Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase

Abstract

X inactivation involves the stable silencing of one of the two X chromosomes in XX female mammals. Initiation of this process occurs during early development and involves Xist (X-inactivespecific transcript) RNA coating and the recruitment of Polycomb repressive complex (PRC) 2 and PRC1 proteins. This recruitment results in an inactive state that is initially labile but is further locked in by epigenetic marks such as DNA methylation, histone hypoacetylation, and MACROH2A deposition. Here, we report that the E3 ubiquitin ligase consisting of SPOP and CULLIN3 is able to ubiquitinate the Polycomb group protein BMI1 and the variant histone MACROH2A. We find that in addition to MACROH2A, PRC1 is recruited to the inactivated X chromosome in somatic cells in a highly dynamic, cell cycle-regulated manner. Importantly, RNAimediated knock-down of CULLIN3 or SPOP results in loss of MACROH2A1 from the inactivated X chromosome (Xi), leading to reactivation of the Xi in the presence of inhibitors of DNA methylation and histone deacetylation. Likewise, Xi reactivation is also seen on MacroH2A1 RNAi under these conditions. Hence, we propose that the PRC1 complex is involved in the maintenance of X chromosome inactivation in somatic cells. We further demonstrate that MACROH2A1 deposition is regulated by the CULLIN3͞ SPOP ligase complex and is actively involved in stable X inactivation, likely through the formation of an additional layer of epigenetic silencing.Bmi1 ͉ SPOP͞CULLIN3 E3 ligase ͉ X inactivation ͉ Polycomb silencing

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

“…Our results mirror reports that SPOP can localize to different types of nuclear bodies, specifically to polycomb bodies and DNA damage foci (Hernández‐Muñoz et al , 2005), and presumably to PML bodies (Kwon et al , 2006; Jung et al , 2007). Substrate may play a role in recruiting SPOP to these nuclear bodies, but in contrast, SPOP can also recruit substrate to a nuclear body (Kwon et al , 2006).…”

“…SPOP is a substrate adaptor of a cullin‐3‐RING ubiquitin ligase (CRL3) and serves to recruit substrates to the CRL3 (Zhuang et al , 2009) for subsequent ubiquitination and degradation (Hernández‐Muñoz et al , 2005; Kent et al , 2006; Kwon et al , 2006; Zhang et al , 2006; Li  et al , 2008). The Drosophila melanogaster homolog of SPOP, Roadkill/HIB, is essential for early development (Kent et al , 2006).…”

Heterochromatin and its Relationship to Cell Senescence and Cancer Therapy

“…MacroH2As contain an N-terminal histone H2A-like domain and a C-terminal "macro domain" of more than 200 residues that is unrelated to other histones. MacroH2A clearly contributes to gene silencing, since it is depleted from active genes, 39 inserted into the inactive X chromosome [39][40][41][42][43] and macroH2A-containing chromatin is resistant to ATP-dependent remodeling proteins and binding of transcription factors in vitro and in vivo. [44][45][46] Moreover, genetic inactivation of macroH2A1.1 and 1.2 in mice caused increased expression of some genes that are normally enriched in macroH2A1.1 and 1.2 and silenced.…”

Specific patterns of histone marks accompany X chromosome inactivation in a marsupial

“…First, histone modifications associated with transcriptional activity (H3K4me2, H3K9ac) are lost (Heard et al 2001;Chaumeil et al 2002;Okamoto et al 2004). Soon after, the inactive X gains specific repressive m a r k s , H 3 K 9 m e 2 / H 3 K 2 7 m e 3 / H 4 K 2 0 m e 1 / H2AK119ub (Heard et al 2001;Mermoud et al 2002;Plath et al 2003;Silva et al 2003;Rougeulle et al 2004;Kohlmaier et al 2004;de Napoles et al 2004;Fang et al 2004;Hernandez-Munoz et al 2005). Finally, it loses acetylation of histone H4 (H4Kac), which is associ-ated with active chromatin (Keohane et al 1996;Heard et al 2001;Chaumeil et al 2002;).…”