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Canonical Wnt Signalling Regulates Nuclear Export of Setdb1 During Skeletal Muscle Terminal Differentiation

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Canonical Wnt Signalling Regulates Nuclear Export of Setdb1 During Skeletal Muscle Terminal Differentiation OPEN Citation: Cell Discovery (2016) 2, 16037; doi:10.1038/celldisc.2016.37 ARTICLE www.nature.com/celldisc Canonical Wnt signalling regulates nuclear export of Setdb1 during skeletal muscle terminal differentiation Sophie Beyer1, Julien Pontis1, Elija Schirwis2,3, Valentine Battisti1, Anja Rudolf2,3, Fabien Le Grand2,3, Slimane Ait-Si-Ali1 1Centre National de la Recherche Scientifique CNRS-Université Paris Diderot, Sorbonne Paris Cité, Epigenetics and Cell Fate UMR7216, Paris, France; 2Institut Cochin, Université Paris-Descartes, Centre National de la Recherche Scientifique (CNRS) UMR8104, Paris, France; 3Institut National de la Santé et de la Recherche Médicale (INSERM) U1016, Paris, France The histone 3 lysine 9 methyltransferase Setdb1 is essential for both stem cell pluripotency and terminal differentiation of different cell types. To shed light on the roles of Setdb1 in these mutually exclusive processes, we used mouse skeletal myoblasts as a model of terminal differentiation. Ex vivo studies on isolated single myofibres showed that Setdb1 is required for adult muscle stem cells expansion following activation. In vitro studies in skeletal myoblasts confirmed that Setdb1 suppresses terminal differentiation. Genomic binding analyses showed a release of Setdb1 from selected target genes upon myoblast terminal differentiation, concomitant to a nuclear export of Setdb1 to the cytoplasm. Both genomic release and cytoplasmic Setdb1 relocalisation during differentiation were dependent on canonical Wnt signalling. Transcriptomic assays in myoblasts unravelled a significant overlap between Setdb1 and Wnt3a regulated genetic programmes. Together, our findings revealed Wnt-dependent subcellular relocalisation of Setdb1 as a novel mechanism regulating Setdb1 functions and myogenesis. Keywords: differentiation; lysine; muscle; methyltransferase; SETDB1/ESET/KMT1E; Wnt signalling Cell Discovery (2016) 2, 16037; doi:10.1038/celldisc.2016.37; published online 18 October 2016 Introduction subpopulation will re-populate the MuSCs niche [1,2]. Terminal differentiation of skeletal muscle is mainly Tissue regeneration requires precise spatio-temporal controlled by a family of specific basic helix–loop–helix regulation of gene expression, which is generally transcription factors (including MyoD and Myogenin) induced by signal transduction pathways. Recon- that cooperate with members of the MEF2 protein struction of the adult skeletal muscle tissue relies on a family in the activation of muscle genes [3]. This pool of resident committed muscle stem cells located myogenic transcriptional network acts together with around the myofibres, called the muscle satellite chromatin-modifying regulators, such as histone lysine cells (MuSCs). Upon damage to the myofibres, methyltransferases (KMTs), histone acetyltransferases quiescent MuSCs are activated and give rise to a and histone deacetylases [4]. These enzymes post- population of transient amplifying myoblasts. Most of translationally modify the amino-terminal tails of the myoblasts then permanently exit the cell cycle, histones, which are emerging from the nucleosomes. In enter terminal differentiation and fuse to form new particular, histone lysine residues can be tri-, di- or myofibres to regenerate the tissue, while a mono-methylated. Depending on the specific histone residue, the degree of methylation and the accessibility fi Correspondence: Fabien Le Grand mediated by other histone modi cations can either Tel: +33 6 67 53 70 91; Fax: +33 1 42 16 57 00; positively or negatively regulate gene expression [5]. E-mail: [email protected] The KMTs play key roles in transcriptional regulation or Slimane Ait-Si-Ali during development and are also emerging as crucial Tel: +33 1 57 27 89 19; Fax: +33 1 57 27 89 11; E-mail: [email protected] players in the control of cellular differentiation, Received 7 September 2016; accepted 19 September 2016 including myogenesis [3, 6]. In particular, histone 3 Wnt3a-mediated nuclear export of Setdb1 2 lysine 9 (H3K9) methylation, which is mainly involved myofibres isolated from the extensor digitorum longus in gene repression, is established in euchromatin by muscles. We used Pax7 (paired box 7 protein) expres- G9a/GLP [7] and Setdb1 [8]. Whereas Suv39h1/2 [9] sion to specifically identify MuSCs (Figure 1a–f), as and Prmd3/16 KMTs [10] are essential for the estab- previously described [29]. We detected Setdb1 protein lishment of H3K9 methylation in heterochromatin. at low levels in quiescent MuSCs in their niche on H3K9 KMTs are involved in the control of gene single fibres immediately after isolation (Figure 1a, top