Pre-Hypertrophic Chondrogenic Enhancer Landscape of Limb and Axial Skeleton Development [scRNA-seq]

The precisely orchestrated differentiation of chondrocytes during skeleton development is a critical determinant of human height and body shape. Disruptions of this process can cause severe skeletal abnormalities. The ultimate size and shape of each of over 200 bones depends on the intricate spatiotemporal regulation of chondrogenic and chondrocyte differentiation genes, but the genomic architecture coordinating these events remains poorly defined. Here we provide a comprehensive map of transcriptional enhancers specifically active in chondrocytes and show that they provide a mechanistic framework through which noncoding genetic variants can influence human stature. We isolated limb and trunk fetal chondrocytes from mice with a Col2a1 fluorescent regulatory sensor and used RNA-seq to identify 780 genes that are specifically expressed during chondrogenesis. To create cell type-specific enhancer maps, we performed ATAC-seq to map open chromatin regions and ChIP-seq for H3K27ac, an enhancer-associated histone modification, and identified 2'704 putative chondrogenic enhancer regions. Most of these enhancers (74%) showed pan-chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. We found that chondrogenic enhancers are enriched for the binding of several chondrogenic transcription factors including SOX9. Moreover, we find that genetic variation overlapping chondrogenic enhancers explains a higher fraction of the heritability of human adult height than the one overlapping non-chondrogenic enhancers. Finally, targeted deletions of identified enhancers at the Fgfr3, Col2a1, Hhip and, Nkx3-2 loci each exhibited a significant reduction of cognate gene expression, therefore demonstrating their functional importance. This data provides a comprehensive mapping of the chondrogenic enhancer repertoire, paving the way to interpreting the role of non-coding sequence polymorphisms in phenotypic variation and bone diseases. Overall design: We devised a Col2a1 fluroescent reporter approach to sort chondrocytes from embryonic trunk and limbs. We validate the specfici of the reproter using scRNAseq. We then analyse Col2a1-GFP positive and negative cells to profile their transcriptome, open chromatin and H3K27ac coverage to define enahncers and asssooicated genes.

骨骼发育过程中软骨细胞的精准协调分化，是决定人类身高与体型的关键因素。该过程发生紊乱可引发严重的骨骼畸形。人体超过200块骨骼的最终大小与形态，依赖于软骨生成与软骨细胞分化相关基因的复杂时空调控，但协调这些事件的基因组架构仍未被充分阐明。本研究构建了软骨细胞特异性活跃的转录增强子（transcriptional enhancers）全景图谱，并证实其可为非编码遗传变异影响人类身高提供机制性框架。我们通过携带Col2a1荧光调控传感器的小鼠，分离其肢体与躯干的胎儿软骨细胞，并利用RNA测序（RNA-seq）鉴定出780个软骨生成阶段特异性表达的基因。为构建细胞类型特异性增强子图谱，我们通过ATAC测序（ATAC-seq）绘制开放染色质区域，并针对增强子相关的组蛋白修饰H3K27乙酰化（H3K27ac）开展染色质免疫共沉淀测序（ChIP-seq），最终鉴定出2704个潜在软骨生成增强子区域。其中大多数增强子（74%）表现出泛软骨生成活性，仅少数群体分别局限于肢体（18%）或躯干（8%）软骨细胞。我们发现软骨生成增强子富集了包括SOX9在内的多种软骨生成转录因子的结合位点。此外，相较于非软骨生成增强子区域，重叠于软骨生成增强子的遗传变异可解释更高比例的成人身高遗传力。最后，针对Fgfr3、Col2a1、Hhip及Nkx3-2基因座的鉴定增强子进行靶向缺失实验，均显著降低了对应基因的表达水平，从而证实了这些增强子的功能重要性。本数据集提供了软骨生成增强子组的全面图谱，为解析非编码序列多态性在表型变异与骨骼疾病中的作用奠定了基础。整体实验设计：我们构建了Col2a1荧光报告系统，用于分选胚胎躯干与肢体来源的软骨细胞；通过单细胞RNA测序（scRNA-seq）验证该报告系统的细胞特异性；随后对Col2a1-GFP阳性与阴性细胞进行转录组、开放染色质及H3K27ac修饰谱分析，以明确增强子及其关联基因。