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The Role of Nuclear Envelope Proteins in Chromatin Organization, Differentiation and Disease Distinct Degenerative Disorders, Referred to As Laminopathies

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The Role of Nuclear Envelope Proteins in Chromatin Organization, Differentiation and Disease Distinct Degenerative Disorders, Referred to As Laminopathies Cecilia Bergqvist The genetic material is highly structured within the nucleus, with transcriptionally inactive heterochromatin enriched at the nuclear The role of nuclear envelope periphery and active euchromatin in the nuclear interior. The nuclear lamina together with several hundred nuclear envelope transmembrane proteins in chromatin proteins (NETs) connect chromatin to the nuclear periphery. Most NETs are tissue-specific and uncharacterized, with mutations linked to organization, differentiation and The role of nuclear envelope proteins in chromatin organization, differentiation and disease differentiation organization, in chromatin proteins of nuclear envelope role The distinct degenerative disorders, referred to as laminopathies. The NET disease primarily studied in this thesis is called Spindle-Associated Membrane Protein 1 (Samp1). We showed that overexpression of Samp1 induced a fast differentiation of human induced pluripotent stem cells and that the binding between two NETs, Samp1 and Emerin, is regulated by Cecilia Bergqvist RanGTP. Another focus of this thesis was the development of a novel method, Fluorescent Ratiometric Imaging of Chromatin (FRIC). FRIC quantitatively monitors the epigenetic state of chromatin in live cells. Using FRIC, we were able to show that Samp1 promotes peripheral heterochromatin organization. FRIC also detected an increased distribution of heterochromatin at the nuclear periphery during neuronal differentiation. In conclusion, FRIC is a useful tool that could serve medical research in elucidating the effects of different chemical agents and NE proteins in chromatin organization. Cecilia Bergqvist ISBN 978-91-7911-230-1 Department of Biochemistry and Biophysics Doctoral Thesis in Neurochemistry with Molecular Neurobiology at Stockholm University, Sweden 2020 The role of nuclear envelope proteins in chromatin organization, differentiation and disease Cecilia Bergqvist Academic dissertation for the Degree of Doctor of Philosophy in Neurochemistry with Molecular Neurobiology at Stockholm University to be publicly defended on Friday 2 October 2020 at 10.00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B. Abstract In eukaryotes the genetic material is separated from the cytoplasm by the nuclear envelope (NE), consisting of the outer and inner nuclear membrane, the nuclear lamina and the nuclear pores. The genetic material is highly structured with transcriptionally inactive heterochromatin enriched at the nuclear periphery and transcriptionally active euchromatin in the nuclear interior. Underlying the inner nuclear membrane is the nuclear lamina (nucleoskeleton) that together with several hundred nuclear envelope transmembrane proteins (NETs) connect chromatin to the nuclear periphery. Most NETs are uncharacterized and expressed in a tissue-specific manner. Mutations in NE proteins are linked to distinct degenerative disorders, referred to as envelopathies or laminopathies. The NET primarily studied in this thesis is called Spindle-Associated Membrane Protein 1 (Samp1). We showed that overexpression of Samp1 induced a fast differentiation of human induced pluripotent stem cells and that the binding between two NETs, Samp1 and Emerin, is regulated by RanGTP. Another focus of this thesis was the development and use of a novel method called Fluorescent Ratiometric Imaging of Chromatin (FRIC). FRIC quantitatively monitors the epigenetic state of chromatin in live cells. Using FRIC, we were able to show that Samp1 promotes peripheral heterochromatin organization. FRIC also detected an increased distribution of heterochromatin at the nuclear periphery during neuronal differentiation. In conclusion, FRIC is a useful tool that could serve medical research in elucidating the effects of different chemical agents and the roles of NE proteins in chromatin organization. Keywords: Nuclear envelope proteins, chromatin organization, epigenetics, differentiation, quantitative image analysis, Samp1. Stockholm 2020 http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-184182 ISBN 978-91-7911-230-1 ISBN 978-91-7911-231-8 Department of Biochemistry and Biophysics Stockholm University, 106 91 Stockholm THE ROLE OF NUCLEAR ENVELOPE PROTEINS IN CHROMATIN ORGANIZATION, DIFFERENTIATION AND DISEASE Cecilia Bergqvist The role of nuclear envelope proteins in chromatin organization, differentiation and disease Cecilia Bergqvist ©Cecilia Bergqvist, Stockholm University 2020 ISBN print 978-91-7911-230-1 ISBN PDF 978-91-7911-231-8 Cover image: A colony of human induced pluripotent stem cells stained for Emerin (cyan) and Lamin A/C (red). Printed in Sweden