expression to regulate cell fate changes [11–13]. panels). After 24 h in 'floating' culture, Setdb1 was Setdb1 (also called Eset and KMT1E) is amplified in highly expressed in activated dividing MuSCs, mainly melanoma [14] and overexpressed in lung cancer [15]. in the nucleus but also in the cytoplasm (Figure 1a, Setdb1 shows distinct subcellular and subnuclear dis- bottom panels). Next, we performed Setdb1 loss-of- tributions [16–20] but the meaning of the different function assays in MuSCs on isolated myofibres and Setdb1 localisations remains elusive. Setdb1 is essential assayed MuSCs regarding stemness (Pax+/MyoD−), in mouse embryonic stem cell (mESC) pluripotency proliferation (Pax+/MyoD+) and terminal differentia- and self-renewal [18, 21, 22] and its knockout is lethal tion (Pax−/MyoD+ or Pax−/Myogenin+). Robust and at the peri-implantation stage [23]. Setdb1 is also acute Setdb1 knockdown (Figure 1b) reduced MuSCs essential for pluripotency and terminal differentiation amplification, as demonstrated by the diminished of many progenitor cell types. Setdb1 is required for the number of cells per fibre 72 h post-transfection survival of spermatogonial progenitor cells in mice [24] (threefold reduction) (Figure 1c). Extended Setdb1 and a mesenchyme-specific Setdb1 knockout resulted knockdown led to a higher proportion of cells in an increase of articular chondrocytes terminal committing to terminal differentiation (Pax7−/MyoD+) differentiation [25]. Furthermore, Setdb1 is crucial for (Figure 1d, red), and confirmed the reduction in the early neurogenesis in mice by promoting proliferation population that undergoes self-renewal (Pax7+/ and cell survival [26]. In contrast, Setdb1 regulates MyoD−) (Figure 1d, green). Among the remaining osteoblast differentiation during bone development MuSCs we observed a significant increase in [27] and is involved in the terminal differentiation of Myogenin-expressing cells after Setdb1 knockdown growth plate chondrocytes [28]. However, it is unclear (Figure 1e, f and Supplementary Figure S1A). how Setdb1 controls these mutually exclusive processes. Together, these results suggested that Setdb1 is To get insights on the apparent opposite functions of positively regulating amplification and negatively Setdb1 in pluripotency versus terminal differentiation, affecting terminal differentiation of MuSCs. we used skeletal muscle cells as a well-established We next conducted a series of in vitro assays using differentiation model for in vitro and ex vivo analyses. the C2C12 mouse skeletal myoblast model. We first Our data show that Setdb1 is required for MuSCs measured total Setdb1 protein levels in proliferating or expansion following activation and suppresses terminal differentiating C2C12 myoblasts (Figure 1g). Setdb1 myoblast differentiation. Furthermore, we demonstrate protein was moderately expressed in proliferating a nuclear export of Setdb1 during terminal differentia- myoblasts, peaked early in differentiating myoblasts tion of myoblasts. Genome-wide studies unravelled that (24 h of differentiation), decreased again at 48 h and Setdb1 relocalisation is dependent on the canonical dropped significantly after 96 h of differentiation Wnt signalling and results in a global release of Setdb1 (Figure 1g and Supplementary Figure S1B). Monitor- from its genomic targets and in the de-repression of a ing the muscle differentiation markers Myogenin, subset of Setdb1 target genes. Transcriptomic studies in Creatine Kinase Muscle (Ckm) and Myosin Heavy myoblasts further showed a significant overlap between Chain (MyHC) ensured proper cell differentiation Setdb1 and Wnt3a regulated genetic programmes. Our (Figure 1g). Besides a significant decrease in Setdb1 results suggest a new regulatory mechanism of Setdb1 total protein after 96 h differentiation, we additionally by the canonical Wnt signalling pathway to control observed an upward shift in the Setdb1 signal at gene expression in muscle cells. this time point (Figure 1g, asterisks). This suggests post-translational modifications of Setdb1 at late Results differentiation. It is possible that cytoplasmic Setdb1 is ubiquitinated and subsequently degraded by the Setdb1 is required for adult skeletal muscle stem cell proteasome in late-differentiated myotubes. amplification To investigate the role of Setdb1 during skeletal We first investigated Setdb1 expression in adult muscle terminal differentiation in vitro,weacutely mouse skeletal muscle satellite cells (MuSCs) on single downregulated Setdb1 using siRNAs at the onset of Cell Discovery | www.nature.com/celldisc Sophie Beyer et al. 3 Cell Discovery | www.nature.com/celldisc Wnt3a-mediated nuclear export of Setdb1 4 differentiation in C2C12 myoblasts. We observed terminal differentiation of myoblasts. To this end, we increased expression of the late muscle differentiation have established a polyclonal cell population over-
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