Enhancer RNAs (eRNA) are noncoding transcripts produced from active enhancers and have potential gene regulatory function. CCAAT enhancer-binding protein alpha (CEBPA) is a transcription factor generally involved in metabolism, cell cycle inhibition, hematopoiesis, adipogenesis, hepatogenesis, and is associated with tumorigenesis. In this study, we demonstrate that an enhancer-associated long noncoding RNA (elncRNA), transcribed from an enhancer located 9 kb downstream from the transcriptional start site (TSS) of CEBPA, positively regulates the expression of CEBPA. As a result, we named this elncRNA “CEBPA regulatory elncRNA downstream 9 kb” or “CRED9.” CRED9 expression level positively correlates with CEBPA mRNA expression across multiple cell lines as detected by RT droplet digital PCR. Knockdown of CRED9 resulted in a reduction of CEBPA mRNA expression in Hep3B cells. Additionally, CRED9 knockdown in Hep3B and HepG2 cells resulted in lower CEBPA protein expression. We also found that knockdown of CRED9 in Hep3B cells caused a 57.8% reduction in H3K27ac levels at the +9 kb CEBPA enhancer. H3K27ac has previously been described as a marker of active enhancers. Taken together, the evidence presented here supports a previously proposed model whereby, in some contexts, eRNA transcripts are necessary to amplify and maintain H3K27ac levels at a given enhancer. Ultimately, this study adds to the growing body of evidence that elncRNA transcripts have important roles in enhancer function and gene regulation.

中文翻译：

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CRED9：一种差异表达的elncRNA调节转录因子CEBPA的表达

增强子 RNA (eRNA) 是由活性增强子产生的非编码转录本，具有潜在的基因调控功能。CCAAT 增强子结合蛋白 α (CEBPA) 是一种转录因子，通常参与代谢、细胞周期抑制、造血、脂肪生成、肝生成，并与肿瘤发生有关。在这项研究中，我们证明了一个增强子相关的长链非编码 RNA (elncRNA)，从位于 CEBPA 转录起始位点 (TSS) 下游 9 kb 处的增强子转录，正向调节 CEBPA 的表达。因此，我们将此 elncRNA 命名为“CEBPA 调控 elncRNA 下游 9 kb”或“CRED9”。通过 RT 液滴数字 PCR 检测，CRED9 表达水平与多个细胞系的 CEBPA mRNA 表达呈正相关。CRED9 的敲低导致 Hep3B 细胞中 CEBPA mRNA 表达的减少。此外，Hep3B 和 HepG2 细胞中的 CRED9 敲低导致 CEBPA 蛋白表达降低。我们还发现 Hep3B 细胞中 CRED9 的敲低导致 +9 kb 时 H3K27ac 水平降低 57.8%CEBPA增强剂。H3K27ac 以前被描述为活性增强剂的标志物。总之，这里提供的证据支持先前提出的模型，在某些情况下，eRNA 转录物对于在给定增强子处放大和维持 H3K27ac 水平是必要的。最终，这项研究增加了越来越多的证据表明 elncRNA 转录本在增强子功能和基因调控中具有重要作用。