X-linked intellectual disability mutations alter the conformational state of a histone demethylase's sensing of chromatin

The H3K4me3 chromatin modification, a hallmark of promoters of actively transcribed genes, is dynamically removed by the KDM5 family of histone demethylases. The KDM5 demethylases have a number of accessory domains, two of which, ARID and PHD1, lie within the catalytic domain. KDM5C, which has a unique role in neural development, harbors a number of mutations adjacent to its accessory domains that cause X-linked intellectual disability (XLID). The roles of these accessory domains remain unknown, limiting an understanding of how XLID mutations affect KDM5C activity. We find that while the ARID and PHD1 domains are required for efficient nucleosome demethylation, the PHD1 domain alone has an inhibitory role in KDM5C catalysis. We further find that binding of the H3 tail to PHD1 is coupled to the recognition of linker DNA by KDM5C. Our data suggests a model in which the PHD1 domain regulates DNA recognition by the ARID domain based on available substrate cues. In this model, recognition of distinct chromatin features is coupled to a conformational rearrangement of the ARID and PHD1 domains, which in turn modulates the positioning of the catalytic domain for efficient nucleosome demethylation. Importantly, we find that XLID mutations adjacent to the ARID and PHD1 domains alter the conformational state of these domains to enhance DNA binding. This results in the loss of specificity in chromatin recognition by KDM5C and renders catalytic activity sensitive to inhibition by linker DNA. Our findings suggest a unifying model by which XLID mutations alter chromatin recognition and enable euchromatin-specific dysregulation of demethylation by KDM5C.

Class I DISARM provides anti-phage and anti-conjugation activity by unmethylated DNA recognition

Bacteriophages impose a strong evolutionary pressure on microbes for the development of mechanisms of survival. Multiple new mechanisms of innate defense have been described recently, with the molecular mechanism of most of them remaining uncharacterized. Here, we show that a Class 1 DISARM (defense island system associated with restriction-modification) system from Serratia sp. provides broad protection from double-stranded DNA phages, and drives a population of single-stranded phages to extinction. We identify that protection is not abolished by deletion of individual DISARM genes and that the absence of methylase genes drmMI and drmMII does not result in autoimmunity. In addition to antiphage activity we also observe that DISARM limits conjugation, and this activity is linked to the number of methylase cognate sites in the plasmid. Overall, we show that Class 1 DISARM provides robust anti-phage and anti-plasmid protection mediated primarily by drmA and drmB, which provide resistance to invading nucleic acids using a mechanism enhanced by the recognition of unmethylated cognate sites of the two methylases drmMI and drmMII.

Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9

CRISPR-Cas9 is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system's function.

Differential Histone-DNA Interactions Dictate Nucleosome Recognition of the Pioneer Transcription Factor Sox

Pioneer transcription factors (PTFs) have the remarkable ability to directly bind to chromatin for stimulating vital cellular processes. Expanding on the recent findings, we aim to unravel the universal binding mode of the famous Sox PTF. Our findings show that the base specific hydrogen bonding (base reading) and the local DNA changes (shape reading) are required for sequence-specific nucleosomal DNA recognition by Sox. Among different nucleosomal positions, base and shape reading can be satisfied at super helical location 2 (SHL2). This indicates that due to distinct histone-DNA interactions, SHL2 acts transparently to Sox binding, where SHL4 permits solely shape reading, and SHL0 (dyad) allows no reading. We also show that at SHL2, Sox binds to its recognition sequence without imposing any major conformational changes, if its consensus DNA sequence is located at the solvent-facing nucleosomal DNA strand. These data explain how Sox have evolved to perfectly adapt for chromatin binding.

Coevolutionary methods enable robust design of modular repressors by reestablishing intra-protein interactions

Genetic sensors with unique combinations of DNA recognition and allosteric response can be created by hybridizing DNA-binding modules (DBMs) and ligand-binding modules (LBMs) from distinct transcriptional repressors. This module swapping approach is limited by incompatibility between DBMs and LBMs from different proteins, due to the loss of critical module-module interactions after hybridization. We determine a design strategy for restoring key interactions between DBMs and LBMs by using a computational model informed by coevolutionary traits in the LacI family. This model predicts the influence of proposed mutations on protein structure and function, quantifying the feasibility of each mutation for rescuing hybrid repressors. We accurately predict which hybrid repressors can be rescued by mutating residues to reinstall relevant module-module interactions. Experimental results confirm that dynamic ranges of gene expression induction were improved significantly in these mutants. This approach enhances the molecular and mechanistic understanding of LacI family proteins, and advances the ability to design modular genetic parts.

Enhanced Specificity Mutations Perturb Allosteric Signaling in CRISPR-Cas9

ABSTRACTCRISPR-Cas9 is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, solution NMR is combined with multi-microsecond molecular dynamics and graph theory-derived models to probe the allosteric role of key enhancement specificity mutations. We show that the mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the HNH domain allosteric structure, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric intercommunication. This differential perturbation of the allosteric signaling reflects the different capabilities of the single mutants to increase Cas9 specificity, with the mutation achieving the highest specificity also strongly perturbing the signaling transfer. These outcomes reveal that the allosteric regulation is critical for the specificity enhancement of the Cas9 enzyme, and are valuable to harness the signaling network to improve the system’s specificity.