Macroh2a Histone Variants Maintain Nuclear Organization And
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© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 1570-1582 doi:10.1242/jcs.199216 RESEARCH ARTICLE MacroH2A histone variants maintain nuclear organization and heterochromatin architecture Julien Douet1,2, David Corujo1,2,*, Roberto Malinverni1,2,*, Justine Renauld3, Viola Sansoni4, Melanija Posavec Marjanović1, Neus Cantariño1, Vanesa Valero1,2, Fabien Mongelard5, Philippe Bouvet5, Axel Imhof4, Marc Thiry3 and Marcus Buschbeck1,2,‡ ABSTRACT literature positions macroH2A proteins as bona fide tumor suppressors (reviewed in Cantariño et al., 2013). In melanoma, Genetic loss-of-function studies on development, cancer and somatic macroH2A expression is lost in advanced stages and re-expression cell reprogramming have suggested that the group of macroH2A of macroH2A proteins suppresses metastatic potential (Kapoor histone variants might function through stabilizing the differentiated et al., 2010). Suppression of macroH2A1 enhanced the cancer stem state by a yet unknown mechanism. Here, we present results cell-like properties in bladder cancer (Park et al., 2015) and led to a demonstrating that macroH2A variants have a major function in massive expansion of undifferentiated carcinoma-like tissue in maintaining nuclear organization and heterochromatin architecture. teratomas grown from xenografted mouse embryonic stem cells Specifically, we find that a substantial amount of macroH2A is (Creppe et al., 2012a). associated with heterochromatic repeat sequences. We further MacroH2A has an ambiguous and still unexplained role in identify macroH2A on sites of interstitial heterochromatin decorated transcriptional regulation. While the genomic distribution of by histone H3 trimethylated on K9 (H3K9me3). Loss of macroH2A macroH2A correlates with suppression of transcriptional activity leads to major defects in nuclear organization, including reduced (Changolkar and Pehrson, 2006; Changolkar et al., 2010; Gamble nuclear circularity, disruption of nucleoli and a global loss of dense et al., 2010), it is in some cases required for signal-induced gene heterochromatin. Domains formed by DNA repeat sequences are activation (discussed in Creppe et al., 2012b). The repressive disorganized, expanded and fragmented, and mildly re-expressed function of macroH2A proteins has been primarily associated with when depleted of macroH2A. At the molecular level, we find that cancer. In both melanoma and breast cancer, macroH2A was, for macroH2A is required for the interaction of repeat sequences with the instance, shown to exert its tumor suppressive function through nucleostructural protein lamin B1. Taken together, our results argue repression of the cell cycle-promoting gene CDK8 (Kapoor et al., that a major function of macroH2A histone variants is to link 2010; Xu et al., 2015). Lavigne and colleagues provided results nucleosome composition to higher-order chromatin architecture. suggesting that rather than being a transcriptional regulator KEY WORDS: Heterochromatin, Histone variant, Nuclear macroH2A proteins stabilize expression states, thus providing organization, DNA repeats robustness and reducing noise (Lavigne et al., 2015). Both macroH2A1 and macroH2A2 co-occupy promoters of INTRODUCTION developmental genes that are bound by polycomb repressive A large number of loss-of-function studies support a physiological complexes (Buschbeck et al., 2009), and large macroH2A-bound role for macroH2A histone variants in the establishment and regions overlap with the histone H3 trimethylated on K27 maintenance of differentiated epigenomes. MacroH2A proteins (H3K27me3) polycomb mark (Gamble et al., 2010). Polycomb- promote differentiation of embryonic and adult stem cells (Creppe bound and H3K27me3-marked regions are frequently referred to as et al., 2012a), are important for proper embryonic development of facultative heterochromatin, which has been defined as a transiently zebrafish (Buschbeck et al., 2009) and form an epigenetic barrier to condensed and transcriptionally silent type of chromatin permissive somatic cell reprograming (Barrero et al., 2013a; Gaspar-Maia et al., to transcriptional activation in response to environmental and 2013; Pasque et al., 2011, 2013). In the context of cancer, the bulk differentiation signals (Trojer and Reinberg, 2007). In contrast, constitutive heterochromatin is characterized by its stable repressive nature. The histone mark H3K9me3 plays an 1Josep Carreras Leukaemia Research Institute (IJC), Campus ICO – Germans Trias i Pujol, Campus Can Ruti, Badalona 08916, Spain. 