Studying Kinetics of Chromatin Assembly with SWATH-MS

DATE: November 23rd, 2016
TIME: 8:00AM PT, 11:00AM ET

The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. The nucleosomes together with the non-histone proteins define a stable chromatin structure. Despite its stability, this structure is disassembled and reassembled during DNA replication, repair, recombination or transcription. During all those processes, defined chromatin regions become accessible to be bound by the required factors, resulting in extensive nucleosome turnover at given genomic loci. The dual nature of chromatin requires a continuous interplay between stable and dynamic structures, which has to be coordinated at the molecular level to maintain the epigenetic information stored in the chromatin structure.
Despite the biological relevance of these processes, little is known about the order of chromatin assembly steps, the molecular mechanisms that coordinate the required cellular machinery in time and the quality control of this assembly. 
To address these questions, our lab uses an in-vitro system that resembles the formation of chromatin on double-stranded DNA. This in-vitro system not only enables us to dissect critical steps of assembly but also to verify predictions we make based on proteomics analysis of captured nascent chromatin (NCC) in living cells.
In order to study the dynamics of chromatin-bound proteins, we applied label free quantitative SWATH-MS (sequential window acquisition of all theoretical fragment-ion spectra) at different assembly times, allowing us to describe distinct aspects of chromatin assembly such as the appearance and disappearance of histone modifications, the levels of histone chaperones, the activity of histone writers/erasers or the concentration of distinct DNA-binding factors. Based on these results, we have accomplished a classification of chromatin factors into functional groups depending on their binding kinetics.

Learning Objective 1: Develop an experimental outline to study chromatin assembly in vitro and in vivo with quantitative proteomics

Learning Objective 2: Establish a SWATH proteomics pipeline combined with statistical data analysis to determine protein binding kinetics