H3 N-terminal Tail Research Articles

MMP-9 drives the melanomagenic transcription program through histone H3 tail proteolysis.

Melanoma is a type of skin cancer that develops in pigment-producing melanocytes and often spreads to other parts of the body. Aberrant gene expression has been considered as a crucial step for increasing the risk of melanomagenesis, but how chromatin reorganization contributes to this pathogenic process is still not well understood. Here we report that matrix metalloproteinase 9 (MMP-9) localizes to the nucleus of melanoma cells and potentiates gene expression by proteolytically clipping the histone H3 N-terminal tail (H3NT). From genome-wide studies, we discovered that growth-regulatory genes are selectively targeted and activated by MMP-9-dependent H3NT proteolysis in melanoma cells. MMP-9 cooperates functionally with p300/CBP because MMP-9 cleaves H3NT in a manner that is dependent on p300/CBP-mediated acetylation of H3K18. The functional significance of MMP-9-dependent H3NT proteolysis is further underscored by the fact that RNAi knockdown and small-molecule inhibition of MMP-9 and p300/CBP impede melanomagenic gene expression and melanoma tumor growth. Together, our data establish new functions and mechanisms for nuclear MMP-9 in promoting melanomagenesis and demonstrate how MMP-9-dependent H3NT proteolysis can be exploited to prevent and treat melanoma skin cancer.

Dynamic chromatin association of IκBα is regulated by acetylation and cleavage of histone H4.

IκBs exert principal functions as cytoplasmic inhibitors of NF-kB transcription factors. Additional roles for IκB homologues have been described, including chromatin association and transcriptional regulation. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds to histones H2A and H4 in the stem cell and progenitor cell compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unknown. Here, we show that serine 32-36 phosphorylation of IκBα favors its binding to nucleosomes and demonstrate that p-IκBα association with H4 depends on the acetylation of specific H4 lysine residues. The N-terminal tail of H4 is removed during intestinal cell differentiation by proteolytic cleavage by trypsin or chymotrypsin at residues 17-19, which reduces p-IκBα binding. Inhibition of trypsin and chymotrypsin activity in HT29 cells increases p-IκBα chromatin binding but, paradoxically, impaired goblet cell differentiation, comparable to IκBα deletion. Taken together, our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular basis for the understanding of the restricted nuclear distribution of p-IκBα in specific stem cell compartments.

Conformational Dynamics of Histone H3 Tails in Chromatin.

Chromatin is a supramolecular DNA-protein complex that compacts eukaryotic genomes and regulates their accessibility and functions. Dynamically disordered histone H3 N-terminal tails are among key chromatin regulatory components. Here, we used high-resolution-magic-angle-spinning NMR measurements of backbone amide 15N spin relaxation rates to investigate, with residue-specific detail, the dynamics and interactions of H3 tails in recombinant 13C,15N-enriched nucleosome arrays containing 15, 30, or 60 bp linker DNA between the nucleosome repeats. These measurements were compared to analogous data available for mononucleosomes devoid of linker DNA or containing two 20 bp DNA overhangs. The H3 tail dynamics in nucleosome arrays were found to be considerably attenuated compared with nucleosomes with or without linker DNA due to transient electrostatic interactions with the linker DNA segments and the structured chromatin environment. Remarkably, however, the H3 tail dynamics were not modulated by the specific linker DNA length within the 15-60 bp range investigated here.

Histone H3 N-terminal mimicry drives a novel network of methyl-effector interactions.

