Understanding the contribution of inositol phosphate signalling to class-1 HDAC complex function Thesis submitted for the degree of Doctor of Philosophy at the University of Leicester by Grace Emily Adams BSc (Hons) College of Medicine, Biological Sciences, and Psychology Department of Molecular and Cell Biology University of Leicester March, 2018 Grace Emily Adams Understanding the contribution of inositol phosphate signalling to class-1 HDAC complex function Abstract: Class 1 histone deacetylases (HDACs) regulate chromatin confirmation and gene expression through recruitment to co-repressor complexes. Recently, it was shown that HDAC3/SMRT binds and is regulated by inositol phosphates (IP) in vitro. Additionally, complex activity of HDAC1/MTA1 and HDAC1/MIDEAS is enhanced by the addition of IPs in vitro, indicating conserved regulation. In this work, I aimed to alter the level of IPs present in the cells through overexpression of a kinase, IPMK, and two phosphatases, PTEN and SopB, and determine the effect on HDAC activity in vivo. In addition, I utilised an IPMK knockout embryonic stem (ES) cell line and generated inducible rescues through a PiggyBac TET system to establish if large scale depletions of IP levels alter HDAC activity. We revealed that manipulation of IP through the overexpression of IPMK, PTEN and SopB does not influence global HDAC activity or histone acetylation levels. Isolation of overexpressed HDAC3/SMRT in concert with these enzymes, revealed differences in HDAC3/SMRT complex activity, however, these differences did not correlate with altered IP levels. Analysis of global HDAC activity, isolated complex activity and HDAC3 target genes in IPMK knockout and rescue ES cell lines further revealed minimal changes. In conclusion, we were unable to show that IPs regulated the activity of class I HDAC complexes in vivo. IPMK, HDAC1 and HDAC3 null mice all exhibit early embryonic lethality suggesting they play essential roles in embryogenesis. Upon differentiation of TET-IPMK cells, embryoid bodies revealed loss of IPMK leads to increased cardiomyocyte markers and decreased formation of neuroectoderm progenitors. Therefore, emphasizing IPMKs important role in gene regulation during embryogenesis. Our data suggests, that this is not through direct regulation of HDAC activity, thus highlighting an undiscovered nuclear role for IPMK. I Acknowledgments I firstly wish to thank my supervisor, Professor Shaun Cowley, for his endless support, guidance, encouragement and faith throughout my time at the University of Leicester. I would also like to thank my co-supervisor Professor John Schwabe for his advice and invaluable discussion during my project. My thanks also go to my committee members Professor Ian Eperon and Dr Tom Webb for their helpful and thought provoking comments and discussion. I would like to extend my gratitude to Dr Peter Watson for his irreplaceable advice, valuable discussion and help in the lab and in establishing the inositol phosphate quantitation method with me. My thanks go to all members of labs 3/37, you’ve made me feel welcome since my first day with your friendship, encouragement and help. Special thanks go to Claire Barnes for sharing the ups and downs of a PhD with me, your support and friendship have kept me sane during this process. Without the unconditional support, love and patience provided by my family, especially my mother, father, brother, sister-in-law and my adorable nephews, this process would have been so much harder, thank you for everything you do for me. Finally, to my husband Cam, you may not understand the contents of this thesis as I do not understand geotechnical engineering; but I could not have got to this stage without your constant love, patience, motivational speeches and simply being there for me. II List of Contents Abstract I Acknowledgments II Table of contents III List of figures VII List of tables XI Abbreviations XII Table of Contents Chapter 1 Introduction ........................................................................................ 1 1.1 Structure and function of chromatin .......................................................................... 1 1.2 Epigenetic regulation of chromatin ............................................................................ 3 1.3 Post translational modifications of histones ............................................................... 4 1.3.1 Sumoylation and Ubiquitinylation .......................................................................... 6 1.3.2 Phosphorylation ..................................................................................................... 7 1.3.3 Crotonylation .......................................................................................................... 8 1.3.4 Methylation ............................................................................................................ 8 1.3.5 Acetylation ............................................................................................................ 10 1.3.6 Readers of histone modifications ......................................................................... 12 1.4 Interplay of epigenetic modifications ....................................................................... 13 1.5 Histone deacetylase (HDAC) family of proteins ........................................................ 14 1.5.1 Classification of histone deacetylases .................................................................. 15 1.5.2 Recruitment of Class I HDACs to DNA via co-repressor complexes ...................... 19 1.5.2.1 Sin3 .......................................................................................................................... 21 1.5.2.2 NuRD ........................................................................................................................ 22 1.5.2.3 CoREST ..................................................................................................................... 23 1.5.2.4 MiDAC ...................................................................................................................... 24 1.5.2.5 SMRT/NCoR ............................................................................................................. 25 1.6 HDACs and transcriptional regulation ....................................................................... 28 1.7 Non-histone targets of HDACs .................................................................................. 29 1.8 Role of class I HDACs in cellular processes ................................................................ 30 1.8.1 Germ-line deletion of class I HDACs in mice ......................................................... 30 1.8.2 Tissue specific deletion of class I HDACs .............................................................. 32 1.9 Inositol phosphates and class I HDACs ...................................................................... 34 1.9.1 Regulation of class I HDACs by inositol phosphates ............................................. 34 III 1.9.2 The inositol phosphate pathway .......................................................................... 38 1.9.3 Inositol phosphates and transcriptional regulation .............................................. 41 1.10 Mouse Embryonic stem cells .................................................................................... 43 1.10.1 Maintenance of pluripotency in ES cells .......................................................... 44 1.10.2 Transcriptional network of pluripotency factors .............................................. 45 1.10.3 Differentiation of ES cells ................................................................................. 47 1.10.4 Modulation of chromatin structure in ES cells ................................................. 50 1.11 Project Aims .............................................................................................................. 52 Chapter 2 Materials and Methods ..................................................................... 53 2.1 Culture of mouse embryonic stem cells .................................................................... 53 2.1.1 Thawing of ES cells ................................................................................................ 53 2.1.2 Passaging of ES cells ............................................................................................. 53 2.1.3 Generation of PiggyBAC TET IPMK Inducible ES cell lines .................................... 54 2.1.4 Freezing ES cells .................................................................................................... 55 2.1.5 Population doubling assay .................................................................................... 55 2.1.6 In Vitro Differentiation of ES cells ......................................................................... 55 2.1.6.1 Differentiation into Embryoid Bodies (EBs) ............................................................. 55 2.1.6.2 Retinoic acid induced differentiation of ES cells ...................................................... 56 2.1.7 Cell cycle Synchronisation of ES cells .................................................................... 56 2.1.8 Media and reagents used for culture of ES cells ................................................... 57 2.2 Culture of Human Embryonic Kidney-293T cells ....................................................... 59 2.2.1 Thawing and maintenance of HEK-293T cells ......................................................