Abstract
The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. It is defined by the histone molecules that constitute the nucleosome, the positioning of the nucleosomes along the DNA and the non-histone proteins that associate with it. These factors help to establish and maintain a largely DNA sequence-independent but surprisingly stable structure. Chromatin is extensively disassembled and reassembled during DNA replication, repair, recombination or transcription in order to allow the necessary factors to gain access to their substrate. Despite such constant interference with chromatin structure, the epigenetic information is generally well maintained. Surprisingly, the mechanisms that coordinate chromatin assembly and ensure proper assembly are not particularly well understood. Here, we use label free quantitative mass spectrometry to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos. The use of a data independent acquisition method for proteome wide quantitation allows a time resolved comparison of in vitro chromatin assembly. A comparison of our in vitro data with proteomic studies of replicative chromatin assembly in vivo reveals an extensive overlap showing that the in vitro system can be used for investigating the kinetics of chromatin assembly in a proteome-wide manner.
Highlights
DNA replication, transcription and repair continuously disturb the conformation of chromatin, which results in a relatively high rate of histone turnover [1] and poses a constant threat to the maintenance of epigenetic information [2, 3]
This observation raises the question of how this structure is assembled, in which order individual factors bind to the DNA, whether distinct intermediates during chromatin assembly exist and which key players mediate chromatin maturation
Medians of SWATH intensities for each protein were calculated and the values for TSA-untreated samples were divided by medians of TSA-treated samples for each time point separately to determine the enrichment upon TSA treatment during chromatin assembly
Summary
DNA replication, transcription and repair continuously disturb the conformation of chromatin, which results in a relatively high rate of histone turnover [1] and poses a constant threat to the maintenance of epigenetic information [2, 3]. Recent systematic studies revealed that mature chromatin adopts a complex molecular structure containing a large variety of binding factors that go way beyond a simple aggregate of DNA and histones [11, 12, 18, 19]. This observation raises the question of how this structure is assembled, in which order individual factors bind to the DNA, whether distinct intermediates during chromatin assembly exist and which key players mediate chromatin maturation. With the recent development of methods like iPOND [10, 26] and NCC [13] to investigate replicative chromatin assembly in vivo and improved techniques of label free MS based quantitation of proteins in complex samples [27] such comparative studies became feasible
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