Systematic analysis of heterochromatin modification readout

von Nadin Zimmermann
Dissertation
Datum der mündl. Prüfung:2016-06-15
Erschienen:2017-06-14
Betreuer:Prof. Dr. Wolfgang Fischle
Gutachter:Prof. Dr. Wolfgang Fischle
Gutachter:Prof. Dr. Holger Stark
Gutachter:Prof. Dr. André Fischer
Gutachter:Prof. Dr. Steven Johnsen
Gutachter:Prof. Dr. Detlef Doenecke
Gutachter:Prof. Dr. Henning Urlaub
Zum Verlinken/Zitieren: http://dx.doi.org/10.53846/goediss-6345

 

 

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Modifications of the building units of chromatin - the histone proteins and DNA - direct the functional readout of the genome and are part of the so called “epigenome” (Fischle et al., 2003b; Kouzarides, 2007). Several studies have shown that specialized proteins recognize these chemical modifications and thereby define the functional state of chromatin (Bartke et al., 2010; Nikolov et al., 2011; Vermeulen et al., 2010). Several individual chromatin-binding factors have been analyzed in detail. Also, proteomics approaches have taken inventory of factors interacting with single chromatin modifications. However, the full set of factors specific to particular modified chromatin domains, such as for example eu-and heterochromatin, is not known yet. In this thesis, I report my findings from a systematic in vitro chromatin affinity purification approach in combination with quantitative mass spectrometry (Nikolov et al., 2011) that significantly expands the list of proteins regulated by heterochromatic modifications, both alone and in combination. The results provide a comprehensive catalogue of proteins specifically regulated in their chromatin binding by specific modification patterns. Moreover, the datasets highlight novel biological functions associated with individual modifications and reveal unpredicted functional relationships of the investigated chromatin modifications. The analysis of the interactomes of combinatorially modified chromatin identifies for the first time proteins whose binding properties to chromatin depend on the cooperative action of two modifications. The results provide novel insights into the communication between two chromatin modifications, namely positive and negative crosstalk. Furthermore, I introduce a new experimental workflow combining chromatin affinity purification and cross-linking mass spectrometry. This workflow maps physical protein-protein interactions sites of chromatin-bound proteins and provides first insights into the hierarchy of protein recruitment to chromatin. Overall, my findings demonstrate that by using a template that mimics the native form of chromatin, which is not accessible by the widely used reductionist approaches, chromatin modifications can be analyzed in multiple dimensions. In combination with the new methods established here, my work will help to improve our understanding of epigenetic regulation of chromatin-associated processes.
Keywords: heterochromatin; histone modification readout; histone modification crosstalk; chromatin affinity purification; chromatin binding interactome
 
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