Hypertranscription of rDNA Responsible for Nucleolar Remodelling is a Doorman for Acquiring Pluripotency

Ribosome biogenesis occurs within the nucleolus, with the initial step being the transcription of ribosomal DNA (rDNA). Although rDNA transcription is limited in somatic cells, it is more active in stem cells. Nevertheless, the mechanisms involved in somatic cell reprogramming remain elusive. Both somatic and stem cell nucleoli exhibit a reticular structure. However, under the electron microscope, we identified an intermediate nucleolar state during reprogramming. This state underwent changes characterised by rDNA hypertranscription, resulting in an enlarged nucleolus, enhanced activity of nucleolus organiser regions (NORs), and a transition from the reticular nucleolar type to an intermediate state of reprogramming, whose three liquid phase boundaries are blurred. Our research revealed that Oct4 was directly targeted to the rDNA enhancer region, promoting its hypertranscription and nucleolar enlargement during reprogramming. Using rDNA transcriptional inhibitors, we proved that nucleolar remodelling and subsequent reprogramming are halted by inhibiting rDNA transcription. But why could rDNA transcriptional activity influence reprogramming? Our findings elucidate that the active nucleoli have the capability to release perinucleolar heterochromatin. By joint analysis of Assay for Transposase-Accessible Chromatin with high throughput Sequencing (ATAC-seq) and RNA-seq, we have characterised the perinucleolar chromatin released by the nucleolus in a reprogramming intermediate state. The released chromatin mainly impacted mesenchymal-to-epithelial transition (MET)-related genes. MET is a stage of silencing of mesenchymal genes, accompanied by the activation of epithelial genes. Concurrently, the morphology of mouse embryonic fibroblast cells (MEFs) transitions from elongated spindle-shaped cells to short roundish forms, exhibiting a propensity to cluster together. MET was considered an early event in reprogramming; our findings suggested that nucleolar remodelling occurred before MET.

chromatin accessibility; iPSC reprogramming; nucleoli; rDNA.