The histone code reader Spin1 controls skeletal muscle development

OPEN Citation: Cell Death and Disease (2017) 8, e3173; doi:10.1038/cddis.2017.468 Official journal of the Cell Death Differentiation Association www.nature.com/cddis The histone code reader Spin1 controls skeletal muscle development Holger Greschik1, Delphine Duteil1, Nadia Messaddeq2, Dominica Willmann1, Laura Arrigoni3, Manuela Sum1, Manfred Jung4,5, Daniel Metzger2, Thomas Manke3, Thomas Günther1 and Roland Schüle*,1,5,6 While several studies correlated increased expression of the histone code reader Spin1 with tumor formation or growth, little is known about physiological functions of the protein. We generated Spin1M5 mice with ablation of Spin1 in myoblast precursors using the Myf5-Cre deleter strain. Most Spin1M5 mice die shortly after birth displaying severe sarcomere disorganization and necrosis. Surviving Spin1M5 mice are growth-retarded and exhibit the most prominent defects in soleus, tibialis anterior, and diaphragm muscle. Transcriptome analyses of limb muscle at embryonic day (E) 15.5, E16.5, and at three weeks of age provided evidence for aberrant fetal myogenesis and identified deregulated skeletal muscle (SkM) functional networks. Determination of genome-wide chromatin occupancy in primary myoblast revealed direct Spin1 target genes and suggested that deregulated basic helix-loop-helix transcription factor networks account for developmental defects in Spin1M5 fetuses. Furthermore, correlating histological and transcriptome analyses, we show that aberrant expression of titin-associated proteins, abnormal glycogen metabolism, and neuromuscular junction defects contribute to SkM pathology in Spin1M5 mice. Together, we describe the first example of a histone code reader controlling SkM development in mice, which hints at Spin1 as a potential player in human SkM disease. Cell Death and Disease (2017) 8, e3173; doi:10.1038/cddis.2017.468; published online 23 November 2017 Spindlin1 (Spin1) is a histone code reader binding histone H3 trimethylated at lysine 4 (H3K4me3) with high affinity.1–3 H3K4me3 association is enhanced by the presence of asymmetrically dimethylated arginine 8 of histone H34. Spin1 is highly expressed in several types of tumors5–7 and affects cell cycle, chromatin segregation, apoptosis, and transformation of cell lines, as well as tumor formation in nude mice.6,8–11 While these studies suggest important roles in cancer, physiological functions of Spin1 have only been subject to initial investigation. Mouse oocytes deficient for maternal Spin1 undergo normal folliculogenesis, but fail to resume meiosis.12 Furthermore, mice with ubiquitous Spin1 ablation die shortly after birth.12 However, tissue-restricted defects accounting for postnatal death have not been reported. Skeletal muscle (SkM) is the most abundant tissue in vertebrates mediating support and movement and contributing to overall metabolism. SkM development is orchestrated by key transcription factors including Pax3 and Pax7, which are also required for muscle stem cell specification,13,14 and the myogenic regulatory factors (MRFs) Myf5, MyoD (Myod1), Mrf4 (Myf6), and myogenin (Myog).13–15 MRFs are tissuespecific basic helix-loop-helix (bHLH) transcription factors acting as homodimers or as heterodimers with other bHLH transcription factors such as the ubiquitously expressed E-proteins E12/E47 (Tcf3), E2-2/ITF2 (Tcf4), and HEB/HTF4 (Tcf12).14 1 SkM fiber formation in mice comprises three successive phases, an embryonic wave from around embryonic day (E) 10.5 to E12.5, a fetal wave from around E14.5 to E17.5, and a postnatal period during which adult fibers are established.14,16–18 Adult myofibers exhibit distinct contractile properties (slow- or fast-twitch), patterns of innervation, and metabolic activities (oxidative or glycolytic), which correlate with the expression of specific myosin heavy chain (MHC) isoforms.19,20 Limb muscle of adult mice is composed of type I (slow, oxidative), type IIa (fast, oxidative), type IIx (fast, glycolytic), and type IIb (fast, glycolytic) fibers.19,20 SkM mass and functions become compromised in disease and numerous gene mutations causing myopathies or muscular dystrophies have been documented.21–28 Interestingly, selected fiber or muscle types preferentially degenerate in certain disease states.29,30 In this study, we crossed mice harboring conditional Spin1 alleles (Spin1p/p) with the Myf5-Cre deleter strain31 to ablate Spin1 in myogenic precursors. Most homozygous Spin1p/p Myf5-Cre (hereafter termed Spin1M5) mice die shortly after birth, while surviving mice display severe growth retardation. Histological, transcriptome, and cistrome analyses provide evidence for aberrant fetal myogenesis and deregulated basic bHLH transcription factor networks around the onset of SkM defects. Furthermore, our observations suggest that altered expression of titin-associated proteins, aberrant glycogen Urologische Klinik und Zentrale Klinische Forschung, Klinikum der Universität Freiburg, Breisacher Str. 66, Freiburg, Germany; 2IGBMC, Department of Functional Genomics and Cancer, Inserm U964, CNRS UMR7104, Université de Strasbourg, Illkirch, France; 3Max-Planck-Institute of Immunology and Epigenetics, Stübeweg 51, Freiburg, Germany; 4Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, Freiburg, Germany; 5Deutsches Konsortium für Translationale Krebsforschung (DKTK), Standort Freiburg, Freiburg, Germany and 6BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-Universität Freiburg, Schänzlestr. 18, Freiburg, Germany *Corresponding author: R Schüle, Urologische Klinik und Zentrale Klinische Forschung, Klinikum der Universität Freiburg, Breisacher Str. 66, Freiburg 79106, Germany. Tel: +49 761 27063100; Fax: +49 761 27063110; E-mail: Received 04.5.17; revised 12.7.17; accepted 28.7.17; Edited by M Agostini Spin1 controls skeletal muscle development H Greschik et al 2 Spin1 E18.5 Spin1 -Pax7 Pax7 -DAPI IF Ctrl (E15) Ctrl IF Ctrl (P0) Spin1R26 Spin1 25 µm Spin1 -MHC Spin1 -MHC -DAPI P0 IF Ctrl (E15) Ctrl Spin1M5 E16.5 IF Ctrl (P0) 25 µm Spin1 Spin1 -Pax7 Pax7 -DAPI Ctrl IF Spin1M5 (E15) 25 µm Spin1 Spin1 -MHC Spin1 -MHC -DAPI IF Spin1M5 (E15) 25 µm Cell Death and Disease Spin1M5 Spin1 controls skeletal muscle development H Greschik et al 3 Figure 1 Loss of Spin1 in SkM results in postnatal lethality. (a) Dropping forelimbs (arrow) indicating a neuromuscular defect observed in ubiquitous Spin1 knockout (Spin1R26) but not Spin1+/+ (Ctrl) fetuses. (b,c) Immunofluorescence (IF) staining of hind limb sections of control fetuses at E15 and of newborn mice (P0) with antibodies directed against Spin1 (green), Pax7 (red), or myosin heavy chain (MHC) (red). Arrowheads mark nuclei of myogenic precursors coexpressing Spin1 and Pax7 (b) or Spin1-positive nuclei in myofibers (c). Arrows depict nuclei devoid of Spin1 staining in myofibers at P0 (c). (d,e) IF staining of hind limb section (...truncated)