A large fraction of UCEs are transcribed T UCEs
A large fraction of UCEs are transcribed (T-UCEs) in a tissue specific manner, and are deregulated in several human cancers (Calin et al., 2007, Fabbri et al., 2008, Fassan et al., 2014, Olivieri et al., 2016). Indeed, it has been shown that T-UCEs may also act as long non-coding RNAs (lncRNAs) regulating other RNAs (Calin et al., 2007, Liz et al., 2014). The main molecular mechanism of T-UCEs activity described so far is the “decoy” function. Indeed, T-UCEs sequester microRNAs (miRNAs) from the cytoplasm and eventually regulate cancer cell proliferation (Calin et al., 2007, Galasso et al., 2014, Olivieri et al., 2016). All together these findings provided robust evidence supporting the functional role of T-UCEs in the human genome, and highlighted a link between these genomic elements and human disease. Nevertheless, currently very little is known on the physiological role of this specific class of lncRNAs, as for instance in stem cell biology (Dinger et al., 2008, Feng et al., 2006, Mattick and Makunin, 2005).
Of note, several lncRNAs, including Hotair (Rinn et al., 2007), LincRNA-RoR (Loewer et al., 2010), Dali, MALAT1, Evf-2, and Nkx2.2AS (Chalei et al., 2014, Dinger et al., 2008, Guan et al., 2013, Ng et al., 2012, Ng and Stanton, 2013), are implicated in stemness and cell fate determination, even though their functional characterization is still incomplete. In this scenario, there is a lack of studies that directly investigate the potential role of T-UCE family members in molecular mechanisms orchestrating the balance between proliferation and differentiation of mouse embryonic stem WIN 18446 (ESCs).
Discussion This work provides evidence of a functional role of T-UCEs in regulating the finely tuned balance between pluripotency and differentiation in mouse ESCs. So far, the T-UCEs have been mostly linked to cancer, whereas their physiological role is still poorly understood. Based on the hypothesis that the T-UCE::miR interaction described in cancer cells can similarly occur in ESCs, we focused on uc.170+::mir9 since (1) uc.170+ carries the seed sequence for miR-9, and (2) uc.170+ and mir-9 expression inversely correlate in ESC neural differentiation. Here, we demonstrate that T-UCstem1 and miR-9 functionally interact and show that T-UCstem1::miR-9 interplay regulates ESC proliferation. According to our findings, recent data showed that miR-9 inhibits neural precursor cell and ESC proliferation by targeting Tlx1 (Qu et al., 2010, Zhao et al., 2009) and Lin28b (Xu et al., 2009, Zhong et al., 2010), respectively. Our results that Lin28b overexpression rescues the proliferation defects of T-UCstem1 KD ESCs support the conclusion that a T-UCstem1/miR-9/Tlx1-Lin28b axis controls cell-cycle progression in ESCs. Besides its pro-proliferative activity, T-UCstem1 also acts as a brake for ESC differentiation. Indeed, genome-wide and targeted analysis indicate that, upon T-UCstem1 silencing, FBS/Lif/Feeders ESCs retain expression of key pluripotency factors but concomitantly induce the expression of a large set of developmental genes of the three germ layers (ectoderm, mesoderm, and endoderm). In line with this peculiar molecular signature, FBS/Lif/Feeders T-UCstem1 KD ESCs keep pluripotency features and are able to differentiate in vitro and contribute to chimeric embryos in vivo. On the other hand, in less-permissive culture conditions (low density without feeders) FBS/Lif T-UCstem1 KD ESCs rapidly exit pluripotency and undergo differentiation, pointing to a key role of T-UCstem1 in preserving ESC self-renewal rather than pluripotency. Of note, since T-UCstem1 expression is not fully abrogated in T-UCstem1 KD ESCs, we cannot rule out the possibility that a complete loss of T-UCstem1 expression could give a more dramatic effect. The observation that T-UCstem1 KD ESC self-renewal, but not the proliferation defects, were rescued in 2i culture conditions, suggests different mechanisms of action of T-UCstem1-dependent control of ESC proliferation and self-renewal. Indeed, a large number (∼50%) of all the developmental regulatory genes that are upregulated in T-UCstem1 KD ESCs are bivalent domains-associated genes, which are characterized by a distinctive histone modification signature that combines the activating H3K4me3 and the repressive H3K27me3 marks. These bivalent domains are considered to poise expression of developmental genes, allowing timely activation, while maintaining repression in the absence of differentiation signals (Voigt et al., 2013). Increasing evidence indicates that PRC2 plays a crucial role in maintaining the bivalent domains in ESCs (Aranda et al., 2015) by ensuring a proper and robust differentiation. Withdrawal of PRC2 activity from ESCs results in global gene derepression of bivalent-associated genes (Azuara et al., 2006, Boyer et al., 2006, Lee et al., 2006) and spontaneous differentiation (Boyer et al., 2006, Endoh et al., 2008). PRC2 interacts with many lncRNAs in ESCs (e.g., HOTAIR, Malat1, and Gtl2), and these facilitate its recruitment to chromatin (Zhao et al., 2010). Furthermore, recent findings indicate that non-coding RNAs recruit PRC2 complex to chromatin either in cis or in trans, thereby causing changes in chromatin composition (Holoch and Moazed, 2015). Our findings indicate that T-UCstem1 is a new player in this complex scenario and provide evidence of a direct involvement of T-UCstem1 in switching the balance of these histone modifications in ESCs. Indeed, we show that T-UCstem1 directly interacts both with PRC2 complex and the bivalent domain-associated genes Nestin, Gata6, and Foxa2, and that this interaction may stabilize/guide PRC2 activity in determining the typical histone modifications at these bivalent domains. Thus, we propose a model wherein PRC2 is displaced in the absence of T-UCstem1, and this results in increased H3K4me3/H3K27me3 ratio on bivalent promoters of differentiation genes, which eventually induces their expression. Notably, we demonstrate that, besides the regulation on bivalent genes localized on different chromosomes (i.e., Nestin, Gata6, and Foxa2), T-UCstem1 also controls the expression of the neighbor bivalent gene Nr2f1 (Laursen et al., 2013), thus suggesting that the T-UCstem1 tethers PRC2 both in cis and in trans. Of note, considering the low abundance of T-UCstem1 and the ∼400 bivalent genes that are deregulated in the T-UCstem1 KD ESCs, we speculate that they might represent both direct and indirect targets of T-UCstem1.