Biochemical investigations of multivalent chromatin reading domains

Abstract

In eukaryotes, the negatively charged nuclear DNA wraps around cationic histone proteins to form nucleosomes and compact the genetic information. Histones carry several post-translational modifications (PTMs) that appear in combinatorial patterns. These marks are interpreted by non-covalent interactions with proteins containing histone modification interacting domains (HiMIDs), also known as “reader” domains. Thirty years ago, it was proposed that the histone marks act as signals in the regulation of transcription and other chromatin functions. With time, this concept has been refined to suggest that combinatorial patterns of marks represent context-specific signals, termed a 'histone code'. It functions as one of the epigenetic regulatory mechanisms, which control reversible and heritable changes in cellular phenotype. Intermolecular models demonstrate thermodynamic benefits from multivalent engagement of nucleosomes, suggesting their widespread occurrence. However, so far only few multivalent readers are known and dissecting their function has been very challenging. This thesis focuses on HiMIDs with complex roles that simultaneously interact with two histone PTMs or two different substrates. Introducing the theoretical foundation, I discuss the thermodynamic and biological basis of how multivalent interactions can guide effector protein complexes, targeting their functions to distinct regions and chromatin states. Then, I present data from the characterisation of the readers DNMT3A-PWWP, DDX19A, and UHRF1-TTD in the context of multivalent engagement of histone PTMs and biomolecules. Starting with DNMT3A-PWWP, I quantified the binding of the wild-type (WT) and a mutant domain to histone H3K36me2/3 peptides, showing negligible differences, while my colleagues showed that the mutant has drastically reduced binding to DNA and nucleosomal substrates. I, then, studied the R-loop helicase DDX19A to demonstrate a very strong binding to H3K27me3 peptides in the nanomolar range, complementing the findings of a complex functional study. The latter showed that interaction with H3K27me3 is necessary for robust DDX19A-mediated R-loop resolution, and LSD1-target gene silencing. With UHRF1-TTD, I discovered and quantified its preferential binding to H3K4me1-K9me2/3 peptides vs H3K9me2/3 alone and engineered mutants with specific and differential binding changes leading to the discovery of a novel Kme1 read-out mechanism, based on the interaction of R207 methylene groups with the H3K4me1 methyl group and on counting the H-bond capacity of H3K4. High-throughput sequencing (HTS) data revealed strong TTD binding at chromatin sites with H3K4me1 peaks and broad H3K9me2/3 signal, which are enriched on enhancers and promoters of cell-type specific genes at the flanks of cell-type specific transcription factor binding sites. Data from the full-length protein in mouse and human cells evidenced the physiological role of the H3K4me1-K9me2/3 double marks in TTD-mediated UHRF1 recruitment. To further illustrate this point, I investigated UHRF1-dependent silencing of repeat elements (RE). To this end, I developed RepEnTools, improving the previously available programmes for RE enrichment analysis in chromatin pulldown studies by leveraging new tools, with carefully chosen and validated settings, enhancing accessibility, and adding some key functions. RepEnTools analyses showed that chromatin binding of hUHRF1-TTD and full-length mUHRF1 was strongly enriched on different REs promoters with the H3K4me1-K9me3 double mark where UHRF1 represses their expression. The data suggest a novel functional role for the H3K4me1-K9me3 signal of the histone code that is both sequence independent and conserved in two distinct mammals. Taken together, the work presented here is consistent with and supports the histone code theory, best illustrated by UHRF1-TTD which binds a specific double mark that has a biological meaning going beyond the meaning of the individual marks. In this thesis, I presented various mechanisms that influence epigenomic regulation, including chromatin 3D-architecture, accessibility, transcription factor recruitment, and chromatin marks. Especially in the context of UHRF1-TTD functions, I discussed how DNA, RNA, histones, and covalent modifications thereof interweave to produce the signalling network necessary throughout the lifetime of the mammalian cell, during differentiation, development and every other phase of life. Thus, within the three-dimensional scaffold of chromatin structures these biomolecules and their modifications collectively form the context-specific network of effectors and maintainers of the epigenomic modifications. The ways in which they influence transcription and translation are only now becoming unravelled. Hence, the recent data suggest the existence of not just a histone code, but a 3D-chromatin modification code, which dictates how biomolecules and their modifications collectively implement epigenomic regulation by interactions along the chromatin and through 3D space. As shown in these projects, readers commonly use the mechanism of multivalent interactions to interpret such contextual signals and guide epigenomic effectors to their targets. The tools and workflows that were developed and applied in this work can be employed to reveal more instances of refined read-out among HiMIDs. Additionally, I leveraged my experience with fluorescence spectroscopy and made contributions to another two published studies. The first study demonstrated that the DNMT3A-ADD Zn-finger domain, which is a known H3K4me0 reader, also binds to a domain from the MECP2 protein. The association was quantified, and the specificity demonstrated with a binding deficient triple mutant. This interaction offers complex additional regulation options to DNMT3A and MECP2, in interplay with the histone code. The second study focused on SETD2, a H3K36me3 depositing enzyme, and the mechanism of its preference for a designed “super substrate” peptide. By elegantly combining computational simulations and experimental data, the study demonstrated that an H3 peptide substrate predominantly exists in an extended conformation in solution, while the super substrate forms a hairpin conformation. Upon binding to the enzyme, the hairpin is opened and the super substrate adopts a similar conformation as the canonical substrate. These results highlighted the dynamic nature of solubilised peptides' conformations, their impact on protein-protein interactions, and the significance of dynamic conformational changes in interactions.