rRNA biogenesis regulates mouse 2C-like state by 3D structure reorganization of peri-nucleolar heterochromatin

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.

Nucleolar-based Dux repression is essential for embryonic two-cell stage exit

“…We found that iPol I ESCs or 2C-like cells do not display typical nucleolar stress markers, and iPol I ESCs still activate Dux in the absence of p53, albeit with slightly reduced levels. In contrast, p53 activation upon longer or more severe nucleolar stress in ESCs may also significantly contribute to Dux activation ( Sun et al 2021 ; Yu et al 2021 ). Instead, our data on acute, milder nucleolar disruption agree with previous nucleolar gene positioning studies, which demonstrated in yeast that an ectopic rDNA repeat can silence its chromosomal region ( Zuccotti et al 2005 ) and that 5S rDNA sequences are sufficient to induce nucleolar association and silencing of a reporter gene in ESCs ( Fedoriw et al 2012 ).…”

DUX: One Transcription Factor Controls 2-Cell-like Fate

“…During the mESC to 2CLC transition, a lower TAD strength and TAD insulation have been observed [39]. Consistent with this is the disruption of chromatin organization by the depletion of CCCTC-binding factor (CTCF)/Cohesin or by culturing with CX-5461 upregulate Dux expression and promote a 2-cell-like program [13,14]. It is worth noting that the attenuation of H3K9me3 and H3K27me3 in CX-5461-treated cells is associated with the reinforcement of a repressed chromatin state [65].…”

“…Thus, differences in ribosome biogenesis can cause specialized mRNA subsets to be translated, which indirectly determine cell identity. Recent studies indicate that the inhibition of rRNA biogenesis can promote the state transition from ESCs to 2CLCs [11,13]. CX-5461, an RNA Pol I inhibitor, can decrease rRNA biogenesis and disrupt the nucleolar integrity and formation of peri-nucleolar heterochromatin (PNH).…”

“…CX-5461, an RNA Pol I inhibitor, can decrease rRNA biogenesis and disrupt the nucleolar integrity and formation of peri-nucleolar heterochromatin (PNH). Upon the disruption of nucleolar integrity, the loss of NCL/TRIM28 complex from PNH causes changes in epigenetic modification, leading to the release of Dux (Figure 4) [13]. Transcription factor LIN28 has been reported to bind to RNAs in the nucleolus, and its loss impeded ribosome assembly and recapitulated CX-5461-induced 2CLCs molecular phenotypes [11].…”

“…A transcriptome analysis of mouse preimplantation embryos revealed that the activation of unique transcripts takes place at the 2-cell stage but is undetectable at any other stages. These transcripts include, but are not limited to, zinc finger and SCAN domain containing 4 (Zscan4) [9][10][11][12][13][14], zinc finger protein 352 (Zfp352) [9], 2-cell-stage, variable group, member 1/3 (Tcstv1/3) [9], predicted gene 4340 (Gm4340) [11], TD and POZ domain containing 1-5 (Tdpoz1-5) [14] and procollagen-proline, 2-oxoglutarate 4-dioxygenase, 2 of 13 alpha II polypeptide (P4ha2) [10], most of them able to generate chimaeric transcripts linked to murine endogenous retrovirus with leucine tRNA primer (MERVL) element. The MERVL-one class of endogenous retroviral elements (ERVs) and hundreds of genes driven by the 5 LTR of MERVL are upregulated specifically at the 2-cell stage.…”