Nuclear organization is dynamic and originates from the fundamental subunit of chromatin, the nucleosome. Post-translational modification of nucleosomal histones, particularly within intrinsically disordered histone tail regions, provides a dynamic regulatory mechanism of accessibility for chromatin-templated processes. While the epigenomic impacts of lysine acetylation and serine phosphorylation in the histone H3 tail are well-known, how these charge-altering post-translational modifications (PTMs) alter nucleosomal tail conformational dynamics remains incompletely characterized. Given that the functional implications of these PTMs are, at least in part, a consequence of modified nucleosome conformation, systematically cataloging the impact of histone PTMs on nucleosome dynamics provides crucial insight into both baseline cellular activity and epigenetic dysregulation that occurs in disease. Previously, our lab demonstrated that arginine citrullination mimetics lead to regional increases in H3 tail dynamics within nucleosome core particles. Here, we performed nuclear magnetic resonance spin relaxation experiments to investigate the effects of lysine acetylation and serine phosphorylation on H3 tail picosecond-nanosecond (ps-ns) dynamics. Using lysine-to-glutamine and serine-to-glutamate mutations as acetyllysine and phosphoserine mimetics, respectively, we found that these PTMs increase ps-ns conformational dynamics regionally around the PTM site, with a position-dependent effect. Additionally, we show that the type of PTM influences the extent of these increases: in general, the effect of mimetics trends in the order of phosphorylation ≤ acetylation < citrullination, suggesting a tunable method for altering histone tail dynamics. Taken together, these results illustrate the role of nucleosome conformational dynamics in conveying the effects of epigenomic PTMs, elucidating a mechanism of the histone language.

Citrullination is a charge-modifying post-translational modification whereby proteinogenic arginine is converted to the non-coded amino acid citrulline by calcium-activated protein arginine deiminases (PADs; EC 3.5.3.15). The five known PAD enzymes in humans (PADs 1, 2, 3, 4, and 6) are differentially expressed and have distinct targets, including histones. While some PAD histone citrullination sites are known, a comprehensive investigation of all histone tail arginines targeted by catalytically active PADs 1-4 is lacking. Here, we sought to identify PAD citrullination sites in histone tails, both within histone peptides and in reconstituted nucleosomes. Toward this objective, we utilized a real-time 1H-15N NMR spectroscopy-based assay. By monitoring both arginine and citrulline backbone amide peak intensities over time, we identified sites of citrullination in 15N-labeled histone tails within peptides and reconstituted nucleosome core particles. We found that PADs 1, 2, and 4 citrullinate all directly observable histone tail arginines to varying degrees. This is distinct from PAD3, which only moderately citrullinates H2A and H4 arginine residues and does not modify H3 tail arginines. Together, these data suggest a level of histone arginine specificity by each PAD. Furthermore, histone tail citrullination is altered within nucleosomes compared to isolated peptides, which we interpret to reflect changes in conformation and accessibility. We speculate that citrullination increases nucleosomal histone tail dynamics, with implications for crosstalk between sites of histone citrullination and other important sites of regulation by PTMs (including lysines) within and between tails.

Integral membrane proteins (IMPs) are fully embedded in the lipid bilayer and are often affected by their interactions with the surrounding lipids. Variation in the lipid environment can result in changes to hydrophobic matching, membrane lateral pressure, and electrostatic potentials, which can disrupt IMP folding and function. Here, we investigate the lipid-dependent behavior of the small multidrug resistance transporter EmrE. EmrE is an IMP native to E. coli and functions to expel toxic compounds from the bacterial cytoplasm. We know from previous studies that EmrE exhibits large variations in binding affinity, alternating access rate (the key step for transport of substrate across the membrane) and net transport rate depending on the transported substrate. Prior literature suggests that EmrE function may also be lipid dependent. In this study, we use NMR and isothermal titration calorimetry to study the impact of lipids on EmrE structure, substrate binding, and alternating access rate. We selected a set of lipids that vary in acyl chain saturation, acyl chain length, headgroup size, and charge. Our results show that EmrE retains highly similar structure, binding affinity, and alternating access rates across all lipid environments tested. Changes in binding enthalpy and entropy are observed in different lipids, but enthalpy-entropy compensation results in much smaller impacts on overall substrate affinity. Thus, despite its small size, key features of EmrE important for transport activity are only modestly perturbed by the lipid environment when substrate is present, with small-molecule substrates having a much greater impact on key steps in the EmrE transport cycle. Impacts of lipid on the transporter in the absence of small-molecule substrates may also affect EmrE transport activity, but are not examined here.

