RNA-seq for wild type and beta-actin KO MEFs in presence and in KD of Meg3

In eukaryotic cells, beta-actin is crucial for nuclear functions. It influences genome organization and regulates promoter-enhancer interactions as part of the BAF (SWI/SNF) chromatin remodeling complex. beta-actin also binds to the hyperphosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. A lack of beta-actin results in altered gene expression, affecting key cellular processes such as differentiation. This study examined whether beta-actin regulates long non-coding RNA (lncRNA) levels. Using bulk RNA-seq and qPCR analyses on total RNA from WT mouse embryonic fibroblasts (MEFs), beta-actin heterozygous (HET) MEFs, and beta-actin knockout (KO) MEFs, we found that the expression of several lncRNAs is influenced by beta-actin depletion. Notably, the lncRNA Meg3 showed increased expression in a dosage-dependent manner. ChIRP-seq analysis revealed changes in Meg3 genomic association and its enrichment at or near enhancer sites following beta-actin depletion, along with increased H3K27 acetylation. These changes led to disrupted promoter-enhancer interactions, as indicated by the Activity by Contact (ABC) model, due to a sponging effect by Meg3. This resulted in the repression of genes involved in the metabolic biosynthetic pathways for chondroitin, heparan, dermatan sulfate, and phospholipases, affecting their synthesis. We suggest that Meg3 disrupts local genome organization (or DNA looping) and negatively impacts gene expression through its chromatin sponging effect, interfering with promoter-enhancer interactions.

在真核细胞中，β-肌动蛋白（beta-actin）对核功能至关重要。作为BAF（SWI/SNF）染色质重塑复合物的核心组分之一，它可调控基因组组织并参与启动子-增强子相互作用的调控。此外，β-肌动蛋白还可结合RNA聚合酶II（RNA polymerase II）的高磷酸化羧基末端结构域（CTD）。β-肌动蛋白的缺失会引发基因表达异常，影响细胞分化等关键细胞生命过程。
本研究旨在探究β-肌动蛋白是否可调控长链非编码RNA（lncRNA）的表达水平。研究团队针对野生型（WT）小鼠胚胎成纤维细胞（MEFs）、β-肌动蛋白杂合（HET）小鼠胚胎成纤维细胞及β-肌动蛋白敲除（KO）小鼠胚胎成纤维细胞的总RNA，开展了批量RNA测序（bulk RNA-seq）与定量聚合酶链反应（qPCR）分析，结果显示多种长链非编码RNA的表达水平受β-肌动蛋白耗竭的显著影响。值得注意的是，长链非编码RNA Meg3的表达呈现出剂量依赖性升高的趋势。
染色质RNA纯化分离测序（ChIRP-seq）分析结果表明，β-肌动蛋白耗竭后，Meg3的基因组结合特性发生改变，其在增强子位点或其邻近区域的富集程度显著升高，同时伴随H3K27乙酰化水平的上调。基于接触活性（ABC）模型的分析显示，上述变化因Meg3的海绵效应破坏了启动子-增强子相互作用，最终抑制了软骨素、硫酸乙酰肝素、硫酸皮肤素代谢生物合成通路及磷脂酶相关基因的表达，进而影响这些物质的合成过程。
本研究提出，Meg3可通过染色质海绵效应破坏局部基因组组织（或DNA环化），并负向调控基因表达，干扰启动子-增强子之间的相互作用。