The reader ability of PHD fingers is largely limited to the recognition of the histone H3 N-terminal tail. Distinct subsets of PHDs bind either H3K4me3 (a transcriptional activator mark) or H3K4me0 (a transcriptional repressor state). Structural studies have identified common features among the different H3K4me3 effector PHDs, including (1) removal of the initiator methionine residue of H3 to prevent steric interference, (2) a groove where arginine-2 binds, and (3) an aromatic cage that engages methylated lysine-4. We hypothesize that some PHDs might have the ability to engage with non-histone ligands, as long as they adhere to these three rules. A search of the human proteome revealed an enrichment of chromatin-binding proteins that met these criteria, which we termed H3 N-terminal mimicry proteins (H3TMs). Seven H3TMs were selected, and used to screen a protein domain microarray for potential effector domains, and they all had the ability to bind H3K4me3-interacting effector domains. Furthermore, the binding affinity between the VRK1 peptide and the PHD domain of PHF2 is ∼3-fold stronger than that of PHF2 and H3K4me3 interaction. The crystal structure of PHF2 PHD finger bound with VRK1 K4me3 peptide provides a molecular basis for stronger binding of VRK1 peptide. In addition, a number of the H3TMs peptides, in their unmethylated form, interact with NuRD transcriptional repressor complex. Our findings provide in vitro evidence that methylation of H3TMs can promote interactions with PHD and Tudor domain-containing proteins and potentially block interactions with the NuRD complex. We propose that these interactions can occur in vivo as well.

MMP-2 is a novel histone H3 N-terminal protease necessary for myogenic gene activation

BackgroundSelective proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during eukaryotic development, generating a cleaved histone H3 (H3cl) product within a small, but significant, portion of the genome. Although increasing evidence supports a regulatory role for H3NT proteolysis in gene activation, the nuclear H3NT proteases and the biological significance of H3NT proteolysis remain largely unknown.ResultsIn this study, established cell models of skeletal myogenesis were leveraged to investigate H3NT proteolysis. These cells displayed a rapid and progressive accumulation of a single H3cl product within chromatin during myoblast differentiation. Using conventional approaches, we discovered that the canonical extracellular matrix (ECM) protease, matrix metalloproteinase 2 (MMP-2), is the principal H3NT protease of myoblast differentiation that cleaves H3 between K18-Q19. Gelatin zymography demonstrated progressive increases in nuclear MMP-2 activity, concomitant with H3cl accumulation, during myoblast differentiation. RNAi-mediated depletion of MMP-2 impaired H3NT proteolysis and resulted in defective myogenic gene activation and myoblast differentiation. Supplementation of MMP-2 ECM activity in MMP-2-depleted cells was insufficient to rescue defective H3NT proteolysis and myogenic gene activation.ConclusionsThis study revealed that MMP-2 is a novel H3NT protease and the principal H3NT protease of myoblast differentiation. The results indicate that myogenic signaling induces MMP-2-dependent H3NT proteolysis at early stages of myoblast differentiation. Importantly, the results support the necessity of nuclear MMP-2 H3NT protease activity, independent of MMP-2 activity in the ECM, for myogenic gene activation and proficient myoblast differentiation.

Combinatorial Histone H3 Modifications Are Dynamically Altered in Distinct Cell Cycle Phases.

The cell cycle is a highly regulated and evolutionary conserved process that results in the duplication of cell content and the equal distribution of the duplicated chromosomes into a pair of daughter cells. Histones are fundamental structural components of chromatin in eukaryotic cells, and their post-translational modifications (PTMs) benchmark DNA readout and chromosome condensation. Aberrant regulation of the cell cycle associated with dysregulation of histone PTMs is the cause of critical diseases such as cancer. Monitoring changes of histone PTMs could pave the way to understanding the molecular mechanisms associated with epigenetic regulation of cell proliferation. Previously, our lab established a novel middle-down workflow using porous graphitic carbon (PGC) as a stationary phase to analyze histone PTMs, which utilizes the same reversed-phase chromatography for gradient separation as canonical proteomics coupled with online mass spectrometry (MS). Here, we applied this novel workflow for high-throughput analysis of histone modifications of H3.1 and H3.2 during the cell cycle. Collectively, we identified 1133 uniquely modified canonical histone H3 N-terminal tails. Consistent with previous findings, histone H3 phosphorylation increased significantly during the mitosis (M) phase. Histone H3 variant-specific and cell-cycle-dependent expressions of PTMs were observed, underlining the need to not combine H3.1 and H3.2 together as H3. We confirmed previously known H3 PTM crosstalk (e.g., K9me-S10ph) and revealed new information in this area as well. These findings imply that the combinatorial PTMs play a role in cell cycle control, and they may serve as markers for proliferation.

