A multiple super-enhancer region establishes inter-TAD interactions and controls Hoxa function in cranial neural crest [RNA-seq]

Enhancer-promoter interactions preferentially occur within boundary-insulated topologically associating domains (TADs), limiting inter-TAD interactions. Enhancer clusters in linear proximity, termed super-enhancers (SEs), ensure high target gene expression levels. Little is known about SE topological regulatory impact during craniofacial development. Here, we identified 2232 genome-wide putative SEs in mouse cranial neural crest cells (CNCCs), 147 of which target genes establishing CNCC positional identity during face formation. In second pharyngeal arch (PA2) CNCCs, a multiple SE-containing region, partitioned into Hoxa Inter-TAD Regulatory Element 1 and 2 (HIRE1 and HIRE2), establishes long-range inter-TAD interactions selectively with Hoxa2, that is required for external and middle ear structures. HIRE2 deletion in a Hoxa2 haploinsufficient background results in microtia. HIRE1 deletion phenocopies the full homeotic Hoxa2 knockout phenotype and induces PA3 and PA4 CNCC abnormalities correlating with Hoxa2 and Hoxa3 transcriptional downregulation. Thus, SEs can overcome TAD insulation and regulate anterior Hoxa gene collinear expression in a CNCC subpopulation-specific manner during craniofacial development. Overall design: RNA expression profiling of cranial neural crest cell subpopulations (PA2 and PA3 at E10.5, Pinna at E12.5 and E14.5) of wildtype and HIRE1 knockout genotypes using RNA-seq.

增强子-启动子相互作用优先发生在边界绝缘的拓扑关联结构域（topologically associating domains, TADs）内部，从而限制了跨TAD的相互作用。线性位置邻近的增强子簇被称为超级增强子（super-enhancers, SEs），可确保靶基因的高表达水平。目前对于颅面发育过程中超级增强子的拓扑调控作用仍知之甚少。本研究在小鼠颅神经嵴细胞（cranial neural crest cells, CNCCs）中鉴定出2232个全基因组范围的潜在超级增强子，其中147个可靶向调控在面部形成过程中建立颅神经嵴细胞位置身份的靶基因。在第二鳃弓（PA2）的颅神经嵴细胞中，一段包含多个超级增强子的区域被划分为Hoxa跨TAD调控元件1和2（HIRE1与HIRE2），该区域可与Hoxa2基因发生选择性的长距离跨TAD相互作用，而这一相互作用对于外耳和中耳结构的形成是必需的。在Hoxa2单倍体不足的背景下敲除HIRE2会导致小耳畸形（microtia）。敲除HIRE1则可模拟完整的同源异型Hoxa2基因敲除表型，并诱导第三、第四鳃弓颅神经嵴细胞异常，该异常与Hoxa2和Hoxa3的转录下调相关。综上，超级增强子能够突破TAD的绝缘限制，并在颅面发育过程中以颅神经嵴细胞亚群特异性的方式调控前部Hoxa基因的共线性表达。整体实验设计：利用RNA测序（RNA-seq）对野生型及HIRE1基因敲除基因型的颅神经嵴细胞亚群（E10.5时期的第二、第三鳃弓细胞，以及E12.5、E14.5时期的耳郭细胞）进行转录表达谱分析。