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

具体目标：肥胖诱导的代谢性炎症导致非酒精性脂肪性肝病（NAFLD）转变为脂肪性肝炎（NASH）[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骨髓多样性的高分辨率命运图[9-11]。最近的进展强调了在肥胖驱动的肝脏炎症中RNA水平上基因表达的转录后调节的重要性[12，13]。N6-甲基腺苷（m6 A）甲基化代表真核生物mRNA的最普遍和最丰富的表观遗传修饰。m6 A甲基化被m6 A甲基转移酶（写入器）、m6 A脱甲基酶（擦除器）和m6 A特异性结合酶（读取器）动态安装、擦除和识别[14，15]。高度m6 A修饰的转录物通常导致较低的mRNA稳定性，导致mRNA水平降低，从而极大地影响多种细胞和生物学反应[16-18]。阅读器蛋白识别m6 A修饰的序列并决定靶mRNA的命运。我们最近发现，骨髓谱系限制性缺失m6 A书写蛋白甲基转移酶样3（stomach L3）导致m6 A修饰的mRNA转录物的差异表达，特别是DNA损伤诱导转录物4（DDIT 4），其负调节mTOR/NF κ B活化，导致免疫细胞浸润减少，恢复肝脏稳态，改善肝脏代谢异常，并在饮食诱导的肥胖/NAFLD期间减少炎症[19]。虽然巨噬细胞L3在肥胖相关的NAFLD中起着重要作用，但它如何与不同的m6 A阅读器竞争和合作尚未确定，甲基化反应和RNA结构基序的特异性也尚未确定。在骨髓细胞亚群中m6 A甲基化的分子和细胞效应，除了降低mRNA的稳定性，还没有阐明在晚期纤维化。 DEAD盒RNA解旋酶DDX 21在核糖体生物合成、RNA编辑、RNA转运和转录中起着多方面的作用。DDX 21被报道与m6 A writer蛋白相互作用。对公开可用的RNA-CLIP测序数据的分析表明，靶mRNA分子上的DDX 21结合区域和m6 A甲基化位点之间存在重叠。我们的初步结果表明，骨髓系中DDX 21缺陷的小鼠（类似于胃L3缺陷）可显著预防饮食诱导的脂肪性肝炎和纤维化。scRNA-seq分析揭示了两种不同的骨髓群体，肝脏驻留（KC-H）巨噬细胞和单核细胞衍生的巨噬细胞（Mo-M），其在食物和饮食喂养的小鼠之间具有相反的比例动员。DDX 21的缺乏恢复了饮食喂养小鼠中KC-H的比例，同时降低了Mo-M的比例，沿着炎性标志物的减少，表明DDX 21在肥胖相关NAFLD期间在肝脏骨髓细胞中发挥了新的作用。因此，我们假设骨髓细胞亚群中mRNA m6 A甲基化与DDX 21相关，导致肥胖相关NAFLD期间的肝脏炎症（图1）。 目的1研究胃L3在肥胖相关NAFLD中髓系细胞驱动的肝脏炎症中的作用。1.1)确定KC-H和Mo-M亚群中的胃L3对肝脏炎症的贡献。1.1.1）在饮食诱导的NAFLD期间，在胃L3缺陷型KC-H和Mo-M细胞中以单核苷酸分辨率获得动态mRNA m6 A的全转录组图谱。1.1.2）。确定其中肌L3缺乏导致代谢性炎症保护的髓系亚群。这一目标将确定骨髓亚群中的m6 A通路和下游基因，这些基因有助于肥胖相关NAFLD期间的肝脏炎症。目的2探讨RNA解旋酶DDX 21与m6 A甲基化在髓系细胞驱动的肝脏炎症中的作用机制。2.1)测定DDX 21对mRNA上m6 A甲基化的识别。2.2)确定DDX 21在髓系中促进肥胖相关脂肪性肝炎和纤维化的作用。这一目标将从机制上揭示DDX 21作为一种新的m6 A调节剂，以及肥胖相关NAFLD的潜在治疗靶点。 本申请将阐明肥胖相关NAFLD下m6 A甲基化在肝脏炎症中的潜在作用的新型调控生物。DDX 21在肝脏骨髓亚群中预防肝脏炎症的关键作用的鉴定将为操纵DDX 21提供理论基础，并为在代谢性肝病中应用靶向m6 A调节剂的小分子提供基础。

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.