Regulation and Roles of the Nucleolus in Embryonic Stem Cells: From Ribosome Biogenesis to Genome Organization

Abstract

The nucleolus is the largest compartment of the eukaryotic cell's nucleus. It acts as a ribosome factory, thereby sustaining the translation machinery. The nucleolus is also the subnuclear compartment with the highest transcriptional activity in the cell, where hundreds of ribosomal RNA (rRNA) genes transcribe the overwhelming majority of RNAs. The structure and composition of the nucleolus change according to the developmental state. For instance, in embryonic stem cells (ESCs), rRNA genes display a hyperactive transcriptional state and open chromatin structure compared with differentiated cells. Increasing evidence indicates that the role of the nucleolus and rRNA genes might go beyond the control of ribosome biogenesis. One such role is linked to the genome architecture, since repressive domains are often located close to the nucleolus. This review highlights recent findings describing how the nucleolus is regulated in ESCs and its role in regulating ribosome biogenesis and genome organization for the maintenance of stem cell identity.

Keywords: ESCs; NAD; genome organization; hypertranscription; nucleolus; rRNA genes.

Figure 1

Chromatin States of rRNA Genes in ESCs and Differentiated Cells The hyperactive state of the nucleolus in ESCs is characterized by the lack of silent rRNA genes and elevated ribosome biogenesis. In contrast, upon exit from pluripotency, a fraction of rRNA genes acquire epigenetic silencing, ribosome biogenesis is reduced, and clusters of heterochromatin blocks surround the nucleolus and the nuclear periphery. The red arrows indicate the link between the nucleolar chromatin state and the rest of the genome. Representative electron microscopy images show the distinct chromatin organization between an mESC and a neural progenitor cell (NPC) 8 days after differentiation. Generally, ESCs display a single, large nucleolus (Nu), whereas differentiated cells have more nucleoli of smaller size compared with ESCs. The contrast procedure reveals in dark large and condensed heterochromatic structures (Het) particularly evident close to the nucleolus of the NPC.

Figure 2

Mechanisms Contributing to the Establishment of the Hyperactive State of the Nucleolus in ESCs Hypertranscription of rRNA genes was shown to be favored by the binding of CHD1 (chromodomain helicase DNA-binding protein 1) (Guzman-Ayala et al., 2015) and LINE1-nucleolin RNA complex (Percharde et al., 2018) with rRNA genes. On the other hand, mechanisms for rRNA gene repression are impaired. The recruitment of TIP5 (TTF1-interacting protein 5) to rRNA genes is abrogated by the impairment of IGS-rRNA processing and consequent lack of formation of mature pRNA (promoter-associated RNA). The association of TIP5 with IGS-rRNA impairs TIP5 recruitment to rRNA genes and their epigenetic silencing (Leone et al., 2017; Savić et al., 2014). DDX18 (DEAD-box helicase 18) was shown to sequester PRC2 (polycomb repressive complex 2) in the outer layer of the nucleolus and impairs its formation (Zhang et al., 2020). This mechanism prevents the deposition of the repressive H3K27me3 mark onto rRNA genes. Hyperactivation of rRNA genes promotes ribosome biogenesis. However, ESCs require a low global protein synthesis rate. The enhanced ribosome biogenesis in ESCs might result in a surplus of free ribosomes, which can be used to allow rapid elevation of translation rate in response to differentiation signals (Golob et al., 2008; Sampath et al., 2008).

Figure 3

Types of Genomic Contacts with the Nucleolus Schema representing nucleolar-associated domains (NADs). Genomic contacts with the nucleolus have been identified by biochemical purification of the nucleoli and contacts with rRNA genes (HIC, 4C-seq) or rRNA transcripts (SPRITE). Some NADs were described to overlap with lamina-associated domains (LADs). The double arrow indicates the relocation of some NADs to the nuclear lamina after mitosis. Green lanes represent chromosomes bearing rRNA genes. Interchromosomal DNA contacts between chromosomes containing rRNA genes have been reported, mirroring the coalescence of rRNA genes from different chromosomes in the same nucleolus.