From global to targeted chromatin proteomics: mapping the control unit of cellular identity

Ugur, Enes (2023): From global to targeted chromatin proteomics: mapping the control unit of cellular identity. Dissertation, LMU München: Fakultät für Biologie

Abstract

Cellular identity is established and maintained by the chromatome, which consists of transcriptional, epigenetic and structural regulators of the chromatin proteome. Serving as a control hub, the chromatome processes incoming signaling cues and modifies the transcriptional program, resulting in a specific cellular phenotype. To fully understand cell-type specific gene regulation, multi-level chromatome analysis is necessary. Chromatin-associated proteins can be explored using mass spectrometry (MS)-based proteomic methods to assess (i) total protein abundances, (ii) chromatin-associated individual complexes, (iii) global or (iv) locus-specific chromatin compositions, and (v) nucleotide and histone (post-translational) modifications. Global chromatin proteomics trails behind other areas of proteomics in terms of data comprehensiveness, accuracy, and throughput. The main aim of this work was the development of an MS-based proteomic method, Chromatin Aggregation Capture followed by Data Independent Acquisition (ChAC-DIA), which enables the comprehensive identification and accurate quantification of chromatin regulators, including those present in low quantities, across different pluripotency stages. ChAC-DIA identified 2-3 times more chromatin-associated proteins with enhanced accuracy and efficiency, required 100-fold less sample material, and halved the MS data acquisition time compared to prior methods. By applying ChAC-DIA an extensive atlas was constructed that encompasses proteomes, chromatomes, and chromatin affinities across the three key phases of pluripotency. The data served not only to verify bona fide pluripotency regulators such as REX1, OCT6 and SOX1, but also to identify new phase-specific factors like JADE1/2/3, QSER1, SUV39H1/2 and FLYWCH1. Moreover, this study offers a straightforward strategy for distinguishing between translation-driven changes in chromatin binding and alterations in nuclear localization or chromatin affinity. Using this approach, we observed that certain heterochromatic proteins, such as HP1β, KAP1, and SUV39H1, exhibited enhanced chromatin affinities towards the exit from pluripotency, which we could demonstrate to be a conserved feature in both mouse and human. In subsequent collaborative endeavors, chromatin proteomics was applied to study epigenetic regulations in several biological contexts. In three distinct projects, chromatin immunoprecipitation combined with MS was employed to analyze the interaction networks of the naive pluripotency marker DPPA3, the histone H3 lysine 9 trimethyl (H3K9me3) reader HP1β and the methylcytosine dioxygenase TET1. Moreover, the KAP1-dependent ubiquitinome was investigated, the composition of HP1β-driven phase-separated droplets was studied, and a proteomic workflow was developed to screen for the efficient incorporation of non-canonical amino acids into target proteins. This work also provided a detailed protocol for probing locus-specific chromatin composition as well as full proteome analyses upon genetic perturbations targeting epigenetic modifiers in an acute myeloid leukemia cell culture model and embryonic stem cells at various pluripotency stages. In a last collaboration, it was tested whether the histone methyltransferases SUV39H1/2 primarily contribute to H3K9me3 formation in visceral endoderm descendants. In summary, this work provides a powerful method to study the global chromatome of any model in development and disease, sheds new light on dynamic rearrangements of pluripotency governing regulatory complexes and contributes to a broad range of epigenetic research by harnessing multi-level chromatome analyses.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Protein-nucleic acid interaction, Chromatin and Epigenetics, Gene Regulation, Mass spectrometry, Proteomics
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 7. Dezember 2023
• 1. Bericht­erstatter:in: Leonhardt, Heinrich