; prevent; prophylactic; recruit; response; single-cell RNA sequencing; small molecule; small molecule inhibitor; therapeutic target; transcriptome; transcriptomics

SPECIFIC AIMS: Obesity-induced metabolic inflammation results in the transition of nonalcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH) [1-3]. Myeloid cell populations such as macrophages have been established as the main driver of metabolic inflammation [4-10]. Single-cell RNA sequencing (scRNA-seq) studies have provided a high-resolution fate map of myeloid diversity in NAFLD associated with distinct epigenomes, transcriptomes, and functions [9-11]. Recent advances emphasize the importance of post-transcriptional regulation of gene expression at the RNA level in obesity-driven hepatic inflammation [12, 13]. N6-methyladenosine (m6A) methylation represents the most prevalent and abundant epigenetic modification of eukaryotic mRNA. m6A methylation is dynamically installed, erased, and recognized by m6A methyltransferases (writers), m6A demethylases (erasers), and m6A specific binding enzymes (readers) [14, 15]. Highly m6A modified transcripts generally result in lower mRNA stability leading to reduced mRNA levels and thus greatly influence diverse cellular and biological responses [16-18]. The reader proteins recognize the m6A-modified sequences and determine the fate of the target mRNA. We recently identified that myeloid lineage-restricted deletion of m6A writer protein methyltransferase like 3 (METTL3) results in differential expression of m6A modified mRNA transcripts, particularly DNA Damage Inducible Transcript 4 (DDIT4) that negatively regulates mTOR/NFkB activation resulting in decreased immune cell infiltration, recovered liver homeostasis, improved hepatic metabolic abnormalities, and reduced inflammation during diet-induced obesity/NAFLD [19]. Although macrophage METTL3 plays an essential role in obesity-associated NAFLD, how it competes and collaborates with distinct m6A readers has not been identified, nor has the specificity of the methylation reaction and RNA structural motif determined. The molecular and cellular effects of m6A methylation in myeloid cell subsets, in addition to reducing mRNA stability, have yet to be elucidated in advanced fibrosis. DEAD-box RNA helicase DDX21 plays multifaceted roles in ribosome biogenesis, RNA editing, RNA transport, and transcription. DDX21 has been reported to interact with m6A writer proteins. Analysis of publicly available RNA-CLIP sequencing data indicates the overlap between DDX21 binding regions and m6A methylation sites on target mRNA molecules. Our preliminary results showed that mice with DDX21 deficiency in myeloid lineage, analogous to METTL3 deficiency, resulted in significant prevention of diet-induced steatohepatitis and fibrosis. The scRNA-seq analysis revealed two distinct myeloid populations, liver-resident (KC-H) macrophages and monocyte-derived macrophages (Mo-M), that have opposite proportional mobilization between chow and diet-fed mice. The lack of DDX21 restored the proportion of KC-H in diet-fed mice while reducing the proportion of Mo-M along with reduced inflammatory markers, indicating DDX21 plays a novel role in liver myeloid cells during obesity-associated NAFLD. Therefore, we hypothesize that mRNA m6A methylation in myeloid cell subsets, in association with DDX21, leads to hepatic inflammation during obesity-associated NAFLD (Fig.1). Aim 1 Establish the role of METTL3 in myeloid cell-driven hepatic inflammation during obesity-associated NAFLD. 1.1) Determine the contributions of METTL3 in KC-H and Mo-M subsets to hepatic inflammation. 1.1.1) Obtain the transcriptome-wide map of dynamic mRNA m6A at single-nucleotide resolution in METTL3-deficient KC-H and Mo-M cells during diet-induced NAFLD. 1.1.2). Identify the myeloid subset in which METTL3 deficiency results in protection from metabolic inflammation. This aim will identify the m6A pathway in myeloid subsets and downstream genes that contribute to hepatic inflammation during obesity-associated NAFLD. Aim 2 Determine the mechanistic relationship between the RNA helicase DDX21 and the m6A methylation to myeloid cell-driven hepatic inflammation. 2.1) Determine DDX21 recognition of m6A methylation on mRNAs. 2.2) Determine the role of DDX21 in myeloid lineage to promote obesity-associated steatohepatitis and fibrosis. This aim will mechanistically uncover DDX21 as a novel m6A regulator, and potential therapeutic target for obesity-associated NAFLD. This application will elucidate novel regulatory organisms underlying the role of m6A methylation in hepatic inflammation under obesity-associated NAFLD. The identification of DDX21 as a key role in liver myeloid subpopulations to prevent hepatic inflammation will provide the rationale for manipulating DDX21 and the basis for applying small molecules targeting m6A modulators in metabolic liver disease.

具体目的：肥胖引起的代谢性炎症导致非酒精性脂肪性肝病（NAFLD）向脂肪性肝炎（NASH）的转变[1-3]。骨髓细胞群如巨噬细胞已被确定为代谢性炎症的主要驱动因素[4-10]。单细胞RNA测序（scRNA-seq）研究提供了与不同表观基因组、转录组和功能相关的NAFLD髓系多样性的高分辨率命运图[9-11]。最近的进展强调了RNA水平上基因表达转录后调控在肥胖驱动的肝脏炎症中的重要性[12,13]。n6 -甲基腺苷（m6A）甲基化是真核生物mRNA中最普遍和最丰富的表观遗传修饰。m6A甲基化被m6A甲基转移酶（写入器）、m6A去甲基化酶（擦除器）和m6A特异性结合酶（读取器）动态安装、擦除和识别[14,15]。高度m6A修饰的转录本通常会导致mRNA稳定性降低，从而导致mRNA水平降低，从而极大地影响各种细胞和生物反应[16-18]。阅读器蛋白识别m6a修饰的序列并决定目标mRNA的命运。我们最近发现髓系谱系限制性缺失m6A书写蛋白甲基转移酶样3 （METTL3）导致m6A修饰mRNA转录物的差异表达，特别是负调控mTOR/NFkB激活的DNA损伤诱导转录物4 (DDIT4)，导致免疫细胞浸润减少，恢复肝脏稳态，改善肝脏代谢异常，减少饮食诱导肥胖/NAFLD bb0期间的炎症。尽管巨噬细胞METTL3在肥胖相关的NAFLD中起着重要作用，但它如何与不同的m6A读取器竞争和协作尚未确定，甲基化反应的特异性和RNA结构基序也未确定。髓细胞亚群中m6A甲基化的分子和细胞效应，除了降低mRNA稳定性外，在晚期纤维化中尚未被阐明。