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    • It remains unclear how LINE RNA Nucleolin

    2021-05-10

    It remains unclear how LINE1 RNA-Nucleolin-Kap1 are targeted to the Dux cluster. LINE1-Nucleolin-Kap1 may have other interacting partners with DNA binding specificity. For example, YY1 has been implicated in targeting a repressive complex containing Nucleolin to the human DUX4 cluster (Gabellini et al., 2002), although we did not find a role for YY1 in Dux repression in ESCs (Figure 5C). Alternatively, KRAB-ZFP transcription factors are known to recruit Kap1 to repress TEs (Lupo et al., 2013, Wolf et al., 2015), a function that might have been coopted for Dux silencing. Finally, our data leave open the possibility that transcribed LINE1 DNA loci contribute in cis to Dux repression or rRNA activation via higher-order chromosome interactions (Figure 7L). Furthermore, while LINE1 is specifically detected at Dux and rRNA, it is likely to have other genomic targets that remain to be discovered. Our data suggest that the LINE1-Nucleolin-Kap1 complex has both an activating function (at rDNA) and a repressive function (at Dux). While this might seem paradoxical, Nucleolin and Kap1 have been shown to have both activating and repressive effects. In addition to its role as activator of rDNA, Nucleolin has been reported to repress the CVT-313 australia of genes, such as cMyc (González et al., 2009), MMP13 (Samuel et al., 2008), or the D4Z4 repeat (Gabellini et al., 2002). Conversely, the co-repressor Kap1 also has activating functions at both the single gene level (Singh et al., 2015) and globally (Bunch et al., 2014). It is possible that the reduction in nascent rRNA synthesis in Kap1 mutant ESCs contributes their self-renewal defect, in addition to the previously reported de-repression of endogenous retroviruses (Rowe et al., 2010). Moreover, the localization of Dux loci to Nucleolin-positive peri-nucleolar regions in ESCs, but not 2C-like cells, provides a potential rationale at the level of nuclear 3D organization for the coordinate roles of LINE1/Nucleolin/Kap1 in activation of rDNA versus repression of Dux. LINE1 is expressed throughout pre-implantation development (Figure 1B) (Fadloun et al., 2013), which implies that its presence alone may not suffice to repress Dux and the 2C program. However, analysis of RNA-seq data from early embryos reveals that Nucleolin and Kap1 are sharply induced at the late 2-cell stage, correlating with Dux silencing (Figure S7K). Thus, it is possible that, by promoting ZGA at the 2-cell stage, Dux induces several of its own repressors, which can then bind LINE1 RNA and silence the Dux loci. Moreover, there are large-scale changes to the organization of the nucleolus and its associated heterochromatin during these stages (Borsos and Torres-Padilla, 2016) that may pertain to the silencing of the Dux/2C program. It has been proposed that the retrotransposition activity of LINE1 is essential for early mouse development, possibly by making cDNA copies of sperm-derived RNAs (Beraldi et al., 2006, Sciamanna et al., 2009). Using inhibitors that block LINE1 mobility (Figure S2G) (Jones et al., 2008), we found that retrotransposition is not involved in the regulation of 2C gene expression nor of ESC self-renewal (Figures S2H and S2I). Moreover, LINE1 ORF1 protein, which is required for retrotransposition, is predominantly cytoplasmic (Figure S1A), in contrast to the nuclear localization of LINE1 RNA. While we cannot at present exclude a non-canonical function for LINE1 ORF1 or ORF2 proteins in the developmental roles reported here, our data indicate that it acts as a chromatin-associated RNA that binds Nucleolin and Kap1 to regulate gene expression. The partnership between LINE1 and Nucleolin in the regulation of rRNA synthesis indicates that LINE1 contributes to ESC hypertranscription (Percharde et al., 2017a). This notion is supported by the fact that LINE1 KD leads to decrease in total RNA levels, nascent transcription, nascent translation and self-renewal. Although we do not detect LINE1 RNA binding at ribosomal protein genes, the synthesis of rRNA and ribosomal proteins genes is highly coordinated (Laferté et al., 2006). In addition, we have recently shown that reductions in translational output in ESCs rapidly induce a decrease in nascent transcription of highly expressed genes, including rRNA and ribosomal protein genes (Bulut-Karslioglu et al., 2018). Our data support a model whereby LINE1-mediated induction of rRNA synthesis leads to global increases in ribosomal biogenesis, enabling rapid growth of the early embryo. We speculate that the LINE1-Nucleolin partnership may play roles in other stem/progenitor cells, where it might not necessarily act to repress Dux but may still promote ribogenesis and proliferation.