Genome-wide chromatin accessibility and transcriptome profiling shows that minimal epigenome changes lead to coordinated transcriptional dysregulation of hedgehog signaling in Danforth's short tail mice (RNA-seq)

Danforth's short tail (Sd) mice provide an excellent model for investigating the underlying etiology of human caudal birth defects, which affect 1 in 10,000 live births. Sd animals exhibit aberrant axial skeleton, urogenital, and gastrointestinal development similar to human caudal syndromes including urorectal septum malformation, caudal regression, VACTERL association, and persistent cloaca. Previous studies have shown that the Sd mutation results from an endogenous retroviral (ERV) insertion upstream of the Ptf1a gene causing its ectopic expression at E9. Though the genetic lesion has been determined, the resulting epigenomic and transcriptomic changes driving the phenotype have not been investigated. Here, we performed ATAC-seq experiments on isolated E9.5 tailbud tissue revealing localized changes in chromatin accessibility in Sd/Sd mutant embryos. Interestingly, chromatin changes were localized to a small genomic sequence overlapping a Ptf1a enhancer region, which is conserved in both mouse and humans. Furthermore, mRNA-Seq experiments revealed increased transcription of Ptf1a target genes and, importantly, the downregulation of the hedgehog pathway genes. Reduced Sonic hedgehog signaling (Shh) was confirmed by both in-situ hybridization and immunofluorescence experiments indicating that the Sd mutation may result, in part, from downregulated Shh signaling. Taken together these data indicate that human caudal dysgenesis disorders may result from dysregulation of hedgehog signaling pathways. Thus it will be important to investigate how epigenome and transcriptome alterations result in hedgehog pathway disruption. RNA-seq was performed on DNA isolated from the tailbuds of E9.5 WT and homozygous Danforth mutant mice (n = 3 per group). Tailbuds from 2 mice were pooled when necessary to ensure sufficient input material.丹福斯短尾（Danforth's short tail, Sd）小鼠是研究人类尾部出生缺陷潜在病因的优秀模型，该类缺陷在每10000例活产婴儿中出现1例。Sd模型小鼠表现出轴骨架、泌尿生殖系统及胃肠道发育异常，与人类尾部综合征表型相似，这类综合征包括尿直肠隔畸形、尾部退化综合征、VACTERL联合征以及泄殖腔残留。既往研究表明，Sd突变源于Ptf1a基因上游的内源性逆转录病毒（endogenous retroviral, ERV）插入，导致该基因在胚胎第9天（E9）出现异位表达。尽管已明确该遗传损伤位点，但驱动该表型的表观基因组与转录组变化尚未被研究。本研究对分离得到的胚胎第9.5天（E9.5）尾芽组织开展ATAC-seq实验，结果显示Sd/Sd突变胚胎中存在局部染色质可及性改变。值得注意的是，染色质改变仅局限于一段小型基因组序列，该序列与Ptf1a增强子区域重叠，且该区域在小鼠与人类中均保守。此外，mRNA-Seq实验结果显示，Ptf1a靶基因的转录水平上调，且尤为重要的是，刺猬信号通路相关基因出现表达下调。通过原位杂交（in-situ hybridization）与免疫荧光（immunofluorescence）实验，均证实声波刺猬信号（Sonic hedgehog, Shh）通路活性降低，这表明Sd突变的部分成因可能为Shh信号通路下调。综合上述数据可知，人类尾部发育不全综合征可能由刺猬信号通路失调所导致。因此，探究表观基因组与转录组改变如何引发刺猬信号通路紊乱具有重要意义。本研究对分离自E9.5野生型（wild type, WT）与纯合丹福斯突变模型小鼠的尾芽组织开展RNA-seq实验（每组n=3）。若所需起始材料不足，则将2只小鼠的尾芽组织混合后进行实验。