bioRxiv preprint doi: https://doi.org/10.1101/437343; this version posted February 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Histone acetyltransferase inhibition rescues differentiation of emerin-deficient 2 myogenic progenitors 3 Katherine A. Bossone, B.S.1,2, Joseph A. Ellis, B.S.2 and James M. Holaska, Ph.D.1,2* 4 5 1Department of Biomedical Sciences, Cooper Medical School of Rowan University 6 2Department of Pharmaceutical Sciences, University of the Sciences 7 *Correspondence: [email protected]; Tel: 856-956-2746 8 9 Author contributions: Conceptualization, methodology, and validation: J.M.H.; Formal 10 analysis: J.M.H. and K.A.B.; Investigation: K.A.B. and J.A.E.; Resources: J.M.H.; Data 11 curation: J.M.H., K.A.B., and J.A.E.; Writing- original draft: J.M.H.; Writing- reviewing and 12 editing: J.M.H., K.A.B., and J.A.E.; Visualization: K.A.B. and J.M.H.; Supervision: J.M.H.; 13 Project administration: J.M.H., K.A.B., and J.A.E. Funding acquisition: J.M.H. 14 15 Acknowledgements: We thank the members of the Holaska laboratory for the many 16 helpful discussions regarding these studies and preparation of this manuscript. This 17 study was supported by the National Institute of Arthritis and Musculoskeletal and Skin 18 Diseases of the National Institutes of Health under Award Number R15AR069935 (to 19 J.M.H.). The content is solely the responsibility of the authors and does not necessarily 20 represent the official views of the National Institutes of Health. 21 Number of words in abstract: 147 22 Number of words in manuscript (excluding abstract, references, table titles and 23 figure legends): 3,073 24 Corresponding Author: James M. Holaska, Department of Biomedical Sciences, bioRxiv preprint doi: https://doi.org/10.1101/437343; this version posted February 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. H4K5ac dynamics in differentiation 2 25 Cooper Medical School of Rowan University, MEB 534, 401 South Broadway, Camden, 26 NJ 08103. Email: [email protected] 27 Running title: H4K5ac dynamics in differentiation 28 Ethical Publication Statement: We confirm that we have read the Journal’s position on 29 issues involved in ethical publication and affirm that this report is consistent with those 30 guidelines. 31 Conflicts of interests: The authors declare no competing or financial interests 32 bioRxiv preprint doi: https://doi.org/10.1101/437343; this version posted February 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. H4K5ac dynamics in differentiation 3 33 Histone acetyltransferase inhibition rescues differentiation of emerin-deficient 34 myogenic progenitors 35 36 ABSTRACT 37 Introduction: Emery-Dreifuss Muscular Dystrophy (EDMD) is a disease characterized 38 by skeletal muscle wasting, major tendon contractures, and cardiac conduction defects. 39 Mutations in the gene encoding emerin cause EDMD1. Our previous studies suggested 40 emerin activation of Histone Deacetylase 3 (HDAC3) to reduce Histone 4-Lysine 5 41 (H4K5) acetylation (ac) is important for myogenic differentiation. Methods: 42 Pharmacological inhibitors (Nu9056, L002) of histone acetyltransferases targeting 43 acetylated H4K5 were used to test if increased acetylated H4K5 was responsible for the 44 impaired differentiation seen in emerin deficient myogenic progenitors. Results: Nu9056 45 and L002 rescued impaired differentiation in emerin deficiency. SRT1720, which inhibits 46 the NAD+-dependent deacetylase Sirtuin 1 (SIRT1), failed to rescue myotube formation. 47 Discussion: We conclude emerin regulation of HDAC3 activity to affect H4K5 48 acetylation dynamics is important for myogenic differentiation. Targeting H4K5ac 49 dynamics represents a new strategy for ameliorating the skeletal muscle wasting seen in 50 EDMD1. 51 52 53 Keywords: Cell signaling, Emerin, Emery-Dreifuss Muscular Dystrophy, Myogenic 54 differentiation 55 bioRxiv preprint doi: https://doi.org/10.1101/437343; this version posted February 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. H4K5ac dynamics in differentiation 4 56 INTRODUCTION 57 The nuclear envelope is composed of two lipid bilayers, the outer nuclear membrane, 58 which is contiguous with the endoplasmic reticulum, and the inner nuclear membrane1. 