MYH1G-AS 是一种与染色质相关的 lncRNA，可调节鸡的骨骼肌发育。
MYH1G-AS is a chromatin-associated lncRNA that regulates skeletal muscle development in chicken.
机构信息
State Key Laboratory of Livestock and Poultry Breeding, Guangdong Laboratory for Lingnan Modern Agriculture, College of Animal Science, South China Agricultural University, Guangzhou, China.
Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, Key Laboratory of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture and Rural Affairs, National-Local Joint Engineering Research Center for Livestock Breeding, Guangzhou, China.
出版信息
Cell Mol Biol Lett. 2024 Jan 4;29(1):9. doi: 10.1186/s11658-023-00525-x.
BACKGROUND
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.
METHODS
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 N-methyladenosine (mA) 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.
RESULTS
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 mA 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.
CONCLUSIONS
Our results reveal a new pattern of the regulation of lncRNA expression at diverse levels and help expound the regulation of mA methylation on chromatin status.
背景
骨骼肌的发育对于动物的生长和健康至关重要。最近，长链非编码 RNA（lncRNA）被发现通过多种作用与染色质相互作用。然而，lncRNA 如何作为染色质相关 RNA 来调节骨骼肌发育还知之甚少。在这里，我们旨在研究染色质相关 RNA（MYH1G-AS）在骨骼肌发育过程中的调控作用。
方法
我们通过 RNA 测序（RNA-seq）与高通量测序的转座酶可及染色质分析（ATAC-seq）相结合，提供了不同肌纤维的 RNA 谱和染色质可及性的全面见解。双荧光素酶报告基因检测和染色质免疫沉淀（ChIP）检测用于分析 MYH1G-AS 的转录调控机制。通过单碱基延伸和连接基于 qPCR 扩增方法（SELECT）检测 ALKBH5 介导的 MYH1G-AS N6-甲基腺苷（m6A）去甲基化。通过原代肌母细胞和慢病毒/胆固醇修饰的反义寡核苷酸（ASO）介导的动物模型研究 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 下拉和 RNA 免疫沉淀（RIP）检测。具体而言，通过共免疫沉淀（Co-IP）和酵母双杂交分析，分析了 FGF18 与 SWI/SNF 相关基质相关肌动蛋白依赖性染色质调节亚家族 A 成员 5（SMARCA5）蛋白之间的相互作用。
结果
总共鉴定出 45 个差异表达（DE）lncRNA，其启动子区域存在 DE ATAC-seq 峰，被归类为开放染色质相关 lncRNA。鉴定出一种骨骼肌特异性 lncRNA（MSTRG.15576.9；MYH1G-AS），它是开放染色质相关 lncRNA 之一。MYH1G-AS 的转录受转录因子（TF）SMAD3 和 SP2 的协调调控。此外，SP2 抑制 ALKBH5 的转录，减弱 ALKBH5 对 MYH1G-AS 的 m6A 去甲基化作用，从而破坏 MYH1G-AS RNA 的稳定性。MYH1G-AS 加速肌母细胞增殖，但抑制肌母细胞分化。此外，MYH1G-AS 驱动从慢肌纤维向快肌纤维的转变，并导致肌肉萎缩。机制上，MYH1G-AS 抑制 FGF18 蛋白的稳定，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，激活 SMAD4 依赖性途径。
结论
我们的结果揭示了 lncRNA 表达在不同水平上的调控的新模式，并有助于阐述 m6A 甲基化对染色质状态的调控。
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