Development of chemical biology tools to reveal the function and organization of the silenced chromatin state

Effector proteins are recruited to chromatin via transient interactions between their reader domains and histone post-translational modification (PTM) patterns. These interactions form signaling pathways that control gene expression or repression and which can lead to cancer when dysregulated. Moreover, combinations of histone PTMs define particular chromatin states and determine 3-dimensional genome organization. Chromatin represents a key intersection point in the complex signaling network of a eukaryotic cell and the mechanisms underlying the readout of PTMs are of great interest, but still not well understood.

Recent studies revealed that histone PTMs can be installed in an asymmetrical configuration, that is, the two copies of histone proteins within a nucleosome do not obligatorily carry the same PTMs. Such asymmetry greatly expands the combinatorial space of histone marks, yet little is known about their biological relevance. In addition, dependent on histone PTM patterns, particular chromatin states -such as the repressive heterochromatin- compartmentalize into membraneless condensates via liquid-liquid phase separation (LLPS). Although it is not clear whether LLPS is a general organizational principle, it certainly constitutes a new paradigm in understanding critical chromatin-dependent mechanisms and diseases.

Chemical biology and biophysics are key for the investigation of chromatin organization and to monitor chromatin-effector interactions in time and space. First, for in vitro studies of the molecular processes encrypted in the histone code, the facile chemical access to pure modified nucleosomes is crucial. However, their preparation is not straightforward. Second, investigations of chromatin dynamics on the molecular scale depend on the ability to site-specifically label protein effectors and chromatin. Thus, new and versatile protein modification methods together with bright and stable dyes are of key importance. Finally, cell permeable probes to investigate the in vivo dynamic behaviour and localization of the genome in live cells are currently lacking. This PhD project approaches these multiple challenges. First, I extended a recently developed crosslinking strategy, which gives control over the supramolecular assembly of nucleosomes, to the synthesis of nucleosomes with asymmetrical PTM patterns on histone H3 and H4. These precious substrates revealed mechanistic details of stem-cell regulation by key methyltransferases, PRC2 and Set8. Second, I report the use of hypervalent iodine reagents for the effective cysteine-selective labeling of proteins, such as histone octamers. The strategy enables to doubly functionalize the substrates via formation of an azide-containing vinylbenziodoxolone intermediate. Using this method, a triplet state quencher was introduced in proximity to a fluorophore, reducing the photobleaching rates and enhancing the fluorescence lifetime in single-molecule studies. Finally, I describe the design of peptide-based LLPS probes for live cell chromatin visualization. Conceived to stain heterochromatin foci, I expect the probe to be easily tunable to other chromatin states, enabling the 3D dynamic detection of chromatin organization in living cells.

Together, this thesis contributes to the deciphering of the multifaced chromatin PTM readout and provides the chemical biology and biophysics community with novel tools that will allow to further investigate chromatin-dependent processes.