To further investigate mechanisms of rXCI
To further investigate mechanisms of rXCI, we generated female ESCs with a homozygous RlimKO. We found that these Waiter Rule undergo XCI in vivo but that XCI in vitro is strongly influenced by culture conditions, including both method of differentiation and O2 levels. Our results demonstrate Rlim-dependent and Rlim-independent pathways for XCI exist in ESCs and, together with published data, profoundly change current models of X dosage compensation.
Discussion Investigating XCI in female ESCs, we found that in an in vivo context, XCI in ESCs lacking Rlim occurs with efficiencies similar to those in WT ESCs (Figure 1), but in vitro Rlim-independent XCI is highly sensitive to the differentiation protocol, as well as cell culture conditions (Figures 2 and 4). In particular, XCI in RA-differentiated ESCs is strictly Rlim-dependent (Figure 2), indicating that this type of differentiation is not compatible with Rlim-independent XCI. The formation of EBs more closely mimics the situation in blastocysts, and the finding that RLIM levels slightly increase during EB differentiation (Figure 2A) (Marks et al., 2015) is reminiscent of the increase in Rlim mRNA levels observed in early blastocysts, when the ICM forms (Wang et al., 2016). Moreover, we found that culturing differentiating ESCs in 7.5% O2 levels had a general positive effect on Rlim-independent XCI efficiency (Figure 4). In utero, mammalian embryos are naturally exposed to low 2%–8% O2 levels (Fischer and Bavister, 1993), and atmospheric O2 levels negatively influence development, global gene expression, and XCI in cultured embryos or ESCs (Harvey et al., 2004, Lengner et al., 2010, Orsi and Leese, 2001). However, even in 7.5% O2, the kinetics of XCI in RlimKO cells is still slower than in WT ESCs (Figure S3A), suggesting that these remain suboptimal XCI conditions. An alternative possibility is that the Rlim-independent XCI occurs more slowly upon induction of ESC differentiation. In this scenario, the presence of RLIM facilitates the more rapid XCI kinetics in WT ESCs. Combined, our results indicate elevated XCI efficiencies by the Rlim-independent pathway under conditions that more closely parallel conditions found in vivo. Thus, it will be interesting to identify the factor or factors and conditions that orchestrate rXCI in epiblasts. In contrast to epiblast cells of embryos undergoing rXCI, RLIM expression is maintained in differentiating ESCs (Figure 2A). Thus, the Rlim-dependent pathway likely contributes to XCI to varying degrees in vitro, depending on differentiation, culture conditions, and likely the specific ESC model used. The findings that (1) REX1 levels are not significantly affected by the Rlim deletion in PGK12.1 ESCs (Figure 3A), (2) REX1 levels rapidly drop to undetectable levels within 24 hr of RA differentiation in ESCs (Gontan et al., 2012) (data not shown), and (3) the development of Xist clouds and H3K27me3 foci upon RA differentiation is strictly Rlim-dependent (Figures 1B and 1C) indicate that at least some functions of Rlim for XCI occur independent of REX1. However, the findings that REX1 levels in F121 ESCs are affected by the presence or absence of RLIM (Gontan et al., 2012) and that Rlim-independent XCI in these ESCs is generally less efficient when compared to PGK12.1 ESCs (Figures 4A–4D) suggests that the cellular repertoire of expressed competency factors (e.g., E3 ligases) in different female ESC models has an important impact on XCI in vitro. Rlim340f ESCs exhibit very low XCI activity, and our results suggest that expression of the truncated RLIM340 might be a contributing factor, because it traps REX1 in the nucleus (Figure 3), and overall REX1 levels are strongly affected by RLIM in F121 ESCs (Gontan et al., 2012), but not in PGK12.1 ESCs (Figure 3). Because RLIM regulates a variety of factors by both RING-finger-dependent and RING-finger-independent mechanisms (Her and Chung, 2009, Krämer et al., 2003, Ostendorff et al., 2002), it is likely that the activities of other nuclear proteins are altered in Rlim340f ESCs, with a potential effect on XCI. Moreover, the specific epigenetic background in individual ESC lines may contribute to XCI activity, because RlimKO ESC lines undergo XCI with variable efficiencies. Although it is clear that RLIM promotes XCI, our results indicate that in vivo, additional factors must be involved in proper counting of X chromosomes, as previously proposed (Barakat et al., 2011, Jonkers et al., 2009). However, secondary roles for Rlim in rXCI in counting X’s in mice with X chromosome abnormalities cannot be ruled out. In this context, it is important to point out the possibility that the gain-of-function activity of RLIM340 might contribute to the skewed inactivation of the X harboring the mutated Rlim allele in Rlim340f heterozygous ESCs (Jonkers et al., 2009).