Profiling of upstream enhancers of SHOX in idiopathic short stature and functional characterization of the cis-regulatory landscape of SHOX

SHOX mutations and deletions of the downstream regulatory region have been reported in cases with idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). Recently, a deletion and duplication of upstream enhancers have been described in ISS. Here, we aimed to evaluate the contribution of upstream copy number variations (CNVs) to the pathogenesis of ISS, to validate the enhancer role of the upstream enhancers in human cells, and to characterize the chromatin architecture of the cis-regulatory landscape of SHOX. CNV analysis of three upstream conserved non-coding elements (CNEs), CNE-5, CNE-3 and CNE-2, in 501 ISS referrals with no established molecular diagnosis revealed two deletions and one duplication. Enhancer activity of the upstream CNEs was corroborated by luciferase assays in human osteosarcoma U2OS cells. In addition, all three CNEs overlap with reported H3K27ac ChIP-seq marks in human embryonic limb buds. To characterize the chromatin interaction profile of the SHOX region, chromosome conformation capture (4C-seq) was performed in chicken embryo limb buds and in U2OS cells, revealing interactions of the upstream CNEs with the SHOX promoter. Moreover, the 4C-seq interaction maps and H3K27ac marks indicated that the cis-regulatory landscape of SHOX encompasses 1 Mb, suggesting additional cis-regulatory elements controlling SHOX.In conclusion, we showed that upstream CNVs of SHOX are rare in ISS and have incomplete penetrance. Chromatin interaction maps, luciferase assays and H3K27ac marks further support an enhancer function for the upstream CNEs. Finally, we demonstrated that the cis-regulatory landscape of SHOX is larger than previously anticipated potentially harboring novel cis-regulatory elements, which may be involved in the pathogenesis of molecularly unsolved ISS cases.. Overall design: 4C-seq samples forshox gene promoter in different samples: HH28 chicken limbs and human U2OS cells

已有研究报道，特发性身材矮小（idiopathic short stature, ISS）及勒里-韦尔软骨发育不全（Leri-Weill dyschondrosteosis, LWD）患者中存在SHOX基因突变及其下游调控区缺失。近期，有研究在ISS患者中报道了上游增强子的缺失与重复事件。本研究旨在评估上游拷贝数变异（copy number variations, CNVs）在ISS发病机制中的贡献，验证上游增强子在人类细胞中的增强子功能，并解析SHOX顺式调控区域的染色质结构。
我们对501例未明确分子诊断的ISS转诊患者的3个上游保守非编码元件（conserved non-coding elements, CNEs）——CNE-5、CNE-3及CNE-2——开展CNV分析，共检出2例缺失及1例重复事件。通过在人类骨肉瘤U2OS细胞系中进行荧光素酶实验，证实了上述上游CNEs的增强子活性。此外，这3个CNEs均与人类胚胎肢芽中已报道的H3K27ac 染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）信号峰重叠。
为解析SHOX区域的染色质相互作用图谱，我们分别在鸡胚肢芽及U2OS细胞系中开展了染色体构象捕获（4C-seq）实验，结果显示上游CNEs可与SHOX基因启动子发生相互作用。此外，4C-seq相互作用图谱及H3K27ac信号峰均表明，SHOX的顺式调控区域跨度达1 Mb，提示存在额外的调控SHOX表达的顺式调控元件。
综上，本研究证实SHOX上游CNVs在ISS患者中较为罕见，且呈现不完全外显率。染色质相互作用图谱、荧光素酶实验及H3K27ac信号峰进一步支持了上游CNEs的增强子功能。最后，我们证明SHOX的顺式调控区域范围较此前预期更广，可能包含新的顺式调控元件，这些元件或参与分子诊断未明确的ISS患者的发病过程。
整体实验设计：针对不同样本中SHOX基因启动子的4C-seq样本：HH28期鸡胚肢芽及人类U2OS细胞系。