Evf2 enhancer lncRNA-enhancer gene guidance through direct RNA binding and RNP recruitment domains

During mouse embryonic brain development, the Evf2 ultraconserved enhancer lncRNA (e-lncRNA) guides the Dlx5/6UCE enhancer to ~129 sites across chr6. However, previous work identified only 4 transcriptionally regulated targets associated with Evf2 enhancer-gene guided sites (EGGs), raising questions about the significance of the majority of Evf2-EGGs. Here, single cell transcriptomics shows that Evf2-regulated EGGs on chr6 coincide with subpopulation-specific Evf2 transcriptional targets, revealing far greater alignment between EGGs and transcriptional effects than previously reported. Evf2 regulation of EGGs to gene bodies (GB 5kb) divides chr6 into short-range (<10Mb), highly activated and long/super long-range (10-129Mb), moderately repressed genes. However, chr6 clustering of Evf2 transcriptionally regulated targets in populations where Evf2 is first activated, supports that transcriptional outcomes of Evf2-EGGs are not limited to gene body guidance, but may result from EGGs as far as 3Mb, uncoupling shift distance from transcriptional effects. Analysis of directly bound Evf2 RNA binding sites on chr6 (6 RBSs), divides chr6 into 4 regions, consistent with a role for RBS spacing in Evf2-EGG mediated, chromosome-spanning transcriptional regulation. In addition to chr6 Evf2 RBSs, 141 genome-wide RBSs are identified, 69 exactly aligning (>25bp) with at least one RBS, In addition to chr6 Evf2 RBSs, 141 genome-wide RBSs are identified, 69 exactly aligning (>25bp) with at least one RBS. RBS DNA identities are associated with transcription factor enrichment rather than DNA looping, as shown by HiCseq (chromosome capture of dual crosslinked) of E13.5GEs. While 80/147 RBSs are involved in inter-or intra-chr interactions, RBS chromosomal domains are differentially enriched for intra- and inter-chromosomal interactions, and domain specific Evf2-regulated RNP binding. Together with heterogeneous Evf2 regulation of protein pool/condensates (PPCs) of diverse Evf2 ribonucleoproteins (RNPs) (Smarcc2, an RNA binding chromatin remodeler, and Nono, an RNA paraspeckle organizer, Sox2 (a homeodomain transcription factor), RNP recruitment at RBSs and transcriptional targets, we propose a model whereby direct e-lncRNA binding, combinatorial Evf2-RNP recruitment, and homeodomain transcription factor DNA motif recognition/binding/activity, enables multi-modal EGG selectivity and transcriptional outcomes. Transcriptional effects of the Evf2 enhancer long non-coding RNA (e-lncRNA) are studied by comparing E13.5 mouse brain ganglionic eminences (GEs) from mutant mice lacking Evf2 (Evf2TS/TS) and