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Contains fulltext : 151683.pdf (Publisher’s version ) (Open Access) The human body consists of billions of cells. These cells are not all the same, but they differ in shape, size and function. This is exemplified by the extreme differences between skin, muscle and neuron cells. The specific characteristics of a cell type are termed the phenotype (or phenotypes) of this cell. Different cell types thus display different phenotypes. Every cell consists largely of proteins. These proteins, which shape the cell, catalyze reactions, and facilitate communication, are essential for cellular function. Proteins are produced based on a blueprint encoded in the DNA, a blueprint which contains all information needed to produce all proteins. To produce a protein, the DNA blueprint is read and copied, a process known as transcription. Interestingly, every cell in the body contains the exact same DNA blueprint and should be capable of producing all proteins. However, DNA is compacted within the nucleus of a cell in order to safely store it. As a consequence, some parts of the DNA are easier to read and copy, whereas other parts are less accessible. Thus, the compaction of the DNA influences transcription. Proteins that are encoded in compacted and less accessible parts of DNA will be produced in far lower amounts, or not at all. DNA is compacted uniquely for every cell type, resulting in an unique set of expressed proteins. DNA is compacted using proteins called histones, and the sum of histone proteins and DNA is termed chromatin. In this thesis, I study the proteins that influence the compaction of DNA and thereby affect transcription. These proteins often combine and work together in so called protein complexes. It is essential to be able to identify these protein complexes in order to study their biological function. To this end, we use mass spectrometry, an advanced instrument able to identify proteins by precisely measuring their mass. In chapter 1, chromatin and chromatin modifications that influence transcription are introduced. Next, chapter 2 presents an overview of different mass spectrometry methods that can be used to identify protein-protein interactions. Current mass spectrometry methods robustly identify protein-protein interactions; however, they cannot be used to determine exact amounts of protein present. In chapter 3, a novel technique is introduced that can measure the amounts of all proteins present in a protein complex. After establishing this method, protein complexes involved in chromatin compaction and transcriptional regulation are studied . In chapter 3, protein complexes that compact chromatin and repress transcription, namely the PRC2 and NuRD complexes, are studied. In chapter 4, a family of protein complexes that activates transcription at loosely compacted part of chromatin, the so called SET1/MLL complexes, is characterized. Using this novel method, we were able to identify proteins present in large amounts that likely have important general functions within the complex as well as proteins present in low amounts which likely have more specialized functions in a subset of the complexes. In order to detect protein-protein interactions, we always purify our protein of interest in order to identify all proteins that co-purify with that specific protein. Typically, the protein of interest is labeled in order to purify it. The most commonly used tag is the large protein GFP, which can disrupt protein function. Therefore, we develop a labeling technique in chapter 5 that is based on very small chemical molecules that can be specifically incorporated into a protein of interest. This chemical group can subsequently be used to enrich the protein using so-called “click” chemistry. To validate whether this technique could be used to study protein-protein interactions, a subunit of the NuRD complex was tagged with this chemical molecule. The click chemistry-based purification of this protein and subsequent protein identification using mass spectrometry indicated a partial enrichment of the NuRD complex. Though this result emphasizes the need to further develop this enrichment approach, it nonetheless indicates that the approach is useful for purification of a protein of interest. Histones, which together with DNA form chromatin, are crucial for proper compaction of DNA. DNA is duplicated just before mitosis such that both daughter cells have a single copy of the DNA blueprint. In this duplication process, histones are evicted from DNA and afterwards inserted again. In chapter 6, in an effort to characterize all proteins involved in this process, we purified histone H3.1 and comprehensively identified protein-protein interactions. We identified 20 different protein complexes handling histone H3.1, most of which contain different enzymatic activities and are likely involved in a wide variety of processes. Radboud Universiteit Nijmegen, 04 maart 2016 Promotor : Vermeulen, M. 168 p. |
