Conserved Cis-Acting Range Extender Elements Mediate Long-Range Developmental Enhancer Activity in Mammals

Long-range developmental enhancers can regulate cell-type specific target gene expression over distances that exceed hundreds of thousands of base pairs in mammals. Although higher-order chromatin organization provides support for such long-range enhancerpromoter interactions, the genetic factors that regulate these interactions during mammalian development remain largely unexplored. To address this central question, we replaced a limb enhancer, located >800,000 base pairs from its target promoter, with equivalent limb enhancers of similar strength from other genomic loci. We used this replacement strategy to generate six knock-in mouse strains, with enhancers from four distinct genomic loci. All enhancers drove strong expression in developing limb buds when placed adjacent to a reporter gene. However, when transplanted to a distal location, all four enhancers failed to activate remote gene expression and distal limb outgrowth, suggesting that proximally-active enhancers cannot activate transcription remotely. Including a highly conserved, but previously uncharacterized, sequence found adjacent to a long-range limb enhancer rescued gene expression and limb outgrowth. This cis-acting Range EXtender (REX) element does not have classical enhancer activity but is necessary and sufficient for enhancerpromoter communication over very long distances in vivo. The REX element contains highly conserved [C/T]AATTA consensus sequences that match binding preferences of LIM-homeodomain (LHX) transcription factors. We used multiomic single-cell ATAC-seq/RNA-seq profiling to identify [C/T]AATTA homeodomain motifs next to thousands of long-range limb enhancers. The presence of these motifs positively correlates with EP distance genome-wide. Our data support a model where REX elements facilitate target gene activation by remote enhancers over very long distances in mammalian genomes. Whole hindlimb was dissected from an E11.5 FVB embryo, dissociated into single cells, lysed into nuclei and then single-cell multiomics (scRNA-seq and scATAC-seq) was performed. For allele-specific ATAC-seq, both hindlimb ZPAs of individual E11.5 embryos from various enSWAP knock-in mice were dissected and slowly frozen using Mr. Frosty. Embryo genotypes were determined and single heterozygotes were chosen and bulk ATAC-seq was performed on the collected tissue.在哺乳动物中，长程发育增强子可在数十万碱基对以上的距离范围内调控细胞类型特异性的靶基因表达。尽管高阶染色质组织为这类长程增强子-启动子（enhancer-promoter）相互作用提供了结构基础，但在哺乳动物发育过程中调控这些相互作用的遗传因子仍未得到充分研究。为解答这一核心科学问题，我们将距离其靶启动子超过80万碱基对的肢体增强子，替换为来自其他基因组位点、活性强度相近的同源肢体增强子。通过该替换策略，我们构建了6株敲入（knock-in）小鼠品系，其增强子分别来自4个不同的基因组位点。当将这些增强子置于报告基因邻近位置时，所有增强子均可在发育中的肢芽中驱动强烈的报告基因表达。但当将这些增强子移植到远端位点后，4种增强子均无法激活远端基因表达及远端肢体发育，这表明近端活性增强子无法远程激活转录。将一段高度保守但此前未被注释的、位于长程肢体增强子邻近区域的序列纳入后，可恢复靶基因表达与肢体发育。该顺式作用序列命名为范围拓展因子（Range EXtender, REX），其本身不具备经典增强子活性，但在体内可介导长距离的增强子-启动子相互作用，且是该过程所必需且充分的条件。REX元件包含高度保守的[C/T]AATTA共有序列，该序列与LIM同源结构域（LIM-homeodomain, LHX）转录因子的结合偏好性相匹配。我们通过多组学单细胞ATAC测序/RNA测序（scATAC-seq/scRNA-seq）分析，在数千个长程肢体增强子邻近区域鉴定出了[C/T]AATTA同源结构域基序。全基因组范围内，这些基序的存在与增强子-启动子间距呈正相关。我们的研究结果支持如下模型：在哺乳动物基因组中，REX元件可协助远程增强子在超远距离下激活靶基因表达。从E11.5龄FVB小鼠胚胎中分离完整后肢，解离为单细胞并裂解获取细胞核，随后进行单细胞多组学（scRNA-seq与scATAC-seq）测序。对于等位基因特异性ATAC测序，我们从不同enSWAP敲入小鼠的单个E11.5胚胎中分离双侧后肢极化活动区（zone of polarizing activity, ZPA），使用Mr. Frosty程序缓慢冷冻保存。鉴定胚胎基因型后，选取单杂合子个体，对收集的组织进行批量ATAC测序（bulk ATAC-seq）。