Data from: Evf2 lncRNA/BRG1/DLX1 interactions reveal RNA-dependent inhibition of chromatin remodeling

Transcription-regulating long non-coding RNAs (lncRNAs) have the potential to control the site-specific expression of thousands of target genes. Previously, we showed that Evf2, the first described ultraconserved lncRNA, increases the association of transcriptional activators (DLX homeodomain proteins) with key DNA enhancers but represses gene expression. In this report, mass spectrometry shows that the Evf2-DLX1 ribonucleoprotein (RNP) contains the SWI/SNF-related chromatin remodelers Brahma-related gene 1 (BRG1, SMARCA4) and Brahma-associated factor (BAF170, SMARCC2) in the developing mouse forebrain. Evf2 RNA colocalizes with BRG1 in nuclear clouds and increases BRG1 association with key DNA regulatory enhancers in the developing forebrain. While BRG1 directly interacts with DLX1 and Evf2 through distinct binding sites, Evf2 directly inhibits BRG1 ATPase and chromatin remodeling activities. In vitro studies show that both RNA-BRG1 binding and RNA inhibition of BRG1 ATPase/remodeling activity are promiscuous, suggesting that context is a crucial factor in RNA-dependent chromatin remodeling inhibition. Together, these experiments support a model in which RNAs convert an active enhancer to a repressed enhancer by directly inhibiting chromatin remodeling activity, and address the apparent paradox of RNA-mediated stabilization of transcriptional activators at enhancers with a repressive outcome. The importance of BRG1/RNA and BRG1/homeodomain interactions in neurodevelopmental disorders is underscored by the finding that mutations in Coffin–Siris syndrome, a human intellectual disability disorder, localize to the BRG1 RNA-binding and DLX1-binding domains.

转录调控型长链非编码RNA（long non-coding RNAs，简称lncRNAs）具备调控数千个靶基因位点特异性表达的潜能。此前本团队已证实，首个被报道的超保守长链非编码RNA Evf2，可增强转录激活因子（transcriptional activators，即DLX同源结构域蛋白）与关键DNA增强子的结合，但同时会抑制基因表达。本研究通过质谱分析法（mass spectrometry）证实，在发育中小鼠前脑中，Evf2-DLX1核糖核蛋白（ribonucleoprotein，简称RNP）包含SWI/SNF家族染色质重塑因子（SWI/SNF-related chromatin remodelers）中的Brahma相关基因1（BRG1, SMARCA4）与Brahma关联因子（BAF170, SMARCC2）。Evf2 RNA与BRG1在核斑中共定位，并可增强BRG1与发育中小鼠前脑中关键DNA调控增强子的结合。尽管BRG1可通过不同结合位点分别与DLX1及Evf2直接相互作用，但Evf2可直接抑制BRG1的ATP酶（adenosine triphosphatase，简称ATPase）活性与染色质重塑活性。体外实验证实，RNA与BRG1的结合以及RNA对BRG1 ATP酶/重塑活性的抑制均存在非特异性，这表明实验环境是RNA依赖型染色质重塑抑制过程中的关键影响因素。综合上述实验结果，本研究提出了一种全新模型：RNA可通过直接抑制染色质重塑活性，将活化的增强子转化为抑制型增强子，这一模型也解释了一个看似矛盾的现象——RNA可在增强子处稳定转录激活因子，却最终产生抑制性的表达结果。研究发现，人类智力障碍疾病科芬-西里斯综合征（Coffin–Siris syndrome）患者的BRG1突变位点恰好位于其RNA结合结构域与DLX1结合结构域中，这进一步凸显了BRG1与RNA、BRG1与同源结构域蛋白的相互作用在神经发育疾病中的重要性。