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

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. Hi-C analysis of the 3D chromatin structure in Md, PA2 and the Pinna at developmental stages E10.5, E12.5 and E14.5 Hi-C analysis of the 3D chromatin structure in mESCs

增强子-启动子相互作用优先发生在受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尚不清楚。
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个可调控在面部形成过程中确立颅神经嵴细胞位置特性的靶基因。在第二咽弓（pharyngeal arch 2, PA2）的颅神经嵴细胞中，一个包含多个超级增强子的区域被划分为Hoxa家族TAD间调控元件1和2（Hoxa Inter-TAD Regulatory Element 1 and 2, HIRE1和HIRE2），该区域可与Hoxa2发生选择性的长距离跨TAD相互作用，而该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。敲除HIRE1则可重现完整的Hoxa2基因敲除的同源异形表型，并诱导第三、四咽弓的颅神经嵴细胞异常，该异常与Hoxa2和Hoxa3的转录下调相关。综上，在颅面发育过程中，超级增强子可突破TAD的绝缘效应，并以颅神经嵴细胞亚群特异性的方式调控Hoxa家族基因的前向共线性表达。本研究对发育阶段E10.5、E12.5和E14.5下的Md、PA2及耳郭（Pinna）的三维染色质结构，以及小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）的三维染色质结构进行了Hi-C分析。