Intestinal differentiation involves cleavage of histone H3 N-terminal tails by multiple proteases.

The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.

DIPG-39. NOVEL PROTEOMIC ANALYSIS REVEALS EPIGENETIC THERAPEUTIC TARGETS IN PEDIATRIC GLIOMA

INTRODUCTIONDiffuse midline glioma is a highly morbid pediatric cancer. Up to 80% harbor Histone H3K27M mutation, which alters Histone H3 post-translational modifications (PTMs) and genomic enrichment patterns, affecting chromatin structure and transcription. We previously identified tumorigenic patterns of H3K27Ac/bromodomain co-enrichment and pre-clinical efficacy of bromodomain inhibition (JQ1) in DMG. Here, we employ a novel proteomics approach developed at our institution to further elucidate the impact of H3K27M mutation on glioma epigenetic signatures and treatment response.METHODSEpiproteomic analysis was performed on pediatric glioma cells (H3K27 WT n=2, H3K27M n=2) to characterize 95 distinct Histone H3 N-terminal tail modification states. Cells were treated with JQ1 or DMSO, and collected at 0h, 24h, 48h, Histones extracted from isolated nuclei and immunopurified, then analyzed by LC-MS/MS. Results were integrated with RNA-Seq and ChIP Seq (H3.3K27M, H3.3, H3K27Ac, H3K27me3, H3K4me1, H3K4me3) from the same cell lines. Pediatric glioma tissues (H3K27M WT n=3, H3K27M n= 9) were similarly analyzed to validate cell line results.RESULTSCell PTM profiles cluster by H3 mutation status on unsupervised analysis. Significant differential PTM abundance and genomic enrichment H3K27M, H3.3 WT, H3K27Me3 and H3K27Ac was observed between mutant and wild type cell lines with epigenetic-targeted therapy, correlating with cell transcriptomes.CONCLUSIONSHistone H3 tail analysis reveals the effects of H3K27M mutation and bromodomain inhibition on the tumor epigenetic landscape, providing insight into mechanisms of tumorigenesis and therapy response. Further investigation of the utility of these signatures as biomarkers for diagnosis and monitoring treatment response are therefore underway.

Distinct Structures and Dynamics of Chromatosomes with Different Human Linker Histone Isoforms

The repeating structural unit of metazoan chromatin is the chromatosome, a nucleosome bound to a linker histone, H1. There are 11 human H1 isoforms with diverse cellular functions, but how they interact with the nucleosome remains elusive. Here, we determined the cryoelectron microscopy (cryo-EM) structures of chromatosomes containing 197bp DNA and three different human H1 isoforms, respectively. The globular domains of all three H1 isoforms bound to the nucleosome dyad. However, the flanking/linker DNAs displayed substantial distinct dynamic conformations. Nuclear magnetic resonance (NMR) and H1 tail-swapping cryo-EM experiments revealed that the C-terminal tails of the H1 isoforms mainly controlled the flanking DNA orientations. We also observed partial ordering of the core histone H2A C-terminal and H3 N-terminal tails in the chromatosomes. Our results provide insights into the structures and dynamics of the chromatosomes and have implications for the structure and function of chromatin.

Acetylation-modulated communication between the H3 N-terminal tail domain and the intrinsically disordered H1 C-terminal domain.

Linker histones (H1s) are key structural components of the chromatin of higher eukaryotes. However, the mechanisms by which the intrinsically disordered linker histone carboxy-terminal domain (H1 CTD) influences chromatin structure and gene regulation remain unclear. We previously demonstrated that the CTD of H1.0 undergoes a significant condensation (reduction of end-to-end distance) upon binding to nucleosomes, consistent with a transition to an ordered structure or ensemble of structures. Here, we show that deletion of the H3 N-terminal tail or the installation of acetylation mimics or bona fide acetylation within H3 N-terminal tail alters the condensation of the nucleosome-bound H1 CTD. Additionally, we present evidence that the H3 N-tail influences H1 CTD condensation through direct protein-protein interaction, rather than alterations in linker DNA trajectory. These results support an emerging hypothesis wherein the H1 CTD serves as a nexus for signaling in the nucleosome.

