The authors then showed that
The authors then showed that Spt6 interacts directly with the PRC2 subunit Suz12. This interaction occurs in solution but not on chromatin, suggesting that Spt6 may prevent PRC2 recruitment by binding to Suz12. They then mapped the domains in both Spt6 and Suz12 that are involved in this interaction and found that Suz12 interacts with Spt6 via a region previously shown to interact with Ezh2, the catalytic subunit of PRC2. These results suggest that Spt6 may prevent the assembly of PRC2 by interfering with the Suz12-Ezh2 interaction. This model was further supported by experiments showing that increasing amounts of Spt6 can compete with Ezh2 for binding to Suz12. These biochemical data are consistent with the authors’ model where Spt6 would impede PRC2 recruitment to enhancers and SEs (Figure 1). Unfortunately, the authors were not able to directly prove their model, as attempts to express siRNA-resistant Spt6 variants in Spt6-depleted Ginsenoside Rb1 were not conclusive. The authors therefore turned to an artificial system where Suz12 is tethered upstream of a promoter driving a luciferase reporter via a fusion with the Gal4 DNA-binding domain. This Gal4-Suz12 fusion recruits Ezh2, leading to H3K27 trimethylation and repression of luciferase activity in 293T cells. All these parameters were reduced upon transient transfection of Spt6, but not by transfection of a truncated version of Spt6 unable to interact with Suz12. These experiments show that, at least in this artificial setting, the Spt6-Suz12 interaction can counteract PRC2 function.
The work by Wang et al. makes a solid case for a role of Spt6 in ESC maintenance. The work also sheds light on a new aspect of Spt6 function at enhancers, connecting Spt6’s function at enhancers to PRC2 and suggesting an antagonistic role in PRC2 assembly. These findings raise some fundamental questions. For instance, what provides the specificity for Spt6 recruitment at OSN-bound SEs? In other words, what makes Spt6 bind OSN-bound SEs, but not OSN-bound repressed elements? Something beyond the presence of OSN has to provide the specificity. This may include the presence of additional TFs, some specific chromatin environment, or perhaps the presence of non-coding transcription at SEs. This last possibility is appealing since Spt6 is known to interact with elongating RNA polymerase II (Yoh et al., 2007).
Introduction Female mammalian embryogenesis and reproduction critically depend on a process called X chromosome inactivation (XCI), which silences one of the two sex chromosomes to achieve dosage compensation. XCI serves as a paradigm to study the epigenetic regulation, whereby gene expression states are maintained independent of DNA sequence. In mice, an imprinted form of XCI (iXCI) is initiated in embryos at the 4-cell stage, silencing exclusively the paternal X (Xp), and this XCI pattern is maintained in extraembryonic tissues. However, epiblast cells, which give rise to the embryo proper, experience a major epigenetic switch around implantation: these cells reactivate the Xp and undergo a random form of XCI (rXCI), in which the Xp or the maternal X (Xm) is inactivated in each cell with equal probability (Payer, 2016). Both forms of XCI require the long non-coding Xist RNA, which forms clouds on the inactive X chromosome (Xi) from which it is transcribed, leading to X silencing. The X-linked gene Rlim (also known as Rnf12) has emerged as a critical mediator of Xist activity. Rlim encodes a ubiquitin ligase (E3) (Ostendorff et al., 2002) that is involved in transcriptional regulation (Bach et al., 1999, Gontan et al., 2012, Güngör et al., 2007) and shuttles between the nucleus and the cytoplasm (Jiao et al., 2013). In mice, a maternally transmitted Rlim knockout (KO) allele (Δm) results in early lethality of female embryos in a sex-specific parent-of-origin effect due to a failure to maintain iXCI and Xist clouds (Shin et al., 2010, Wang et al., 2016). In contrast, loss of Rlim in female epiblast cells has minimal effect on the rXCI process. RLIM protein levels are downregulated specifically in epiblast cells of implanting embryos, consistent with the lack of rXCI phenotype in Rlim mutant females (Shin et al., 2014). These data identify Rlim-dependent and Rlim-independent mechanisms of XCI in vivo that separately act in pre-implantation embryos and epiblasts, respectively. However, Rlim is crucial for XCI in female embryonic stem cells (ESCs) differentiated in culture (Barakat et al., 2011, Barakat et al., 2014).