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  • During mouse embryogenesis RLIM protein is detectable throug


    During mouse embryogenesis, RLIM protein is detectable throughout preimplantation development, consistent with its functions in iXCI maintenance. However, in contrast to differentiating ESCs in culture (Figure 2A), RLIM protein levels are downregulated in nuclei of epiblast cells of implanting embryos to levels that are undetectable by immunofluorescence (Shin et al., 2014). RLIM levels continue to remain low at early post-implantation stages through E7.5. At E8.5, RLIM protein levels are slowly upregulated in specific embryonic cell types, and by E11.5, RLIM protein is widely detectable in many tissues (Ostendorff et al., 2006) (data not shown). Thus, functions of Rlim in Xi maintenance at later embryonic stages and/or in mature tissue types are likely. The developmental expression pattern, combined with the finding of Rlim-dependent and Rlim-independent XCI pathways in ESCs in vitro, suggests a model for X dosage compensation in which Rlim occupies a major role to maintain Xist clouds and iXCI in cells of female embryos before X chromosome reactivation (XCR) (Figure 4F). Although this role continues in extraembryonic tissues, RLIM is specifically downregulated in the epiblast lineage shortly before implantation, thereby likely contributing to XCR, followed by induction of rXCI by an Rlim-independent pathway (Figure 4F). This scenario is consistent with findings that Rlim is essential for the maintenance of iXCI but dispensable for rXCI in epiblast cells. Moreover, it explains the precocious rXCI in epiblast cells of Δ/Δ Scrambled 10Panx outgrowths (Figure 1A), because with lack of iXCI, XCR is not required before induction of rXCI. Thus, iXCI in early female embryos and rXCI in epiblast cells are regulated by distinct pathways, and the existence of Rlim-dependent and Rlim-independent pathways for XCI in female ESCs is likely the consequence of persistent RLIM expression upon differentiation in vitro.
    Experimental Procedures
    Introduction Embryonic stem cells (ESCs) are capable of self-renewal and differentiation into all cell types of the body, which is conferred by the coordination of key factors, including transcription factors (TFs), polycomb complexes, microRNAs, and histone modifiers (Tee and Reinberg, 2014, Li and Belmonte, 2017). Such factors also include ATP-dependent chromatin remodeling complexes that hydrolyze ATP to change the conformation of chromatin, thereby modulating the access of TFs to chromosomal DNA (Kadoch and Crabtree, 2015). The mammalian switch/sucrose nonfermentable (SWI-SNF) complex, also called the BAF (Brg or Brahma-associated factors) complex, represents one subfamily of the ATP-dependent chromatin remodeling superfamily and forms polymorphic assemblies of up to 15 subunits with different functional specificity based on subunit composition (Kadoch and Crabtree, 2015). BAF complexes have been shown to be essential for mammalian pre- to post-implantation development (Ho and Crabtree, 2010, Panamarova et al., 2016),and play important roles in controlling the self-renewal and pluripotency of ESCs (Ho and Crabtree, 2010). However, the function of only a small number of BAF complex subunits has been studied in ESCs and in the early embryo, and how BAF complexes mechanistically control cell fate decisions is not well understood. The BAF45 subunit is encoded by a family of four genes (BAF45a, BAF45b, BAF45c, and BAF45d) that have different expression patterns (Kadoch and Crabtree, 2015). These proteins contain two plant homeodomain (PHD) fingers that may target the BAF complex to genomic loci bearing specific histone marks (Kadoch and Crabtree, 2015). In the mouse, BAF45a is essential for the maintenance of hematopoietic stem cells (Krasteva et al., 2017) and for the self-renewal of neural progenitors and is replaced by BAF45b/c as neural progenitors differentiate (Kadoch and Crabtree, 2015), whereas BAF45c is critical for heart and muscle development (Lange et al., 2008). BAF45d, also called Dpf2, is the only ubiquitously expressed BAF45 subunit (Mertsalov et al., 2000) and, so far, has been implicated in the programmed cell death response after deprivation of interleukin-3 from myeloid cells (Gabig et al., 1994). However, the biochemical interaction of DPF2 with pluripotency TFs in ESCs (Pardo et al., 2010, van den Berg et al., 2010) suggests a function of this BAF subunit in pluripotent cells, which has not been examined to date.