Partial Replacement of Nucleosomal DNA with Human FACT Induces Dynamic Exposure and Acetylation of Histone H3 N-Terminal Tails.

SummaryThe FACT (facilitates chromatin transcription) complex, comprising SPT16 and SSRP1, conducts structural alterations during nucleosome unwrapping. Our previous cryoelectron microscopic (cryo-EM) analysis revealed the first intermediate structure of an unwrapped nucleosome with human FACT, in which 112-bp DNA and the phosphorylated intrinsically disordered (pAID) segment of SPT16 jointly wrapped around the histone core instead of 145-bp DNA. Using NMR, here we clarified that the histone H3 N-terminal tails, unobserved in the cryo-EM structure, adopt two different conformations reflecting their asymmetric locations at entry/exit sites: one corresponds to the original nucleosome site buried in two DNA gyres (DNA side), whereas the other, comprising pAID and DNA, is more exposed to the solvent (pAID side). NMR real-time monitoring showed that H3 acetylation is faster on the pAID side than on the DNA side. Our findings highlight that accessible conformations of H3 tails are created by the replacement of nucleosomal DNA with pAID.

Deacetylation of H4 lysine16 affects acetylation of lysine residues in histone H3 and H4 and promotes transcription of constitutive genes

ABSTRACT Histone modification map of H4 N-terminal tail residues in Saccharomyces cerevisiae reveals the prominence of lysine acetylation. Previous reports have indicated the importance of lysine acetylation in maintaining chromatin structure and function. H4K16, a residue with highly regulated acetylation dynamics has unique functions not overlapping with the other H4 N- terminal acetylable residues. The present work unravels the role of H4K16 acetylation in regulating expression of constitutive genes. H4K16 gets distinctly deacetylated over the coding region of constitutively expressed genes. Deacetylation of H4K16 reduces H3K9 acetylation at the cellular and gene level. Reduced H3K9 acetylation however did not negatively correlate with active gene transcription. Significantly, H4K16 deacetylation was found to be associated with hypoacetylated H4K12 throughout the locus of constitutive genes. H4K16 and K12 deacetylation is known to favour active transcription. Sas2, the HAT mutant showed similar patterns of hypoacetylated H3K9 and H4K12 at the active loci, clearly implying that the modifications were associated with deacetylation state of H4K16. Deacetylation of H4K16 was also concurrent with increased H3K56 acetylation in the promoter region and ORF of the constitutive genes. Combination of all these histone modifications significantly reduced H3 occupancy, increased promoter accessibility and enhanced RNAPII recruitment at the constitutively active loci. Consequently, we found that expression of active genes was higher in H4K16R mutant which mimic deacetylated state, but not in H4K16Q mimicking constitutive acetylation. To summarize, H4K16 deacetylation linked with H4K12 and H3K9 hypoacetylation along with H3K56 hyperacetylation generate a chromatin landscape that is conducive for transcription of constitutive genes.

Characterization of the plant homeodomain (PHD) reader family for their histone tail interactions

