Long noncoding RNAs transcribed downstream of the human β-globin locus regulate β-globin gene expression
Long noncoding RNAs transcribed downstream of the human β-globin locus regulate β-globin gene expression
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DOI：
10.1093/jb/mvab130
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发表时间：
2021-12-08
影响因子：
2.7
通讯作者：
Ito, Takashi
中科院分区：
文献类型：
--
作者：
Higashi, Miki;Ikehara, Tsuyoshi;Ito, Takashi
已知人类β -珠蛋白基因位点上的5个β样珠蛋白基因（epsilon、G γ、A γ、delta和β）在特定的发育阶段表达，但其潜在机制的细节仍有待揭示。在这里，我们使用体外转录测定来阐明控制该基因表达的机制。我们首先使用RT-qPCR检测HeLa细胞的核RNA，在β -珠蛋白基因编码区下游4.4 kb开始的5.2 kb区域内发现了长链非编码RNA （lncRNAs）。我们使用引物延伸实验研究了K562细胞的核RNA，并确定了这些lncrna的转录起始位点（tss）。为了阐明它们的功能作用，我们在K562细胞中对这些lncrna进行了敲低（KD）。羟基脲（HU）诱导K562细胞分化，增加血红蛋白肽的产生，这些lncrna的KD增强了这种作用，也增强了HU诱导的γ -珠蛋白表达上调。为了证实这些结果，我们进行了体外转录试验。非编码单链rna抑制β -珠蛋白的表达，而β -珠蛋白被gata1上调。此外，在体外实验中，lncRNAs与gata1无序列特异性相互作用，抑制gata1与靶DNA应答元件的结合。我们的研究结果表明-珠蛋白基因位点下游的lncrna是调节珠蛋白基因表达的关键因素。
The five beta-like globin genes (epsilon, G gamma, A gamma, delta and beta) at the human beta-globin gene locus are known to be expressed at specific developmental stages, although details of the underlying mechanism remain to be uncovered. Here we used an in vitro transcription assay to clarify the mechanisms that control this gene expression. We first tested nuclear RNA from HeLa cells using RT-qPCR and discovered a long noncoding RNAs (lncRNAs) within a 5.2-kb region beginning 4.4 kb downstream of the beta-globin gene coding region. We investigated nuclear RNA from K562 cells using a primer-extension assay and determined the transcription start sites (TSSs) of these lncRNAs. To clarify their functional role, we performed knockdown (KD) of these lncRNAs in K562 cells. Hydroxyurea (HU), which induces differentiation of K562 cells, increased haemoglobin peptide production, and the effect was enhanced by KD of these lncRNAs, which also enhanced upregulation of the gamma-globin expression induced by HU. To confirm these results, we performed an in vitro transcription assay. Noncoding single-stranded RNAs inhibited beta-globin expression, which was upregulated byGATA1. Furthermore, lncRNAs interacted withGATA1without sequence specificity and inhibited its binding to its target DNA response element in vitro. Our results suggest that lncRNAs downstream of the beta-globin gene locus are key factors regulating globin gene expression.