Infoscience

Thesis

Heterochromatin protein 1 recruitment and chromatin conformational dynamics at the single-molecule level

Chromatin is the template on which DNA-associated transactions take place in eukaryotic organisms. Nucleosomes consisting of the four histones H2A, H2B, H3 and H4 each organize  150bp of DNA and constitute a first layer of chromatin. The three-dimensional organization of chromatin as well as histone post-translational modifications (PTMs) regulate recruitment of chromatin-associated effector proteins (effectors). Heterochromatin protein 1 (HP1) is an effector associated with silenced genome regions. HP1 recognizes histone H3 trimethylated at lysine 9 (H3 K9me3) and can dimerize. This results in a protein with two binding domains allowing multivalent engagement of target chromatin. HP1 can further promote chromatin condensation and inter-fiber contacts. The effector p53 binding protein (53BP1) is a key regulator in the DNA damage repair pathway. It is known to target a trio of PTMs; H4 dimethylated at K20 (H4 K20me2), H2A(.X) ubiquitylated at K15 (H2A.X K15ub) and H2A.X phosphorylated at S139 (H2AX S139ph). Although details about the function of the individual domains of these proteins have been uncovered, little is known about the binding mechanism of the holoproteins and how this affects chromatin conformation. The aim of this thesis was to develop chromatin engineering and single-molecule fluorescence based methods to; i) Interrogate recruitment kinetics of HP1 to post-translationally modified chromatin. ii) Understand how HP1 binding alters the conformational dynamics of chromatin secondary structure. iii) Prepare histones carrying the PTM signature recognized by 53BP1. We established an assay to monitor binding kinetics of HP1α to modified chromatin using co-localization single-molecule microscopy (CoSM). H3 K9me3 octamers, labeled HP1α, dimerized HP1α and labeled array DNA formed the basis for this. With this, we found that HP1a multivalency induced by dimerization functions as a platform to enhance HP1a binding to target chromatin up to 9-fold by both accelerating association and prolonging retention. This was further corroborated by FRAP measurements using specific mutants in live mouse fibroblasts. Chromatin conformational dynamics were investigated by ensemble and single-molecule FRET (smFRET). Multiple combinations of FRET positions allowed us to obtain multi-perspective information on the conformational changes in chromatin upon compaction. This showed distinct steps in the local folding of chromatin. Histone acetylation of histone H4 prevented the last steps of this folding pathway. HP1-mediated compaction promotes earlier steps of compaction while maintaining conformational dynamics. Finally, towards similar studies with 53BP1, we devised schemes for synthesis of histones containing the target PTMs. Histones with the individual PTMs were prepared by ligation and desulfurization. H2A.X with both an N-terminal ubiquitin and C-terminal phosphorylation was prepared through a convergent route using recombinant SUMO and a split intein from Nostoc Punctiforme as orthogonal recombinant protection groups. Together the work described in this thesis combines advanced protein and chromatin engineering with CoSM and smFRET. This resulted in mechanistic insight into the spatio-temporal regulation of HP1 recruitment, chromatin conformational dynamics, templates for similar investigations with 53BP1, and tools for further investigation of nucleosome function in the context of chromatin.

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