BackgroundPlant homeodomain (PHD) fingers are central “readers” of histone post-translational modifications (PTMs) with > 100 PHD finger-containing proteins encoded by the human genome. Many of the PHDs studied to date bind to unmodified or methylated states of histone H3 lysine 4 (H3K4). Additionally, many of these domains, and the proteins they are contained in, have crucial roles in the regulation of gene expression and cancer development. Despite this, the majority of PHD fingers have gone uncharacterized; thus, our understanding of how these domains contribute to chromatin biology remains incomplete.ResultsWe expressed and screened 123 of the annotated human PHD fingers for their histone binding preferences using reader domain microarrays. A subset (31) of these domains showed strong preference for the H3 N-terminal tail either unmodified or methylated at H3K4. These H3 readers were further characterized by histone peptide microarrays and/or AlphaScreen to comprehensively define their H3 preferences and PTM cross-talk.ConclusionsThe high-throughput approaches utilized in this study establish a compendium of binding information for the PHD reader family with regard to how they engage histone PTMs and uncover several novel reader domain–histone PTM interactions (i.e., PHRF1 and TRIM66). This study highlights the usefulness of high-throughput analyses of histone reader proteins as a means of understanding how chromatin engagement occurs biochemically.

Proteomic Analysis of Saccharomyces cerevisiae Response to Oxidative Stress Mediated by Cocoa Polyphenols Extract.

The present study addressed the protective effects against oxidative stress (OS) of a cocoa powder extract (CPEX) on the protein expression profile of S. cerevisiae. A proteomic analysis was performed after culture preincubation with CPEX either without stress (−OS) or under stress conditions (+OS) (5 mM of H2O2). LC-MS/MS identified 33 differentially expressed proteins (–OS: 14, +OS: 19) that were included By Gene Ontology analysis in biological processes: biosynthesis of amino acids, carbohydrate metabolism and reactive oxygen species metabolic process. In a gene-knockout strains study, eight proteins were identified as putative candidates for being involved in the protective mechanism of cocoa polyphenols against OS induced by H2O2. CPEX was able to exert its antioxidant activity in yeast mainly through the regulation of: (a) amino acids metabolism proteins by modulating the production of molecules with known antioxidant roles; (b) stress-responsive protein Yhb1, but we were unable to fully understand its down-regulation; (c) protein Prb1, which can act by clipping Histone H3 N-terminal tails that are related to cellular resistance to DNA damaging agents.

Dynamics of the nucleosomal histone H3 N-terminal tail revealed by high precision single-molecule FRET.

Chromatin compaction and gene accessibility are orchestrated by assembly and disassembly of nucleosomes. Although the disassembly process was widely studied, little is known about the structure and dynamics of the disordered histone tails, which play a pivotal role for nucleosome integrity. This is a gap filling experimental FRET study from the perspective of the histone H3 N-terminal tail (H3NtT) of reconstituted mononucleosomes. By systematic variation of the labeling positions we monitored the motions of the H3NtT relative to the dyad axis and linker DNA. Single-molecule FRET unveiled that H3NtTs do not diffuse freely but follow the DNA motions with multiple interaction modes with certain permitted dynamic transitions in the μs to ms time range. We also demonstrate that the H3NtT can allosterically sense charge-modifying mutations within the histone core (helix α3 of histone H2A (R81E/R88E)) resulting in increased dynamic transitions and lower rate constants. Those results complement our earlier model on the NaCl induced nucleosome disassembly as changes in H3NtT configurations coincide with two major steps: unwrapping of one linker DNA and weakening of the internal DNA - histone interactions on the other side. This emphasizes the contribution of the H3NtT to the fine-tuned equilibrium between overall nucleosome stability and DNA accessibility.

Structural Basis of H2B Ubiquitination-Dependent H3K4 Methylation by COMPASS.

The COMPASS (complex of proteins associated with Set1) complex represents the prototype of the SET1/MLL family of methyltransferases that controls gene transcription by H3K4 methylation (H3K4me). Although H2B monoubiquitination (H2Bub) is well known as a prerequisite histone mark for COMPASS activity, how H2Bub activates COMPASS remains unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of an extended COMPASS catalytic module (CM) bound to the H2Bub and free nucleosome. The COMPASS CM clamps onto the nucleosome disk-face via an extensive interface to capture the flexible H3 N-terminal tail. The interface also sandwiches a critical Set1 arginine-rich motif (ARM) that autoinhibits COMPASS. Unexpectedly, without enhancing COMPASS-nucleosome interaction, H2Bub activates the enzymatic assembly by packing against Swd1 and alleviating the inhibitory effect of the Set1 ARM upon fastening it to the acidic patch. By delineating the spatial configuration of theCOMPASS-H2Bub-nucleosome assembly, our studies establish the structural framework for understanding the long-studied H2Bub-H3K4me histone modification crosstalk.