wildtypes (Evf2+/+). E13.5 GEs are used in these studies to study the birthplace of embryonic interneurons. scSeq: 10X genomics single cell sequencing platform was performed to determine gene expression at single cell resolution from mutant and wildtype E13.5GEs. ChIPseq : Cut&Run native chromatin immunoprecipitation sequencing method was performed E13.5GEs from mutant and wildtypes to determine the effects of Evf2 on binding by two Evf2 ribonucleoproteins -Smarcc2 and Nono binding genome-wide, using anti-Smarcc2 and anti-Nono and subtraction with anti-IgG profiles as a negative control. ChIRP: Chromatin immunoprecipitation of RNA (ChIRP) on glutaraldehyde crosslinked Evf1/2TSTS E13.5GEs (mice lacking both Evf2 and Evf1, alternatively spliced form) followed by probing with two sets of antisense oligo probes (odd or even probes against the Evf2 transcript). GAPDH antisense oligo probes were used as a negative control. Only overlapping odd and even probe binding regions absent from GAPDH peaks and absent were considered to be Evf1/2 RNA binding sites.在小鼠胚胎脑发育过程中，Evf2超保守增强子长链非编码RNA（ultraconserved enhancer lncRNA，e-lncRNA）将Dlx5/6UCE增强子引导至6号染色体（chr6）上约129个位点。然而，既往研究仅鉴定出4个与Evf2增强子-基因引导位点（enhancer-gene guided sites，EGGs）相关的转录调控靶标，这引发了学界对绝大多数Evf2-EGGs生物学意义的诸多疑问。本研究通过单细胞转录组测序发现，6号染色体上受Evf2调控的EGGs与亚群特异性Evf2转录靶标高度重合，揭示出EGGs与转录调控效应之间的关联程度远高于此前报道。Evf2对EGGs的基因区域（gene bodies，GB 5kb）调控作用将6号染色体划分为两类区域：短程（<10Mb）高激活基因区域，以及长/超长程（10-129Mb）中度抑制基因区域。不过，在Evf2首次激活的细胞群中，受Evf2转录调控的靶标在6号染色体上的聚类分布表明，Evf2-EGGs介导的转录调控效应并不局限于基因区域引导，还可由最远达3Mb的EGGs介导，这打破了调控距离与转录效应之间的固有关联。对6号染色体上直接结合的Evf2 RNA结合位点（RNA binding sites，RBSs）进行分析，可将6号染色体划分为4个区域，这与RBS间隔在Evf2-EGGs介导的跨染色体转录调控中的作用相一致。除6号染色体上的Evf2 RBSs外，本研究还鉴定出141个全基因组范围的RBSs，其中69个可与至少一个RBS精准匹配（匹配长度>25bp）。通过对E13.5胎脑神经节隆起（ganglionic eminences，GEs）的Hi-C测序（双交联染色体捕获技术）分析发现，RBS的DNA序列特征与转录因子富集显著相关，而非DNA环化作用。尽管147个RBSs中有80个参与了染色体内或染色体间相互作用，但RBS所在的染色体结构域在染色体内/间相互作用以及结构域特异性Evf2调控的核糖核蛋白（ribonucleoproteins，RNPs）结合方面存在显著差异。结合多种Evf2核糖核蛋白——Smarcc2（一种RNA结合型染色质重塑因子）、Nono（一种RNA核散斑组织者蛋白）、Sox2（一种同源域转录因子）的蛋白池/凝聚体（protein pool/condensates，PPCs）异质性调控，以及RBS处的RNP招募与转录靶标情况，本研究提出了一个模型：直接的e-lncRNA结合、组合式Evf2-RNP招募，以及同源域转录因子的DNA基序识别、结合与活性调控，共同实现了多模态的EGGs选择性与转录调控效应。本研究通过对比缺失Evf2的突变小鼠（Evf2^TS/TS）与野生型小鼠（Evf2^+/+）的E13.5胎脑神经节隆起（GEs），解析了Evf2增强子长链非编码RNA的转录调控效应；选择E13.5 GEs作为研究对象，旨在探究胚胎中间神经元的起源位点。单细胞测序：采用10X Genomics单细胞测序平台，以单细胞分辨率检测突变型与野生型E13.5 GEs的基因表达谱。染色质免疫共沉淀测序（ChIP-seq）：采用Cut&Run原生染色质免疫共沉淀测序技术，对突变型与野生型E13.5 GEs进行检测，通过抗Smarcc2抗体、抗Nono抗体，并以抗IgG测序数据作为阴性对照，分析Evf2对两种Evf2核糖核蛋白——Smarcc2与Nono全基因组结合的调控作用。RNA染色质免疫共沉淀（ChIRP）：对经戊二醛交联的、同时缺失Evf2与Evf1（一种可变剪接形式）的E13.5 GEs（Evf1/2^TS/TS小鼠）进行ChIRP实验，随后使用两套反义寡核苷酸探针（分别靶向Evf2转录本的奇数或偶数区域）进行杂交捕获；同时以靶向GAPDH的反义寡核苷酸探针作为阴性对照。仅将奇数与偶数探针结合的重叠区域、且不处于GAPDH富集峰中的区域，认定为Evf1/2 RNA结合位点。