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PDF Hosted at the Radboud Repository of the Radboud University Nijmegen PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/147462 Please be advised that this information was generated on 2021-10-11 and may be subject to change. Chromatin Regulatory Complexes from an Interaction Proteomics Perspective an Interaction Proteomics from Regulatory Complexes Chromatin Chromatin Regulatory Complexes from an Interaction Proteomics Perspective H. İrem Baymaz H. İrem Baymaz H. İrem Baymaz 2015 Chromatin Regulatory Complexes from an Interaction Proteomics Perspective H. İrem Baymaz printed by: Proefschriftmaken.nl || Uitgeverij BOXPress Copyright © 2015 H. İrem Baymaz Chromatin Regulatory Complexes from an Interaction Proteomics Perspective Chromatine-Regulerende Complexen vanuit een Interactie-Proteomics Perspectief Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. dr. Th.L.M. Engelen, volgens besluit van het college van decanen in het openbaar te verdedigen op donderdag 26 november 2015 om 12.30 uur precies door Hamdiye İrem Baymaz geboren op 8 maart 1986 te Ankara, Turkije Promoter: Prof. dr. M. Vermeulen Manuscriptcommissie: Prof. dr. J.H.L.M. van Bokhoven Prof. dr. H.T.M. Timmers (UMCU) Dr. L.M. Kamminga Anne ve Babama. Table of Contents List of Abbreviations 9 Chapter 1 11 Introduction Chapter 2 45 Identifying nuclear protein-protein interactions using GFP affinity purification and SILAC-based quantitative mass spectrometry Chapter 3 65 MBD5 and MBD6 interact with the human PR-DUB complex through their methyl-CpG binding domain Addendum to Chapter 3 83 Chapter 4 93 Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression Chapter 5 107 Towards elucidating the stability, dynamics and architecture of the NuRD complex using quantitative interaction proteomics Chapter 6 131 Perspective on unraveling the versatility of ‘co-repressor’ complexes Chapter 7 145 Discussion Appendix Summary 171 Samenvatting 172 Curriculum Vitae 173 List of Publications 174 Acknowledgments 175 List of Abbreviations ATP: Adenosine Triphosphosphate cDNA: complementary DNA CpGI: Cytosine-Guanine Island DDR: DNA Damage Response DNA: Deoxyribonucleic Acid GTF: General Transcription Factor ICR: Imprint Control Region KDM: Lysine-specific Demethylase LMR: Low Methylated Region MBD: Methyl-CpG-binding domain meCpG: methylated Cytosine-Guanine linear dinucleotide NuRD: Nucleosome Remodeling and Histone Deacetylation O-GlcNAc: O-linked beta-N-acetylglucosamine PcG: Polycomb Group PGC: Primordial Germ Cell PIC: Preinitiation Complex PPI: Protein-Protein Interaction PR-Dub: Polycomb Repressive Deubiquitinase PRC: Polycomb Repressive Complex PRE: Polycomb Response Element PTM: Post-Translational Modification RNA pol II: RNA polymerase II RNA: Ribonucleic Acid TF: Transcription Factor TrxG: Trithorax Group TSS: Transcription Start Site WGBS: Whole Genome Bisulphite Sequencing 9 Chapter 1 Introduction “‘But for what purpose was the earth formed?’ asked Candide. ‘To drive us mad.’ replied Martin.” Voltaire, Candide (1759) Chapter 1 12 Introduction Basic concepts in molecular biology in relation to epigenetics Deoxyribonucleic acid (DNA) is the hereditary material in all domains of life except for the cases of some obligate parasites (figure )1 1. DNA is made up of nucleotides that consist of a phosphate group, a sugar (deoxyribose), and either of the 4 bases; adenine, guanine, cytosine or thymine. The double helix structure consists of the phosphate backbone facing 1 outward while the bases form hydrogen bonds (A basepairing with T, and G with C) in- between the 2 antiparallel strands. DNA is able to inherit all the information it carries by replication where it serves as the template. LCA Microsporidia Flagellates Fungi Green Plants Ciliates Animals Bacteria Archae Eucarya Figure 1: Representative sketch of the tree of life. The 3 domains of life are depicted as Bacteria, Archaea and Eucarya, originating from the last common ancestor (LCA). It is still a matter of debate whether and where the obligate parasites with an RNA genome belong in this picture2, especially considering the predominant view in the field that the LCA had DNA as its genetic material.3 Figure adapted from Woese et al, 1990;4 however the branch lengths do not represent any phylogenetic information. All model organisms mentioned in this thesis belong to the Eucarya domain: Saccharomyces cerevisiaea in Fungi; Arabidopsis thaliana in Plants; Caernohabditis elegans, Drosophila melanogaster, Mus musculus and Homo sapiens in Animals (metazoans). The central dogma of molecular biology posits that information stored on DNA is passed on to ribonucleic acid (RNA) molecules in a process called