Unlinking a lncRNA from its associated cis element [Capture-C]

Capture-C using probes at the Cdkn1b promoter and the Lockd promoter Overall design: Many long non-coding (lnc) RNAs are reported to regulate gene expression and protein functions. However, the proportion of lncRNAs with biological activities among the thousands expressed in mammalian cells is controversial. We studied Lockd (LncRNA downstream of Cdkn1b), a 434 bp polyadenylated lncRNA originating 4 kb 3’ to the Cdkn1b gene. Heterozygous and homozygous deletion of the 25 kb Lockd locus reduced Cdkn1b transcription by approximately 35 and 70% respectively in a mouse erythroid cell line. In contrast, homozygous insertion of a polyadenylation cassette 80 bp downstream of the Lockd transcription start site reduced the entire lncRNA transcript level by > 90%, but had no effect on Cdkn1b transcription. The promoter of the Lockd gene contains a DNase hypersensitive site, binds numerous transcription factors (TFs), and physically associates with the Cdkn1b promoter in chromosomal conformation capture (NG Capture-C) studies. Thus, the Lockd gene positively regulates Cdkn1b transcription through an enhancer-like cis element, while the lncRNA itself is dispensable. These findings demonstrate that the biological activities of a lncRNA cannot be inferred from phenotypes that arise after deleting the corresponding gene. Rather, the model of an inert transcript arising from a functional genomic cis element should be considered while investigating the biology of any lncRNA.

以Cdkn1b启动子与Lockd启动子为探针的捕获式染色体构象捕获（Capture-C）实验 实验设计概述：已有研究表明，众多长链非编码RNA（lncRNA）可调控基因表达与蛋白质功能。然而，哺乳动物细胞中表达的数千种lncRNA里，具备生物学活性的占比仍颇具争议。本研究聚焦于Lockd（Cdkn1b下游长链非编码RNA）：这是一段长度为434 bp、经聚腺苷酸化修饰的lncRNA，其转录起始位点位于Cdkn1b基因下游4 kb处。在小鼠红系细胞系中，对25 kb大小的Lockd基因座进行杂合缺失与纯合缺失，分别使Cdkn1b的转录水平降低约35%与70%。与之相反，在Lockd转录起始位点下游80 bp处纯合插入聚腺苷酸化盒，可使整条lncRNA转录本的丰度降低超过90%，但对Cdkn1b的转录水平无显著影响。在染色体构象捕获（NG Capture-C）实验中，Lockd基因的启动子区域含有DNase超敏位点，可结合多种转录因子（transcription factors, TFs），并与Cdkn1b启动子发生物理相互作用。由此可见，Lockd基因通过类增强子顺式作用元件正向调控Cdkn1b的转录，而其自身转录产生的lncRNA并非必需。上述研究结果表明，无法通过删除对应基因后产生的表型来推断某一lncRNA的生物学活性。反之，在研究任意lncRNA的生物学功能时，应考虑到这一模型：功能性基因组顺式作用元件转录产生的是无活性的转录本。