Double Monoubiquitination Modifies the Molecular Recognition Properties of p15PAF Promoting Binding to the Reader Module of Dnmt1.

The proliferating cell nuclear antigen (PCNA)-associated factor p15PAF is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer, and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15PAF is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its N-terminal tail. Here we present an NMR conformational characterization of p15PAF monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15PAF is monomeric, intrinsically disordered, and binds to PCNA as nonubiquitinated p15PAF does but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15PAF shows that the ubiquitin moieties, separated by eight disordered residues, form transient dimers because of the high local effective ubiquitin concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15PAF is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15PAF directly interacts with PCNA and recruits Dnmt1 for maintenance of DNA methylation during replication.

Role of Fine Structural Dynamics in Recognition of Histone H3 by HP1γ(CSD) Dimer and Ability of Force Fields to Describe Their Interaction Network.

Human heterochromatin protein 1 (HP1) is a key factor in heterochromatin formation and maintenance. Its chromo-shadow domain (CSD) homodimerizes, and the HP1 dimer acts as a hub, transiently interacting with diverse binding partner (BP) proteins. We analyze atomistic details of interactions of the HP1γ(CSD) dimer with one of its targets, the histone H3 N-terminal tail, using molecular dynamics (MD) simulations. The goal is to complement the available X-ray crystallography data and unravel potential dynamic effects in the molecular recognition. Our results suggest that HP1(CSD)-BP recognition involves structural dynamics of both partners, including structural communication between adjacent binding pockets that may fine-tune the sequence recognition. For example, HP1 Trp174 sidechain substates may help in distinguishing residues bound in the conserved HP1(CSD) ±2 hydrophobic pockets. Further, there is intricate competition between the binding of negatively charged HP1 C-terminal extension and solvent anions near the ±2 hydrophobic pockets, which is also influenced by the BP sequence. Phosphorylated H3 Y41 can interact with the same site. We also analyze the ability of several pair-additive force fields to describe the protein-protein interface. ff14SB and ff99SB-ILDN* provide the closest correspondence with the crystallographic model. The ff15ipq local dynamics are somewhat less consistent with details of the experimental structure, while larger perturbations of the interface commonly occur in CHARMM36m simulations. The balance of some interactions, mainly around the anion binding site, also depends on the ion parameters. Some differences between the simulated and experimental structures are attributable to crystal packing.

Structure of the primed state of the ATPase domain of chromatin remodeling factor ISWI bound to the nucleosome.

ATP-dependent chromatin remodeling factors of SWI/SNF2 family including ISWI, SNF2, CHD1 and INO80 subfamilies share a conserved but functionally non-interchangeable ATPase domain. Here we report cryo-electron microscopy (cryo-EM) structures of the nucleosome bound to an ISWI fragment with deletion of the AutoN and HSS regions in nucleotide-free conditions and the free nucleosome at ∼ 4 Å resolution. In the bound conformation, the ATPase domain interacts with the super helical location 2 (SHL 2) of the nucleosomal DNA, with the N-terminal tail of H4 and with the α1 helix of H3. Density for other regions of ISWI is not observed, presumably due to disorder. Comparison with the structure of the free nucleosome reveals that although the histone core remains largely unchanged, remodeler binding causes perturbations in the nucleosomal DNA resulting in a bulge near the SHL2 site. Overall, the structure of the nucleotide-free ISWI-nucleosome complex is similar to the corresponding regions of the recently reported ADP bound ISWI-nucleosome structures, which are significantly different from that observed for the ADP-BeFx bound structure. Our findings are relevant to the initial step of ISWI binding to the nucleosome and provide additional insights into the nucleosome remodeling process driven by ISWI.

Histone chaperone exploits intrinsic disorder to switch acetylation specificity

Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.