br Results br Discussion Investigating XCI

Results
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 (R)-baclofen 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.