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Accepted Manuscript Accepted Manuscript Fibrogenic Activity of MECP2 is Regulated by Phosphorylation in Hepatic Stellate Cells Eva Moran-Salvador, Marina Garcia-Macia, Ashwin Sivaharan, Laura Sabater, Marco Y.W. Zaki, Fiona Oakley, Amber Knox, Agata Page, Saimir Luli, Jelena Mann, Derek A. Mann PII: S0016-5085(19)41126-8 DOI: https://doi.org/10.1053/j.gastro.2019.07.029 Reference: YGAST 62784 To appear in: Gastroenterology Accepted Date: 17 July 2019 Please cite this article as: Moran-Salvador E, Garcia-Macia M, Sivaharan A, Sabater L, Zaki MYW, Oakley F, Knox A, Page A, Luli S, Mann J, Mann DA, Fibrogenic Activity of MECP2 is Regulated by Phosphorylation in Hepatic Stellate Cells, Gastroenterology (2019), doi: https://doi.org/10.1053/ j.gastro.2019.07.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Fibrogenic Activity of MECP2 is Regulated by Phosphorylation in Hepatic Stellate Cells ACCEPTED MANUSCRIPT Short title : Mecp2 controls myofibroblast transcriptome Eva Moran-Salvador ###, Marina Garcia-Macia ###, Ashwin Sivaharan ###, Laura Sabater, Marco Y.W. Zaki, Fiona Oakley, Amber Knox, Agata Page, Saimir Luli, Jelena Mann* and Derek A Mann*. Newcastle Fibrosis Research Group, Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK. # these authors contributed equally to the manuscript *joint senior authors Financial support: DM, FO and JM are funded by the UK Medical Research Council (Grants MR/K10019494/1, MK/K001949/1 and MR/R023026/1). National Institute on Alcohol Abuse and Alcoholism (NIAAA) (grant UO1AA018663). The work was also supported in part by GSK contribution of funding to the study to DAM and JM (CRAFT consortium). The research was further supported by the National Institute for Health Research Newcastle Biomedical Research Centre based at Newcastle Hospitals NHSMANUSCRIPT Foundation Trust and Newcastle University. Abbreviations : ACAN, aggrecan; ACTA2, smooth muscle aortic alpha-actin; aHSC, activated hepatic stellate cells; ALT, alanine aminotransferase; ASH1, histone-lysine n-methyltransferase; AST, aspartate aminotransferase; BrdU, bromodeoxyuridine; BRIP1, BRCA1-interacting protein 1; CNC, coding-non-coding gene coexpression; CCl 4, carbon tetrachloride; CCNA2, cyclin A2; Cdc7, cell division cycle 7-related protein kinase; Cdk15, cyclin dependent kinase 15; COL1A1, type IA1 Collagen; CREB, cAMP response element binding; CXCL2, chemokine ligand-2; Des, desmin; DNA2, DNA replication ATP-dependent helicase/nuclease; DUSP5, dual specificity phosphatase 5; ECM, extracellular matrix; Eme1, Essential Meiotic Structure-Specific Endonuclease 1; EZH2, enhancer of zeste homolog 2; PDGF-BB, Platelet-Derived Growth FactorACCEPTED BB; HA, hyaluronic acid; Has1, 2, 3, hyaluronan synthase 1, 2, 3; HDAC6, histone Deacetylase 6; HDAC/Sin3A, histone deacetylase/ paired amphipathic helix protein; HIPK2, homeodomain-interacting protein kinase 2; HSC, hepatic stellate cells; H&E, Haematoxylin and Eosin; IGV, Integrative Genomics Viewer; IL1, 6, Interleukin 1, 6; KEGG, Kyoto Encyclopedia of Genes and Genomes; KC, kupffer cells; linRNAs, long intergenic noncoding RNAs; lncRNA, long non-coding RNA; MCM2-6, maintenance protein complex 2-6; Mecp2, methyl-CpG binding protein 2; mHSC, mouse hepatic stellate cells; MMP2, 9, 13, matrix metalloproteinases 2, 9, 13; MTT, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Myl7, myosin light chain 7; ORC, origin recognition complex; 1 OSTN, osteocrin; PCNA, proliferating cell nuclear antigen; PTCH1, hedgehog receptor Patched 1; POLD1, ACCEPTED MANUSCRIPT polymerase delta 1; PPAR γ, peroxisome proliferator-activated receptor gamma; RASAL1, RAS GTPase activating-like protein 1; RPA2, replication protein A2; rHSC, rat hepatic stellate cells; RNAseq, RNA sequencing; Sepp1, Selenoprotein P; SFRP4, secreted frizzled-related protein 4; siRNA, small interfering RNA; αSMA, alpha smooth muscle actin; TGF-β1, transforming growth factor beta1; TIMP-1, tissue inhibitor of metalloproteinase-1; TNF-α, tumor necrosis factor alpha; TNXB, tenascin XB; TSC1/2, tuberous sclerosis proteins 1/2. Contact information: *Joint senior authors. Corresponding author: Jelena Mann, Institute of Cellular Medicine, Faculty of Medical Sciences, 4th Floor, William Leech Building, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. Tel +44 191 208 3851. E-mail [email protected] Disclosure: Authors report no conflict of interest Author Contributions . EMS, AP, FO, AK and MGM performed experiments, analyzed the data and performed statistical analysis; AS, LS and MZ performed bioinformatic analysis; SL performed histological measurements and digital quantification; JM and DAM designed the study design and contributed to data analysis, supervised the project and wrote the paper. Acknowledgments : We are very grateful to Professor Qian Chang, UW, Madison, USA, for the kind gift of Mecp2 S80A mice. We gratefully acknowledge Graham Smith of the BioinformaticsMANUSCRIPT Support Unit for assistance with bioinformatics analysis. ACCEPTED 2 Abstract ACCEPTED MANUSCRIPT Background & Aims: Methyl-CpG binding protein 2 (MECP2), which binds to methylated regions of DNA to regulate transcription, is expressed by hepatic stellate cells (HSCs) and is required for development of liver fibrosis in mice. We investigated the effects of MECP2 deletion from HSCs on their transcriptome and of phosphorylation of MECP2 on HSC phenotype and liver fibrosis. Methods: We isolated HSCs from Mecp2 -/y mice and wild-type (control) mice. HSCs were activated in culture and used in array analyses of mRNAs and long non-coding RNAs. KEGG pathway analyses identified pathways regulated by MECP2. We studied mice that expressed a mutated form of Mecp2 that encodes the S80A substitution (MECP2S80), causing loss of MECP2 phosphorylation at serine 80. Liver fibrosis was induced in these mice by administration of CCl 4, and liver tissues and HSCs were collected and analyzed. Results: MECP2 deletion altered expression of 284 mRNAs and 244 long non-coding RNAs, including those that regulate DNA replication, are members of the minichromosome maintenance protein complex family, or encode CDC7, HAS2, DNA2 (a DNA helicase), or RPA2 (a protein that binds single-strand DNA). We found MECP2 to regulate the DNA repair Fanconi anemia pathway in HSCs. Phosphorylation of MECP2S80 and its putative kinase, HAS2, were induced during transdifferentiation of HSCs. HSCs from MECP2S80MANUSCRIPT mice had reduced proliferation and livers from these mice had reduced fibrosis following CCl 4 administration. Conclusions: In studies of mice with disruption of Mecp2 or that expressed a form of MECP2 that is not phosphorylated at S80, we found phosphorylation of MECP2 to be required for HSC proliferation and induction of fibrosis. In HSCs, MECP2 regulates expression of genes required for DNA replication and repair. Strategies to inhibit MECP2 phosphorylation at S80 might be developed for treatment of liver fibrosis. Keywords: MCM, lncRNA, myofibroblast, epigenetic factor ACCEPTED 3 ACCEPTED MANUSCRIPT Introduction In the context of a self-limiting injury and acute inflammation, fibrogenesis is an important contributor to the wound repair and regeneration. The purpose of fibrogenesis is to form a temporary extracellular matrix (ECM)-rich barrier known as granulation tissue, which serves to maintain tissue integrity and prevent infection. Once inflammation subsides and effective regenerative processes are underway, fibrogenesis subsides and gives way to fibrolysis leading to the natural breakdown of temporary granulation tissue and its replacement with repaired epithelial and endothelial structures. Where an injury and/or inflammation persists or if regeneration is impaired, such as in the ageing organ, then fibrogenesis fails to subside and instead promotes then net deposition and maturation of fibril-forming collagen-rich ECM. In time, if unabated, non- resolving fibrogenesis leads to the formation of highly cross-linked mature scar tissue that distorts and perturbs normal organ architecture and function. Liver fibrosis is a common pathological process associated with the majority of chronic liver diseases and in the absence of an effective treatment for the underlying cause of liver damage willMANUSCRIPT often progress to end stage cirrhosis and/or hepatocellular carcinoma 1, 2. The relatively recent discovery that fibrogenesis is highly dynamic, with the potential to both regress as well as progress, was an important conceptual milestone, as was the clinical observation made across multiple types of liver disease that fibrosis can spontaneously regress upon effective therapeutic removal of the causative agent. These discoveries have stimulated new investigations into the molecular mechanisms of fibrogenesis and have encouraged the pharmaceutical industry that fibrosis is an attractive and tractable therapeutic target in chronic liver diseases. A further conceptual ACCEPTEDmilestone was the experimental demonstration that, irrespective of cause of liver injury, myofibroblasts are the central cellular drivers
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