RESEARCH ARTICLE Role of SmcHD1 in establishment of epigenetic states required for the maintenance of the X-inactivated state in mice Yuki Sakakibara 1 , Koji Nagao 2, *, Marnie Blewitt 3 , Hiroyuki Sasaki 1 , Chikashi Obuse 2 and Takashi Sado 1,4, * ABSTRACT X inactivation in mammals is regulated by epigenetic modifications. Functional deficiency of SmcHD1 has been shown to cause de-repression of X-inactivated genes in post-implantation female mouse embryos, suggesting a role of SmcHD1 in the maintenance of X inactivation. Here, we show that de-repression of X-inactivated genes accompanied a local reduction in the enrichment of H3K27me3 in mouse embryonic fibroblasts deficient for SmcHD1. Furthermore, many of these genes overlapped with those having a significantly lower enrichment of H3K27me3 at the blastocyst stage in wild type. Intriguingly, however, depletion of SmcHD1 did not compromise the X-inactivated state in immortalized female mouse embryonic fibroblasts, in which X inactivation had been established and maintained. Taking all these findings together, we suggest that SmcHD1 facilitates the incorporation of H3K27me3 and perhaps other epigenetic modifications at gene loci that are silenced even with the lower enrichment of H3K27me3 at the early stage of X inactivation. The epigenetic state at these loci would, however, remain as it is at the blastocyst stage in the absence of SmcHD1 after implantation, which would eventually compromise the maintenance of the X-inactivated state at later stages. KEY WORDS: X inactivation, Chromatin, Epigenetic modifications, Mouse development INTRODUCTION Female mammals, which have twice as many X-linked genes as males do, have evolved a mechanism to compensate for this dosage difference by inactivating one of the two X chromosomes during early development (Lyon, 1961). In the mouse, X inactivation is imprinted in favor of the paternal X chromosome in the extra- embryonic tissues, which give rise to the future placenta and some extra-embryonic membranes (Takagi and Sasaki, 1975), whereas it takes place in a random fashion (with respect to the parental origin of the X chromosome) in the embryonic tissue, which differentiates into all tissues of the fetus. In both imprinted and random X inactivation, silencing of the X chromosome is mediated by X-linked noncoding Xist RNA, which is monoallelically upregulated and coats the X chromosome from which it originates in cis (Clemson et al., 1996; Marahrens et al., 1997; Panning and Jaenisch, 1996; Penny et al., 1996). Targeted disruption has clearly demonstrated that Xist is essential for X inactivation to occur in cis, and the X chromosome deficient for Xist never undergoes inactivation. It is well accepted that Xist RNA forms a platform or scaffold to recruit proteins involved in heterochromatinization or its maintenance on the X chromosome undergoing inactivation (Wutz, 2011). Once the inactivated state of the X chromosome is established, it is stably maintained throughout successive cell divisions in the somatic lineages. It is well known that epigenetic modifications such as DNA methylation and histone modifications play an important role in the stable maintenance of the inactivated state of the X chromosome (Wutz, 2011). The importance of DNA methylation in the maintenance of X inactivation has been known for nearly four decades (Holliday and Pugh, 1975). CpG islands, which are often found in gene regulatory regions such as promoters or enhancers, are heavily methylated on the inactive X but less methylated on the active X. It has been shown that demethylation of CpG islands often leads to sporadic re-activation of genes on the inactive X, suggesting a role of DNA methylation in long-term memory for the stable maintenance of the repressed state. Roles of histone modifications in X inactivation have also been well studied. One of the most prominent features of the inactive X is the enrichment of histone H3 trimethylated at lysine 27 (H3K27me3), which is involved in gene repression and is a mark of facultative heterochromatin (Plath et al., 2003; Silva et al., 2004). Other histone modifications enriched on the inactive X include H2AK119ub1, H3K9me2, H3K9me3 and H4K20me1 (Chadwick and Willard, 2004; de Napoles et al., 2004; Fang et al., 2004; Heard et al., 2001; Keniry et al., 2016; Kohlmaier et al., 2004; Nozawa et al., 2013). In contrast, acetylated histone H3 and H4, both of which are involved in gene activation, are essentially excluded from the inactive X (Jeppesen and Turner, 1993). A role of H3K27me3 in the stable maintenance of the inactive X has been suggested in the extra-embryonic tissues of the mouse embryo based on studies of a loss-of-function mutation of Eed, which is one of the components in polycomb repressive complex 2 (PRC2) responsible for producing H3K27me3 (Wang et al., 2001). Female embryos carrying a paternal X-linked GFP transgene on an Eed-null background exhibit re-activation of the GFP transgene, which was repressed by imprinted X inactivation at earlier stages, in the extra-embryonic tissues. SmcHD1 (structural maintenance of chromosomes containing hinge domain), a noncanonical member of the SMC family of proteins, was initially reported as a protein for which loss of function compromises X inactivation, resulting in female-specific lethality at the midgestation stage (Blewitt et al., 2008). The late timing of lethality suggested that SmcHD1 may be involved in the maintenance of X-linked gene silencing. In an SmcHD1-null mutant, Smchd1 MommeD1/MommeD1 (Smchd1 MD1/MD1 hereafter), re-activation of X-inactivated genes is often accompanied by Received 7 April 2018; Accepted 10 August 2018 1 Medical Institute of Bioregulation, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. 2 Department of Biological Science, Graduate School of Science, Osaka University, Toyonaka, Japan. 3 The Walter and Eliza Hall Institute of Medical Research, 1G Royal Pde, Parkville 3052 VIC, Australia; The Department of Medical Biology, University of Melbourne, Parkville 3052, VIC, Australia. 4 Department of Advanced Bioscience, Graduate School of Agriculture, Kindai University, 3327-204, Nakamachi, Nara, 630-8505, Japan. *Authors for correspondence (nagao@bio.sci.osaka-u.ac.jp; tsado@nara.kindai.ac.jp) K.N., 0000-0003-1418-6988; T.S., 0000-0002-1232-0250 1 © 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev166462. doi:10.1242/dev.166462 DEVELOPMENT