Structural and Functional Analysis of Gene and Protein Sequence Families

National Library Of Medicine

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Abstract

Nucleosomes comprise of 147 bp of DNA wrapped around a histone octamer and are central in coordinating various signaling pathways involved in epigenetic regulation. The molecular recognition of nucleosomes by chromatin factors frequently occurs through the interactions with the nucleosomal and linker DNA, histone tails, and histone globular domains as well as by recognizing their specific covalent modifications. Therefore, elucidating the molecular mechanism of interactions between histone/nucleosome and various chromatin factors is essential for our understanding of the principles of chromatin organization and regulation. Protein interaction networks allow for the large-scale measurements of protein-protein interactions (PPIs) in various cellular compartments of different species, but a comprehensive human protein interactome characterization remains a challenge. Herein, we perform a comprehensive mapping of human histone and nucleosome interactions by systematically analyzing the structural, chemical cross-linking, and high-throughput data. To elucidate the physicochemical properties of human histone/nucleosome interactions, we constructed three human histone interaction networks, including structural, cross-linking, and high-throughput interactomes, and further explored histone interactions at different levels of granularity: protein, domain, and residue levels. To analyze histone-associated pathways, we further identified an additional layer of partners that interact with histone-binding proteins and construct so-called global histone interactomes. We systemically compared three global histone interactomes (structural, cross-linking, and high throughput) and observed a very low fraction of overlap (4%). Next, we systemically analyzed and compared topological properties of global structural, cross-linking, and high-throughput networks. We showed that the node-degree distributions for all three types of histone networks follow a power law, as they show a remarkably strong linear association between the fraction of nodes and node degree on a log-log plot. In a scale-free network, the majority of proteins present as low degree nodes, and only a small number of proteins act as hubs that play critical roles in mediating the network integrity and connectivity. Therefore, the histone interaction network may be relatively tolerant for the defects in low-degree nodes but vulnerable to the perturbations in the hub nodes, for example, between histones and other hub regulatory proteins. Further, we observed a strong power-law decay of values of clustering coefficients with increasing node degree. Such observations indicate a high modularity of those networks that have more dense connections between the nodes within the modules compared to connections between different modules. Using data on binding interfaces extracted from structural and cross-linking interactomes, we systematically characterized the binding sites of all human histone variants. We performed multiple sequence alignments of human histone variants per each histone type and mapped the protein binding sites onto histone sequences, using binding interfaces extracted from structural and cross-linking interactomes. Next, to identify the histone binding hotspots, we mapped the number of unique binding proteins for each residue onto the consensus sequences of the alignment, using binding interfaces extracted from the structural and cross-linking interactomes. Finally, we systemically analyzed the histone binding sites in the context of the full nucleosomes. We collected 26 human nucleosome complex structures from PDB and classified them by functions and map binding interfaces onto the molecular surfaces of nucleosomes. We showed that many binding proteins, with the exception of transcription regulatory proteins, recognize nucleosomes via the localized acidic patches. At the same time, in some cases, histones participate in multivalent binding. a. Elucidation of the effects of histone cancer mutations on histone/nucleosome structure and interactions. We show that many driver mutations lead to a significant decrease in histone-histone, histone-DNA, and histone-partner binding energies, perturbating nucleosome stability and the interactions between nucleosome and different chromatin factors. b. Elucidation of the roles of histone tails in nucleosome recognition by regulatory proteins. Overall, our results point to a potential competitive binding mechanism: Nucleosome-binding proteins compete with DNA if they recognize tails and compete with histone tails for binding to DNA. c. Understanding of the effects of PTMs and mutations on histone tail dynamics and interactions. Last, we perform comprehensive simulations of several representative PTMs and cancer mutations on the H3 tail, including H3K27Me3, H3K27Ac, H3K36Me3, H3K36Ac, H3G34R, and H3G35R. In our simulations, only one PTM or mutation is introduced into the tails each time. Interestingly, we observe that PTMs and mutations can alter histone tail conformations and the binding modes between H3 tails and nucleosomal and linker DNA. Such effects depend on the locations and types of PTMs and mutations. For instance, we find that H3K36Me3 significantly increases the radius of gyration of H3 tails and leads to compaction of H3 tail conformations. Changes of tail conformations also could alter tail binding modes with DNA, and H3K36Me3 is shown to reduce the interactions of tails with linker DNA. In contrast, H3K36Ac leads to more extended conformations of H3 tails as compared with unmodified tails and favors the binding of tails to linker DNA. Finally, we show that two cancer mutations, H3G34R and H3G35R, can reduce the H3 tail dynamics, which leads to the compaction of H3 tail configurations.