The Nucleolar Remodeling is Required for the of Mouse Fibroblasts Reprogramming to iPSC [RNA-Seq]

The nucleolus is a membraneless organelle responsible for ribosome biogenesis, perinuclear heterochromatin formation, and genome stability regulation. However, how cell fate decision occurs, including early embryonic development, ESC differentiation, and tumorigenesis, remains poorly understood at the nucleolar level. It has been observed that large nucleoli and rDNA hyperactivity are common in pluripotent stem cells and tumor cells, while the nucleolus shrinks and rDNA transcriptional activity decrease during lineage commitment. iPSCs nucleolar size and rDNA transcriptional activity are greater than that before reprogramming. It remains unclear how and when the differentiated cell nucleoli convert to the stem cell nucleoli during iPSC reprogramming.In this study, we found that nucleolar remodeling, manifested as enlarged nucleolus, activation of rDNA transcription, enhanced activity of nucleolar organizing regions (NORs), and conversion of reticular nucleolar ultrastructure into low-granular, is an early and stage-specific event that occurs during iPSCs reprogramming. Our study highlights the importance of rDNA transcriptional activity in the early stages of iPSC reprogramming, which is crucial for nucleolar remodeling and regaining stemness. Interfering rDNA transcription hinders nucleolar remodeling, which has disastrous consequences for chromatin remodeling in early stage of iPSC reprogramming and iPSCs establishment. Moreover, our results revealed a nucleolar regulation on chromatin accessibility during iPSC reprogramming and identified some candidate genes (Mybl2, Bard1 and other chromosome related genes) that might be associated with iPSC reprogramming, which may apply to nucleolar remodeling in other cell fated decision. Overall design: Comparative gene expression profiling analysis of RNA-seq data for 4F2A cells(+DOX,48h) that treated by DMSO or CX-5461 for 1 hour at reprogramming begainning.

核仁（nucleolus）是一种无膜细胞器，负责核糖体生物发生、核周异染色质形成以及基因组稳定性调控。然而，包括早期胚胎发育、胚胎干细胞（ESC）分化与肿瘤发生在内的细胞命运决定调控机制，在核仁层面仍尚未被充分阐明。已有研究观察到，多能干细胞与肿瘤细胞中普遍存在大核仁及核糖体DNA（rDNA）过度激活的现象；而在谱系定型过程中，核仁会发生收缩，且rDNA转录活性随之降低。诱导多能干细胞（iPSCs）的核仁尺寸与rDNA转录活性均高于重编程前的细胞。目前仍不清楚在诱导多能干细胞重编程过程中，分化细胞的核仁是如何、何时转变为干细胞型核仁的。本研究发现，核仁重塑——表现为核仁增大、rDNA转录激活、核仁组织区（nucleolar organizing regions, NORs）活性增强，以及网状核仁超微结构向低颗粒化转变——是诱导多能干细胞重编程过程中出现的早期、阶段特异性事件。本研究强调了rDNA转录活性在诱导多能干细胞重编程早期阶段的重要性，其对于核仁重塑以及重获干细胞干性至关重要。干扰rDNA转录会阻碍核仁重塑，这对诱导多能干细胞重编程早期的染色质重塑以及诱导多能干细胞的建立会产生灾难性影响。此外，本研究揭示了诱导多能干细胞重编程过程中核仁对染色质可及性的调控作用，并鉴定出若干可能与诱导多能干细胞重编程相关的候选基因（Mybl2、Bard1及其他染色体相关基因），该发现或可推广至其他细胞命运决定过程中的核仁重塑。整体实验设计：对重编程起始阶段经二甲基亚砜（DMSO）或CX-5461处理1小时的4F2A细胞（+DOX，48小时）的RNA测序数据进行比较基因表达谱分析。