transcription5. RNA molecules are further ‘decoded’ into functional agents called proteins via a process called translation. Although the flow of information was originally postulated to be unidirectional, nature has presented exceptions to the rule6,7. A gene is the structural unit encoding for an RNA molecule8. Messenger RNAs are translated into proteins, while ribosomal RNAs and transfer RNAs are not; but they are required for the process of translation. More recently discovered types of RNA, such as long non-coding RNAs and microRNAs, expand the group of untranslated and functional RNAs, playing important roles in modulating transcription or translation processes9–11. The transcriptome of a cell refers to all the genes that are transcribed (active, expressed) and the proteome refers to all the proteins that are translated and folded correctly into a functionally capable structure. A cell is the most basic unit of life and, all organisms, however complex they may be in the end, arise from a single cell1. More specifically, focusing on the mammalian development, the fertilized egg (zygote) transitions into the blastocyst with an inner cell mass (ICM). The cells within the ICM then give rise to the three germ layers that form all the subsequent organs and tissues of the organism. These germ layers are the endoderm (forming e.g. lungs, liver, gut), the mesoderm (muscular system, heart, blood) and the ectoderm (nervous 13 Chapter 1 system, skin)12. A cell with the potential to give rise to a whole organism is called totipotent. A cell with the potential to give rise to any cell type of an organism is called pluripotent. From pluripotent cells, progenitors of different tissues are derived which have the potential to both self-renew (i.e. proliferate) and further specialize (i.e. differentiate) into the different cell types of specific tissues (figure ).2 In the end, there are around 250 different types of cells in a human12. Starting from a single cell, this level of differentiation is impressive, especially considering the fact that all the different cells have the same DNA. However, it is well-established that besides some common genes (the housekeeping genes) that are required for the basic cellular structures and functions, the transcriptomes and hence the proteomes of different cells types vary remarkably12. Zygote Blastocyst Progenitor cells Dierentiated cells Figure 2: A simplified sketch of cellular differentiation.The zygote transitions into the blastocyst. Cells within the inner cell mass are pluripotent. Progenitors capable of self-renewal and differentiation further differentiate into tissue-specific cells. A defining property of the eukaryotic family of organisms is that DNA is not free-floating in the cell, but confined to the nucleus. Due to the sheer size of DNA (e.g. 2 meters when all human chromosomes are aligned end to end), it’s compacted thoroughly by being wrapped around 4 types of histone proteins that, in pairs, make up an octamer. DNA wrapped around this octamer of histone proteins is called the nucleosome (box 1 and figure ).3 The strands of DNA that enter and exit the nucleosome structure are stabilized by another histone protein, Histone H1, also called the linker histone. Nucleosomes, nucleosome-associated proteins and long noncoding RNAs are collectively called chromatin1. Early visualization studies yielded the ‘beads-on-a-string’ image of DNA wrapped around nucleosomes which represents the 10 nanometer (nm) chromatin fiber. Images with a 30 nm diameter show stacks of polynucleosomes in a higher order chromatin structure, although the exact organization of nucleosomes at this stage and beyond need to be resolved13–15. It is also known that chromosomes are not in a state of uniform compaction in interphase (the longest phase of the cell cycle). Highly dense regions of chromatin are stained more strongly by basic dyes and called heterochromatin while the less dense, less intensely 14 Introduction stained regions are called euchromatin. These classifications predate the discovery of the structure of DNA and nucleosomes. Amazingly, the functional implications articulated at the time hold true to this day, i.e. the euchromatic regions are more transcriptionally active than the heterochromatic regions16. Initially considered a structural requirement for the compaction of DNA, the functional 1 involvement of nucleosomes in all DNA-templated processes became clear soon enough. First of all, chromatin structure is refractory to all processes that require access to DNA such as DNA replication, DNA repair and transcription, hence chromatin manipulation prior to any of these processes is essential15. Secondly,
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