Skeletal muscle development is pivotal for animal growth and health. Recently, long noncoding RNAs (lncRNAs) were found to interact with chromatin through diverse roles. However, little is known about how lncRNAs act as chromatin-associated RNAs to regulate skeletal muscle development. Here, we aim to investigate the regulation of chromatin-associated RNA (MYH1G-AS) during skeletal muscle development. We provided comprehensive insight into the RNA profile and chromatin accessibility of different myofibers, combining RNA sequencing (RNA-seq) with an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). The dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to analyze the transcriptional regulation mechanism of MYH1G-AS. ALKBH5-mediated MYH1G-AS N6-methyladenosine (m6A) demethylation was assessed by a single-base elongation and ligation-based qPCR amplification method (SELECT) assay. Functions of MYH1G-AS were investigated through a primary myoblast and lentivirus/cholesterol-modified antisense oligonucleotide (ASO)-mediated animal model. To validate the interaction of MYH1G-AS with fibroblast growth factor 18 (FGF18) protein, RNA pull down and an RNA immunoprecipitation (RIP) assay were performed. Specifically, the interaction between FGF18 and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SMARCA5) protein was analyzed by coimmunoprecipitation (Co-IP) and a yeast two-hybrid assay. A total of 45 differentially expressed (DE) lncRNAs, with DE ATAC-seq peaks in their promoter region, were classified as open chromatin-associated lncRNAs. A skeletal muscle-specific lncRNA (MSTRG.15576.9; MYH1G-AS), which is one of the open chromatin-associated lncRNA, was identified. MYH1G-AS transcription is coordinately regulated by transcription factors (TF) SMAD3 and SP2. Moreover, SP2 represses ALKBH5 transcription to weaken ALKBH5-mediated m6A demethylation of MYH1G-AS, thus destroying MYH1G-AS RNA stability. MYH1G-AS accelerates myoblast proliferation but restrains myoblast differentiation. Moreover, MYH1G-AS drives a switch from slow-twitch to fast-twitch fibers and causes muscle atrophy. Mechanistically, MYH1G-AS inhibits FGF18 protein stabilization to reduce the interaction of FGF18 to SMARCA5, thus repressing chromatin accessibility of the SMAD4 promoter to activate the SMAD4-dependent pathway. Our results reveal a new pattern of the regulation of lncRNA expression at diverse levels and help expound the regulation of m6A methylation on chromatin status.

中文翻译：

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MYH1G-AS 是一种染色质相关 lncRNA，可调节鸡骨骼肌发育

骨骼肌发育对于动物生长和健康至关重要。最近，人们发现长非编码 RNA (lncRNA) 通过不同的作用与染色质相互作用。然而，人们对 lncRNA 如何作为染色质相关 RNA 来调节骨骼肌发育知之甚少。在这里，我们的目标是研究骨骼肌发育过程中染色质相关 RNA (MYH1G-AS) 的调节。我们将 RNA 测序 (RNA-seq) 与转座酶可及染色质高通量测序 (ATAC-seq) 分析相结合，全面了解不同肌纤维的 RNA 谱和染色质可及性。采用双荧光素酶报告基因实验和染色质免疫沉淀（ChIP）实验分析MYH1G-AS的转录调控机制。通过单碱基延伸和基于连接的 qPCR 扩增方法 (SELECT) 测定评估 ALKBH5 介导的 MYH1G-AS N6-甲基腺苷 (m6A) 去甲基化。通过原代成肌细胞和慢病毒/胆固醇修饰的反义寡核苷酸（ASO）介导的动物模型研究了 MYH1G-AS 的功能。为了验证 MYH1G-AS 与成纤维细胞生长因子 18 (FGF18) 蛋白的相互作用，进行了 RNA pull down 和 RNA 免疫沉淀 (RIP) 测定。具体来说，通过免疫共沉淀 (Co-IP) 和酵母双杂交测定分析了 FGF18 和 SWI/SNF 相关基质相关肌动蛋白依赖性染色质亚家族 A 成员 5 (SMARCA5) 蛋白之间的相互作用。总共 45 个差异表达 (DE) lncRNA，其启动子区域有 DE ATAC-seq 峰，被归类为开放染色质相关 lncRNA。鉴定出骨骼肌特异性 lncRNA（MSTRG.15576.9；MYH1G-AS），它是开放染色质相关 lncRNA 之一。MYH1G-AS 转录受转录因子 (TF) SMAD3 和 SP2 协调调节。此外，SP2 抑制 ALKBH5 转录，削弱 ALKBH5 介导的 MYH1G-AS m6A 去甲基化，从而破坏 MYH1G-AS RNA 稳定性。MYH1G-AS 加速成肌细胞增殖但抑制成肌细胞分化。此外，MYH1G-AS 会驱动慢肌纤维向快肌纤维的转变，并导致肌肉萎缩。从机制上讲，MYH1G-AS 抑制 FGF18 蛋白稳定，减少 FGF18 与 SMARCA5 的相互作用，从而抑制 SMAD4 启动子的染色质可及性，从而激活 SMAD4 依赖性途径。我们的结果揭示了不同水平的lncRNA表达调控的新模式，并有助于阐明m6A甲基化对染色质状态的调控。