by Universitetsservice US-AB, Stockholm 2020 Distributor: Department of Biochemistry and Biophysics, Stockholm University Grandma, perhaps the stars are openings in the sky where our loved ones shine to let us know they are there. Abstract In eukaryotes the genetic material is separated from the cytoplasm by the nuclear envelope (NE), consisting of the outer and inner nuclear membrane, the nuclear lamina and the nuclear pores. The genetic material is highly structured with transcriptionally inactive heterochromatin enriched at the nuclear periphery and transcriptionally active euchromatin in the nuclear interior. Underlying the inner nuclear membrane is the nuclear lamina (nucleoskeleton) that together with several hundred nuclear envelope transmembrane proteins (NETs) connect chromatin to the nuclear periphery. Most NETs are uncharacterized and expressed in a tissue-specific manner. Mutations in NE proteins are linked to distinct degenerative disorders, referred to as envelopathies or laminopathies. The NET primarily studied in this thesis is called Spindle-Associated Membrane Protein 1 (Samp1). We showed that overexpression of Samp1 induced a fast differentiation of human induced pluripotent stem cells and that the binding between two NETs, Samp1 and Emerin, is regulated by RanGTP. Another focus of this thesis was the development and use of a novel method called Fluorescent Ratiometric Imaging of Chromatin (FRIC). FRIC quantitatively monitors the epigenetic state of chromatin in live cells. Using FRIC, we were able to show that Samp1 promotes peripheral heterochromatin organization. FRIC also detected an increased distribution of heterochromatin at the nuclear periphery during neuronal differentiation. In conclusion, FRIC is a useful tool that could serve medical research in elucidating the effects of different chemical agents and the roles of NE proteins in chromatin organization. 1 2 List of publications This thesis is based on the following publications and manuscripts, referred to as Paper I-V in the text: Paper I: Cecilia Bergqvist, Frida Niss, Ricardo A Figueroa, Marie Beckman, Danuta Maksel, Mohammed H Jafferali, Agné Kulyté, Anna-Lena Ström, and Einar Hallberg (2019). Monitoring of Chromatin Organization in Live Cells by FRIC. Effects of the Inner Nuclear Membrane Protein Samp1. Nucleic Acids Research 47, no. 9: e49. https://doi.org/10.1093/nar/gkz123. Paper II: Balaje Vijayaraghavan, Ricardo A. Figueroa, Cecilia Bergqvist, Amit J. Gupta, Paulo Sousa, and Einar Hallberg (2018). RanGTPase Regulates the Interaction between the Inner Nuclear Membrane Proteins, Samp1, and Emerin. Biochimica et Biophysica Acta (BBA) - Biomembranes 1860, no. 6: 1326–34. https://doi.org/10.1016/j.bbamem.2018.03.001. Paper III: Cecilia Bergqvist, Mohammed Hakim Jafferali, Santhosh Gudise, Robert Markus, and Einar Hallberg (2017). An Inner Nuclear Membrane Protein Induces Rapid Differentiation of Human Induced Pluripotent Stem Cells. Stem Cell Research 23: 33–38. https://doi.org/10.1016/j.scr.2017.06.008. Paper IV: Cecilia Bergqvist, Urška Kašnik, and Einar Hallberg. Chromatin reorganization during neuronal differentiation. Manuscript. Paper V: Cecilia Bergqvist, Urška Kašnik, and Einar Hallberg. Investigations of Emery-Dreifuss Muscular Dystrophy mutants of Samp1. Manuscript. Minor parts of the work presented in this thesis have previously been published in my licentiate thesis: Cecilia Bergqvist, The role of nuclear membrane proteins in differentiation and chromatin organization (2016), Stockholm University. ISBN 978-91-7649-632-9. 3 Table of Contents Abstract ...................................................................................................................................... 1 List of publications ..................................................................................................................... 3 Abbreviations ............................................................................................................................. 7 1.1 The nuclear envelope ........................................................................................................... 9 1.1.1 The nuclear lamina ...................................................................................................... 10 Lamin A processing ......................................................................................................... 10 The function of the nuclear lamina .................................................................................. 11 1.1.2 The linker of nucleoskeleton and cytoskeleton (LINC) complex ............................... 12 The function of the LINC complex .................................................................................. 13 1.1.3 Nuclear envelope transmembrane proteins ................................................................. 13 Emerin .............................................................................................................................
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