2Program of Predictive and important role in its establishment and maintenance (Maison and Personalized Medicine of Cancer, Germans Trias and Pujol Research Institute Almouzni, 2004). H3K9me3 is enriched in gene-poor genomic 3 (PMPPC-IGTP), Campus Can Ruti, Badalona 08916, Spain. Cell and tissue biology regions containing repetitive elements and is believed to largely unit, GIGA-Neurosciences, University of Liege, C.H.U. Sart Tilman, Liege 4000, Belgium. 4BioMedical Center and Center for Integrated Protein Sciences Munich, encompass the same genomic locations in different cell types. As Ludwig-Maximilians-University of Munich, Großhaderner Straße 9, Planegg- such, constitutive heterochromatin is considered to be a more static Martinsried 82152, Germany. 5Universitéde Lyon, Ecole normale Supérieure de Lyon, Centre de Recherche en Cancérologie de Lyon, Cancer Cell Plasticity structure than polycomb-marked chromatin. The DNA sequences Department, UMR INSERM 1052 CNRS5286, Centre Léon Bérard, 69008 Lyon, contained in constitutive heterochromatin include telomeric and France. pericentromeric satellite repeats, transposable elements, such as long *These authors contributed equally to this work interspersed DNA elements (LINEs), and long terminal repeats ‡Author for correspondence ([email protected]) (Peters et al., 2003; Rice et al., 2003). An important function of constitutive heterochromatin is to prevent genomic instability and N.C., 0000-0002-9548-3927; A.I., 0000-0003-2993-8249; M.B., 0000-0002- 3218-4567 ensure stable replication of the genome by maintaining transposons and simple DNA repeats as being transcriptionally silent (Bouzinba- Received 2 November 2016; Accepted 7 March 2017 Segard et al., 2006; Peters et al., 2003; Taddei et al., 2001). Journal of Cell Science 1570 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1570-1582 doi:10.1242/jcs.199216 One of the most conserved characteristics of genome organization (Fig. 1A,B). As shown in Fig. 1C, depletion of macroH2A is the accumulation of heterochromatin close to the nuclear lamina, proteins had a major effect on the nuclear organization and shape. which comprises a meshwork of four lamin proteins and a number of Nuclei in control cells show well-shaped nucleoli and regions of transmembrane proteins in mammals (reviewed in Amendola and dense heterochromatin staining in the nucleoplasm and at the van Steensel, 2014; Gruenbaum and Medalia, 2015). Three genes nuclear and nucleolar periphery. Knockdown of macroH2A LMNA, LMNB1 and LMNB2 encode the four major mammalian proteins led to the global loss of dense heterochromatin. This loss lamins: the splice variants lamin A and C, lamin B1 and lamin B2. of dense heterochromatin was most pronounced at the classic sites Mapping techniques, such as DNA adenine methyltransferase of heterochromatin accumulation, the surface of the nucleolus and identification (DamID), have demonstrated that lamins are the nuclear periphery but could also be observed in the nucleoplasm enriched over domains ranging from 10 kb to 10 Mb in size that (Fig. 1C,D). This effect had a very high penetration in the cell are termed lamin-associated domains (LADs; Guelen et al., 2008). In population, and when analyzing more than 100 cells we could not particular those LADs that have high cell-to-cell consistency are identify a single knockdown cell with intact dense heterochromatin inversely linked with gene activity and associated with H3K9me3 (Fig. 1E). In knockdown cells, nucleoli were expanded and had a (Kind et al., 2015). Importantly, in both Caenorhabditis elegans and reticulated appearance with several small fibrillar centers (Fig. 1D). human cells H3K9-specific methylases, and the resulting H3K9me2 Such structural changes in nucleolar organization are indicative of and H3K9me3 marks, are required for the tethering of genomic loci increased transcriptional activity of ribosomal DNA (rDNA), which to the nuclear lamina (Bian et al., 2013; Kind et al., 2013; Towbin we previously reported in macroH2A-depleted cells (Cong et al., et al., 2012). It is still unclear to what extent other marks enriched in 2013). We confirmed the nucleolar expansion by performing LADs, such as H3K27me3, also contribute to tethering (Guelen immunofluorescence experiments with nucleophosmin as a marker et al., 2008; Harr et al., 2015). of nucleoli (Fig. 1F). We observed macroH2A1 staining in the entire While lamin B proteins are primarily localized at the periphery nucleus except in the nucleoli (Fig. 1B). Quantification by interacting with heterochromatin, a substantial fraction of lamin A/C automated area measurements of 2D projections revealed both the is also found in the nucleoplasm and interacts with euchromatic nucleus and nucleolus are increased in size in macroH2A-deficient regions (Gesson et al., 2016). In the nucleoplasm, lamin A/C further cells (Fig. 1G,H). The increase in nucleolar size remained interacts with the surface of polycomb-marked