Challenging chromatin assembly in vitro: leveraging and expanding the use of the Drosophila embryonic extract system to study chromatin structure and function

Kleene, Vera (2024): Challenging chromatin assembly in vitro: leveraging and expanding the use of the Drosophila embryonic extract system to study chromatin structure and function. Dissertation, LMU München: Medizinische Fakultät

Abstract

In eukaryotes, the genome is packaged into chromatin to compact but also regulate the genome. Chromatin consists of the DNA, histones, and non-histone proteins that interact with the chromatin fiber. The structure of chromatin fiber is highly dynamic and depends on multiple modulators. The question what factors influence the protein association with chromatin is therefore of critical import ance. The influencing factors range from the binding modes that contribute to fiber association to the question whether the chromatome is regulated by its metabolic environment. The challenge in studying these questions is the requirement of sufficiently complex systems that allow the detection of transferable and physiologically relevant effects while still being easy-to-manipulate and well-characterized to allow deconvolution of the factors. The preblastoderm Drosophila embryo extract (DREX) chromatin assembly system is currently the best-characterized method to investigate the assembling chromatin fiber. However, it still lacks thorough characterization regarding total protein composition and metabolic state and activity. This study characterizes previously unknown key aspects of the DREX in vitro system including a full proteome analysis with a hitherto unreached depth and the first metabolomics profiling of the extract. Notably, this study also proves that the extract is metabolically active and able to degrade proteins and replenish metabolites. These finding significantly advance the understanding of the extracts protein composition and metabolic state. The obtained data provide a crucial baseline reference that can be used as a tool for future research and facilitates the adaption of the DREX as a model with significant potential for advancing the understanding of metabolic processes. Secondly, the metabolic processes detected within the DREX extract are leveraged to study the link between metabolism and chromatin structure. The DREX chromatin assembly model is challenged using the supplementation of isotopically labeled metabolites. Subsequent mass spectrometry analysis reveals changes in histone modifications, suggesting a strong coupling of metabolism and histone modifications in the extract. Thirdly, this study addresses the effect of synthetic DNA mimic foldamers that mimic DNA shape on the composition of chromatin. Changes in protein composition revealed insights into the partial contributions of different binding modes to the chromatin association of selected proteins. By using flow cytometry and fractionation coupled with proteomics, similar effects and disruption cell cycle progression in vivo were observed. In addition to the mechanistic insights, this provides a proof-of-concept for the DREX assembly system to be used as a method to screen small molecules for pharmacological intervention with chromatin assembly in the future. In conclusion, in this study the in vitro chromatin assembly system DREX is characterized in depth, proteomically and metabolomically, to facilitate future research and findings are leveraged to challenge the system using DNA mimic foldamers, metabolite depletion, and supplementation. Novel insights into the contribution of chromatin protein binding modes and coupling of metabolism and histone modifications during chromatin assembly are gained. This research characterizes and leverages the in vitro system DREX chromatin assembly, expanding the model for adaptation in metabolomics research and establishes it as a pharmacological screening assay with good transferability to in vivo setting.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 24. Oktober 2024
• 1. Bericht­erstatter:in: Imhof, Axel