Whole Genome Bisulfite Sequencing in Pax7-reprogrammed corticotropes AtT-20 cells

Deployment of a cell-specifying enhancer repertoire by the pioneer factor Pax7 The establishment and maintenance of cell identity depends on implementation of stable cell-specific chromatin landscapes. Pioneer transcription factors establish new cell fate competences by triggering chromatin remodeling during development. Here, we used pituitary cell specification to define the salient features of pioneer action. Comparison of purified pituitary cells of different lineages showed that chromatin accessibility differs at enhancers rather than promoters. The pioneer factor Pax7 specifies one pituitary lineage identity by opening a specific repertoire of enhancers that are distinct from the myogenic targets of Pax7. Pax7 binds its pioneer targets rapidly and days before chromatin remodeling and gene activation. Finally, enhancers opened by Pax7-dependent chromatin remodeling exhibit loss of DNA methylation and they acquire long term epigenetic memory. The present work identifies enhancer pioneering as a critical feature for cell fate specification and maintenance. Overall design: Bisulfite conversion of genomic DNA followed by high throughput sequencing (WGBS)

先驱因子Pax7介导细胞特异性增强子组的功能部署。细胞身份的建立与维持，依赖于稳定细胞特异性染色质景观的构建。先驱转录因子（pioneer transcription factor）可在发育进程中通过触发染色质重塑，建立新的细胞命运潜能。本研究以垂体细胞特化为模型，解析先驱因子发挥作用的核心特征。对不同谱系纯化垂体细胞的比较分析显示，染色质可及性的差异主要存在于增强子（enhancer）区域，而非启动子（promoter）区域。先驱因子Pax7通过开放一套区别于其肌源性靶标的特异性增强子组，特化一种垂体细胞谱系身份。Pax7可快速结合其先驱靶标，且结合时间早于染色质重塑与基因激活数天。最终，由Pax7依赖性染色质重塑所开放的增强子区域会发生DNA甲基化丢失，并获得长期表观遗传记忆。本研究证实，增强子先驱调控是细胞命运特化与维持的关键特征。总体实验设计：对基因组DNA进行亚硫酸氢盐转化后，开展全基因组亚硫酸氢盐测序（Whole Genome Bisulfite Sequencing，WGBS）