Integration of 3D genome architecture and local chromatin features uncovers enhancers underlying craniofacial-specific cartilage defects

Aberrations in tissue-specific enhancers underlie many developmental defects. Disrupting a distal noncoding region of the human SOX9 gene causes the Pierre Robin sequence (PRS) characterized by the undersized lower jaw. Such a craniofacial-specific defect has been previously linked to enhancers transiently active in cranial neural crest cells (CNCCs). We demonstrate the PRS region also strongly regulates Sox9 in CNCC-derived Meckel's cartilage (MC), but not in limb cartilages. The MC-specific regulatory function of the PRS region cannot be attributed to the CNCC enhancers that have been inactivated in cartilages, but correlates with its MC-specific interactions with the Sox9 gene. By integrating the enhancer signatures and chromatin topology, we uncovered ~20,000 enhancers that function differentially between MC and limb cartilages and demonstrated their association with human diseases. Our findings highlight the importance of lineage-dependent chromatin topology in instructing enhancer usage and provide critical insights for interpreting the genetic basis of craniofacial pathologies. Overall design: In this study, we systematically identified the enhancer atlas in mouse mandibular Meckel's cartilage (MC) and forelimb cartilage with multiple high-throughput sequencing techiniques including ATAC-seq, H3K27Ac CUT & Tag and Hi-C. By integrating local chromatin features and 3D genome architecture, our analyses uncovered several distal enhancers that specifically regulate Sox9 expression in MC, which could possibly contribute to the incidence of Pierre Robin sequence when disrupted.

组织特异性增强子的异常是诸多发育缺陷的致病基础。敲除人类SOX9(SOX9)基因的远端非编码区，会引发以小下颌为特征的皮埃尔·罗宾序列征(PRS)。这类颅面特异性缺陷此前已被证实与在颅神经嵴细胞(CNCCs)中瞬时活跃的增强子相关。本研究证实，PRS区域可在CNCC来源的梅克尔软骨(MC)中强力调控Sox9的表达，但在肢体软骨中并无此作用。PRS区域在MC中的特异性调控功能，无法归因于软骨中已失活的CNCC增强子，而是与其与Sox9基因的MC特异性染色质互作相关。通过整合增强子特征与染色质拓扑结构，我们共鉴定出约20000个在MC与肢体软骨间功能存在差异的增强子，并证实这些增强子与人类疾病存在关联。本研究结果凸显了谱系依赖性染色质拓扑结构对增强子使用模式的调控作用，同时为阐释颅面病理的遗传基础提供了关键见解。实验设计：本研究采用ATAC-seq、H3K27Ac CUT&Tag及Hi-C等多种高通量测序技术，系统性鉴定了小鼠下颌梅克尔软骨(MC)与前肢软骨的增强子图谱。通过整合局部染色质特征与三维基因组架构，本研究分析鉴定出数个可在MC中特异性调控Sox9表达的远端增强子，这些增强子若发生异常，或可参与皮埃尔·罗宾序列征的发病过程。