Systematic analysis of the interactome of modified chromatin
by Miroslav Nikolov
Date of Examination:2012-10-19
Date of issue:2013-09-13
Advisor:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Peter Rehling
Referee:Prof. Dr. Dirk Görlich
Persistent Address:
http://dx.doi.org/10.53846/goediss-4047
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Abstract
English
Regulation of chromatin composition and structure is crucial for maintaining genome integrity and execution of the wide array of functions related to gene regulation and downstream cell signaling. Chemical modifications of chromatin are at the core of these processes and include methylation of the primary sequence of DNA, as well as various modifications of the proteinaceous chromatin packaging units - the histones. Recent genome-wide mapping approaches have been instrumental for characterization of the location and distribution of these marks relative to the different functional domains and gene regulatory elements in chromatin. The majority of the characterized chromatin modifications function as signaling platforms for recruitment of various protein complexes. Therefore, it is of equal importance to describe these sets of proteins for dissection of the functional consequence of their binding. More importantly, global analysis of the interactomes of functionally associated chromatin modifications might shed light on the proteins required for establishment and operation of specific chromatin domains. In this study, a novel approach for identification of chromatin modification-dependent protein binding was established. In vitro reconstituted oligonucleosomal templates with homogeneous and defined modification status were used for affinity purification from SILAC-labeled nuclear extracts. The interactomes of ten individual chromatin species were investigated and the results provided valuable insight into chromatin biology on several levels. Investigation of the nature of the subproteomes recruited by single modifications provided evidence for their functional role. Additionally, the results offer a comprehensive catalogue of candidate proteins for further dissection of specific chromatin modification molecular readout. This was exemplified here with the demonstration of the recruitment of the SWI/SNF complex to monoubiquitylated H2B-containing chromatin for regulation of transcription of a specific set of genes. Investigation of the interactome of doubly modified chromatin identified a large number of factors whose recruitment presumably depends on the cooperative action of two modifications. Furthermore, the comparative analysis of individual datasets revealed novel relationships between the different modifications. On a global scale, this resulted in the identification of a limited set of proteins that likely play an important role for the function of heterochromatin domains. Lastly, the chromatin affinity purification approach was used for developing a SILAC internal standard method for direct quantitative comparison of recruitment to different chromatin modifications and combinations thereof.
Keywords: chromatin; histone; PTM