Abstract
The nucleolus is the organelle for ribosome biogenesis and sensing various types of stress. However, its role in regulating stem cell fate remains unclear. Here, we present evidence that nucleolar stress induced by interfering rRNA biogenesis can drive the 2-cell stage embryo-like (2C-like) program and induce an expanded 2C-like cell population in mouse embryonic stem (mES) cells. Mechanistically, nucleolar integrity maintains normal liquid-liquid phase separation (LLPS) of the nucleolus and the formation of peri-nucleolar heterochromatin (PNH). Upon defects in rRNA biogenesis, the natural state of nucleolus LLPS is disrupted, causing dissociation of the NCL/TRIM28 complex from PNH and changes in epigenetic state and reorganization of the 3D structure of PNH, which leads to release of Dux, a 2C program transcription factor, from PNH to activate a 2C-like program. Correspondingly, embryos with rRNA biogenesis defect are unable to develop from 2-cell (2C) to 4-cell embryos, with delayed repression of 2C/ERV genes and a transcriptome skewed toward earlier cleavage embryo signatures. Our results highlight that rRNA-mediated nucleolar integrity and 3D structure reshaping of the PNH compartment regulates the fate transition of mES cells to 2C-like cells, and that rRNA biogenesis is a critical regulator during the 2-cell to 4-cell transition of murine pre-implantation embryo development.
Highlights
Two-cell (2C) stage embryonic cells are totipotent cells in an earlier stage of embryo development and can generate all cell types of embryonic and extraembryonic tissues
We first explored whether nucleolar stress produced by inhibiting rRNA biogenesis could induce cell fate reprogramming to 2C stage embryo-like (2C-like) cells (2CLCs) by performing RNA-seq analysis of mouse embryonic stem (mES) cells treated by three inducers of cellular stress, including CX-5461, an RNA polymerase I (Pol I) inhibitor; rotenone, an electron transport chain complex 1 inhibitor, and rapamycin, a mTOR pathway inhibitor (CX-5461 treatment dosage: 2 μM, CX-5461 treatment time: 12 h; rotenone treatment dosage: 1 μM, rotenone treatment time: 12 h; rapamycin treatment dosage: 2 μM, rapamycin treatment time: 12 h)
Gene Expression Omnibus (GEO) accession code GSE26360 for P53 and GEO accession code GSE85632 for DUX. g qRT-PCR showing the expression of Dux or 2C-related genes in Dux silenced mES cells; GM12794: p = 1.21E−13(***), Zscan[4]: p = 3.00E−09(***), MERVL-pol: p = 4.55E−03(**), Dux: p = 9.60E−14(***), two-way ANOVA; N = 3 biologically independent ChIP-qPCR experiment; Data are presented as mean values +/− standard error of the mean (SEM)
Summary
Two-cell (2C) stage embryonic cells are totipotent cells in an earlier stage of embryo development and can generate all cell types of embryonic and extraembryonic tissues. The structure of chromatin is an important epigenetic factor and is closely related to the regulation of gene expression and cell fate transition. One of the mechanisms of Dux expression is dependent on a complex of nucleolin NCL and heterochromatin factor TRIM28 in the PeriNucleolar Heterochromatin (PNH) region[14,24,25]. We find that inhibition of nucleolar rRNA biogenesis triggered nucleolar stress, which activated 2C-like transcriptional program and induced an expanded 2C-like cell population in mES cells with a mechanism involving 3D structure reorganization of the PNH and the Dux expression. Our findings provide a mechanistic perspective of rRNA biogenesis in regulating the homeostasis between 2C-like and mES cells, and highlight that rRNA biogenesis in the nucleolus is a critical molecular switch from ZGA gene expressing 2-cell stage to nucleolus-matured blastocyst stage embryos
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