59 Although the outer and inner nuclear membranes arise from a common membrane, they 60 are functionally distinct membranes. Underlying the inner nuclear membrane is a 61 network of Type V intermediate filament proteins named lamins that provide nuclear 62 rigidity and elasticity2. The inner nuclear membrane contains a large number of unique 63 integral inner nuclear membrane proteins3, many of which show cell-type-specific 64 expression4-11. Inner nuclear membrane proteins function in diverse roles, including 65 nuclear structure, genomic organization, chromatin architecture, gene expression, cell 66 cycle regulation, and cytoskeletal organization12,1. The nuclear lamins and its associated 67 inner nuclear membrane proteins define the nuclear lamina. 68 69 Emerin is a lamin-binding, integral inner nuclear membrane protein. Mutations in the 70 gene encoding emerin cause X-linked Emery-Dreifuss muscular dystrophy (EDMD1), an 71 inherited disorder causing progressive skeletal muscle wasting, irregular heart rhythms, 72 and contractures of major tendons13-16. Evidence suggests the skeletal muscle wasting 73 seen in EDMD is not caused by increased damage to the myofiber, but by impaired 74 differentiation of skeletal muscle stem cells. Supporting this hypothesis, skeletal muscle 75 necrosis and increased skeletal muscle fiber permeability are rarely seen in EDMD 76 patients17. Further, emerin knockout mice (also commonly referred to as emerin-null or 77 emerin-deficient mice) exhibit delayed skeletal muscle regeneration and repair, motor 78 coordination defects, and mild atrioventricular conduction defects18,19. Skeletal muscle 79 from EDMD1 and EDMD2 patients and emerin-deficient mice both showed altered 80 expression of muscle regeneration pathway components20,18. Emerin-deficient myogenic 81 progenitors and emerin-downregulated C2C12 myoblasts exhibit impaired differentiation bioRxiv preprint doi: https://doi.org/10.1101/437343; this version posted February 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. H4K5ac dynamics in differentiation 5 82 and myotube formation21-23 due to aberrant temporal activation of myogenic 83 differentiation genes24 and disruption of key signaling pathways25, suggesting defective 84 muscle regeneration contributes to the EDMD skeletal muscle phenotype22,21,18. 85 86 The coordinated temporal expression of MyoD, Myf5, Pax3 and Pax7, which are 87 important for proper differentiation, was disrupted in emerin-deficient myogenic 88 progenitors26 due to the inability of the genome to properly reorganize during 89 differentiation20,18,25. This supports the hypothesis that emerin-deficient myogenic 90 progenitors fail to undergo the transcriptional reprogramming required for myogenic 91 differentiation. Furthermore, emerin was shown to bind directly to Histone Deacetylase 3 92 (HDAC3) and activate its deacetylase activity27. HDAC3 activity is required for proper 93 dynamic reorganization of MyoD, Myf5, Pax3 and Pax726. Thus, regulation of HDAC3 94 activity by emerin is critical for transcriptional reprogramming during myogenic 95 differentiation. 96 97 We used histone acetyltransferase (HAT) inhibitors targeting HATs mediating H4K5 98 acetylation (e.g., Tip60/KAT5) to further test the hypothesis that acetylation dynamics on 99 lysine 5 of Histone 4 (H4K5) were important for in myogenic differentiation. Here we 100 show increased H4K5 acetylation (H4K5ac) contributes to the impaired differentiation of 101 emerin-deficient myogenic progenitors. Targeting H4K5ac dynamics represents a 102 potential new strategy for ameliorating the skeletal muscle wasting seen in EDMD1. 103 104 bioRxiv preprint doi: https://doi.org/10.1101/437343; this version posted February 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. H4K5ac dynamics in differentiation 6 105 METHODS 106 Pharmacological treatments 107 We previously showed emerin-deficient myogenic progenitors had impaired 108 differentiation and was rescued by activation of HDAC323. To independently test if 109 altered H4K5 acetylation dynamics was responsible for the impaired differentiation of 110 emerin-deficient