Nuclear Envelope Laminopathies: Evidence for Developmentally Inappropriate Nuclear Envelope-Chromatin Associations
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Nuclear Envelope Laminopathies: Evidence for Developmentally Inappropriate Nuclear Envelope-Chromatin Associations by Jelena Perovanovic M.S. in Molecular Biology and Physiology, September 2009, University of Belgrade M.Phil. in Molecular Medicine, August 2013, The George Washington University A Dissertation submitted to The Faculty of The Columbian College of Arts and Sciences of The George Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 31, 2015 Dissertation directed by Eric P. Hoffman Professor of Integrative Systems Biology The Columbian College of Arts and Sciences of The George Washington University certifies that Jelena Perovanovic has passed the Final Examination for the degree of Doctor of Philosophy as of May 5, 2015. This is the final and approved form of the dissertation. Nuclear Envelope Laminopathies: Evidence for Developmentally Inappropriate Nuclear Envelope-Chromatin Associations Jelena Perovanovic Dissertation Research Committee: Eric P. Hoffman, Professor of Integrative Systems Biology, Dissertation Director Anamaris Colberg-Poley, Professor of Integrative Systems Biology, Committee Member Robert J. Freishtat, Associate Professor of Pediatrics, Committee Member Vittorio Sartorelli, Senior Investigator, National Institutes of Health, Committee Member ii © Copyright 2015 by Jelena Perovanovic All rights reserved iii Acknowledgments I am deeply indebted to countless individuals for their support and encouragement during the past five years of graduate studies. First and foremost, I would like to express my gratitude to my mentor, Dr. Eric P. Hoffman, for his unwavering support and guidance, and keen attention to my professional development. This Dissertation would not have been possible without the critical input he provided and the engaging environment he created. I would also like to thank my Dissertation Committee of Dr. Anamaris Colberg-Poley, Dr. Robert J. Freishtat, Dr. Vittorio Sartorelli and Dr. Ashley Hill for challenging and guiding me throughout the development of this Dissertation. I am grateful to my Master thesis mentor, Dr. Nikola Tucic at University of Belgrade, Serbia, who taught me to observe biological phenomena through the lens of evolution and encouraged me to pursue my doctoral degree in the US. Furthermore, I would like to thank Hoffman lab members for their friendship, help and support throughout the years, Center for Genetic Medicine at Children’s National Medical Center and Institute for Biological Sciences at the George Washington University, especially Dr. Linda Werling, Dr. Anne Chiaramello and Marc Wittlif. I owe much gratitude to my parents, Milan and Sladjanka Perovanovic, and brother, Rade, for their continuous love and support throughout my life. Finally, I would like to thank my beloved Nikola for his unconditional love, patience and support. iv Abstract of Dissertation Nuclear Envelope Laminopathies: Evidence for Developmentally Inappropriate Nuclear Envelope-Chromatin Associations The nuclear envelope is important to myogenesis, where the transition of lamin B to lamin A/C during terminal myogenic differentiation is needed for correct muscle development. Gene mutations of inner nuclear envelope components (LMNA, EDM) cause muscular dystrophy phenotypes (Emery-Dreifuss muscular dystrophy [EDMD]; limb-girdle muscular dystrophy). There is considerable allelic and clinical heterogeneity of the laminopathies, where distinct LMNA mutations selectively affect striated muscle, adipose, peripheral nerve, or multiple systems. These nuclear envelope components provide structural support to eukaryotic nuclei, but emerging data suggest key roles in DNA replication, transcription and chromatin organization. Previous studies in our lab have shown that patients with EDMD show specific alterations of MyoD/Rb pathways compared to other dystrophies (Bakay et al., 2006), and further that emerin null mice show a specific block in myogenesis during terminal differentiation of myoblasts (Melcon et al., 2006). In this dissertation it is shown that mutations in LMNA and loss of emerin causing EDMD lead to disruption of developmentally appropriate chromatin-nuclear envelope associations during myogenesis. We scanned for genomic loci interacting with lamin A protein during myogenic differentiation using DamID-seq, and determined the effect of disease-associated amino acid changes on these protein/chromatin interactions (p.R453W Emery-Dreifuss Muscular Dystrophy; p.R482W Familial Partial Lipodystrophy). Lamin A missense mutations caused both promiscuous formation of lamin A associated heterochromatin domains (LAD), and decreased the length of WT v LADs. These epigenetic alterations corresponded with pervasive presence of lamin A p.R452W (EDMD) at myogenic loci that was not seen with WT and p.R482W (FPLD). This finding was validated by DNA methylation studies in EDMD patient myogenic cells (WT vs. p.H222P). Also, appropriate exit from the cell cycle was perturbed by both mutations, with CDK1 and RB1 loci showing failure to attain LAD-mediated heterochromatin formation by DamID-seq and Chromatin Immuprecipitation sequencing ChIP-seq (H3K9me3 heterochromatin mark) analyses. Epigenetic findings in EDMD-AD (lamin A missense) were shared with the clinical phenocopy EDMD-XR (lamin A- associated emerin loss of function), with ChIP-seq of emerin null myogenic cells showing loss of heterochromatin marks at Sox2 and pluripotency pathway members. The failure of epigenetic remodeling at Sox2 was validated by mRNA (in vitro, and in EDMD patient muscle biopsies), and by lamin A p.H222P ChIP-qPCR. The alterations of SOX2 epigenetics may be upstream of the observed myogenic and cell cycle changes, as over- expression of SOX2 inhibited myogenic differentiation in vitro. Our findings suggest that nuclear envelopathies are disorders of developmental epigenetic programming caused by altered chromatin tethering to the nuclear periphery. vi Table of Contents Acknowledgments ............................................................................................................ iv Abstract of Dissertation ..................................................................................................... v List of Figures ................................................................................................................... ix List of Tables ..................................................................................................................... x List of Abbreviations ......................................................................................................... xi 1 Chapter: Introduction ................................................................................................... 1 1.1 Overview ............................................................................................................... 1 1.2 Diseases linked to mutations in LMNA .................................................................. 6 1.3 Molecular role of LMNA in normal and pathogenic conditions ............................ 16 1.4 Current molecular laminopathy models ............................................................... 28 1.5 Significance ......................................................................................................... 29 2 Chapter: Material and Methods ................................................................................. 30 2.1 Cell culture .......................................................................................................... 30 2.2 qRT-PCR ............................................................................................................. 32 2.3 Chromatin immunoprecipitation assay and ChIP-seq ......................................... 34 2.4 ChIP-seq data analysis ....................................................................................... 35 2.5 DamID sequencing .............................................................................................. 35 2.6 Bioinformatics analysis ....................................................................................... 41 2.7 Methylation analysis ............................................................................................ 41 2.8 Expression data .................................................................................................. 42 2.9 Overexpression and myogenic differentiation experiments ................................ 42 3 Chapter: Results ........................................................................................................ 43 3.1 Summary ............................................................................................................. 43 vii 3.2 Introduction ......................................................................................................... 44 3.3 Results ................................................................................................................ 47 3.3.1 Lamin mutations alter genomic LADs of human muscle cells ...................... 47 3.3.2 EDMD mutations show allele-specific alteration of epigenomic programming of myogenic differentiation ................................................................. 56 3.3.3 Delayed E2F cell cycle suppression is predicted by lamin A mutation (EDMD-AD) ............................................................................................... 76 3.3.4 Genome-wide prioritization of ChIP-seq alterations identifies aberrant persistence of pluripotency