Centromeric chromatin is defined by the presence of the histone H3 variant CENP-A, which forms a specialized nucleosome required for kinetochore assembly. Compared to canonical H3 nucleosomes, CENP-A nucleosomes exhibit an open DNA conformation that leaves an additional 13 base pairs of DNA accessible at the entry and exit sites. While the CENP-A αN-helix has previously been implicated in promoting this enhanced DNA breathing, the contributions of the intrinsically disordered N-terminal tail and adjacent latch regions of CENP-A in nucleosome conformation remain unknown. The intrinsically disordered N-terminal regions of histone H3 are known to facilitate interactions with DNA to stabilize overall nucleosome conformation. Here, we systematically tested the contribution of each N-terminal histone region to maintaining H3 histones by utilizing a combination of MNase digestion assays and coarse-grained molecular dynamics simulations of H3/CENP-A chimera histone nucleosomes containing targeted swaps of the tail, latch, and αN-helix regions. Removal or substitution of individual H3 with CENP-A N-terminal regions increased DNA accessibility and nucleosome unwrapping. While any single CENP-A N-terminal region was sufficient to open the canonical nucleosomal DNA conformation, replacement of any single CENP-A N-terminal region with its H3 counterpart was insufficient to restore the wrapped DNA conformation characteristic of canonical H3 nucleosomes. Instead, progressive incorporation of multiple H3-derived regions produced increasingly closed DNA conformations, demonstrating that the H3 tail, latch, and αN-helices act cooperatively to stabilize wrapped nucleosomal DNA. Taken together, these findings demonstrate that the more restricted DNA breathing of canonical nucleosomes arises from coordinated contributions across multiple N-terminal regions and suggest that the multi-region redundancy in the conformational flexibility of the centromeric nucleosome could emphasize the importance of retaining flexibility in the centromeric nucleosome, even upon post-translational modification and binding to structural proteins.

Emerging models of nuclear organization suggest that chromatin forms functionally distinct microenvironments through phase separation. As chromatin architecture is organized at the level of the nucleosome and regulated by histone post-translational modifications, we investigated how these known regulatory mechanisms influence nucleosome phase behavior. By systematically altering charge distribution within the H3 tail, we found that the terminal and central regions modulate the phase boundary and tune nucleosome condensate viscosity differentially, as revealed by microscopy-based assays, microrheology, and simulations. Nuclear magnetic resonance relaxation experiments revealed that H3 tails remain dynamically mobile within condensates, and their mobility correlates with condensate viscosity. These results demonstrate that the number, identity, and spatial arrangement of basic residues in the H3 tail critically regulate nucleosome phase separation. Our findings support a model in which nucleosomes, through their intrinsic properties and modifications, actively shape the local chromatin microenvironment-providing new insight into the histone language in chromatin condensates.

The overexpression and mislocalization of the H3 histone variant centromeric protein A (CENP-A) is a predictive biomarker for several cancers and has been implicated in chromosomal instability. Despite these findings, the variant-specific interactions mediated by CENP-A within centromeric chromatin that contribute to the maintenance of genomic stability are not well understood at the molecular level. The basic subunit of chromatin is the nucleosome, which consists of DNA wrapped around a histone octamer core with intrinsically disordered terminal tail regions protruding from the core.

Chromatin is dynamically reorganized spatially and temporally, and the post-translational modification of histones is a key component of this regulation. The basic subunit of chromatin is the nucleosome core particle, consisting of two copies each of the histones H2A, H2B, H3, and H4 around which ∼147 base pairs of DNA wrap. The intrinsically disordered histone termini, or tails, protrude from the core and are heavily post-translationally modified. Previous studies have shown that the histone tails exist in dynamic ensembles of DNA-bound states within the nucleosome. Histone tail interactions with DNA are involved in nucleosome conformation and chromatin organization. Charge-modulating histone post-translational modifications (PTMs) are poised to perturb the dynamic interactions between histone tails and DNA. Arginine side chains form favorable interactions with DNA and are sites of charge-modulating PTMs such as citrullination. Our current focus is on the H3 tail, the longest histone tail. Four arginine residues are relatively evenly spaced along the H3 tail sequence, suggesting multivalent interactions with DNA poised for regulation by PTMs. In this study, we use NMR nuclear spin relaxation experiments to investigate the contribution of arginine residues to H3 tail dynamics within the nucleosome core particle. By neutralizing arginine via mutation to glutamine, we begin to work towards a comprehensive understanding of the contribution of individual residues to H3 tail dynamics. We find that neutralization of arginine residues results in increased regional mobility of the H3 tails, with implications for understanding the direct effects of arginine citrullination. Altogether, these studies support a role for dynamics within the histone language and emphasize the importance of charge-modulating histone PTMs in regulating chromatin dynamics, starting at the level of the basic subunit of chromatin.

Reconstitution of nucleosomes from recombinant histones and DNA is a widely used tool for studying nucleosome structure, dynamics, and interactions. Preparation of reconstituted nucleosomes allows for the study of nucleosomes with defined compositions. Here, we describe methods for refolding recombinant human histones, reconstituting nucleosome core particles with 147 bp Widom 601 DNA, and purification via sucrose gradient.

Chromatin organization and its dynamic regulation are crucial in governing the temporal and spatial accessibility of DNA for proper gene expression. Disordered chains of nucleosomes comprise the basis of eukaryotic chromatin, forming higher-level organization across a range of length scales. Models of chromatin organization involving phase separation driven by chromatin-associating proteins have been proposed. More recently, evidence has emerged that nucleosome arrays can phase separate in the absence of other protein factors, yet questions remain regarding the molecular basis of chromatin phase separation that governs this dynamic nuclear organization. Here, we break chromatin down into its most basic subunit, the nucleosome core particle, and investigate phase separation using turbidity assays in conjunction with differential interference contrast microscopy. We show that, at physiologically-relevant concentrations, this fundamental subunit of chromatin undergoes phase separation. Individually removing the H3 and H4 tails abrogates phase separation under the same conditions. Taking a reductionist approach to investigate H3 and H4 tail peptide interactions in-trans with DNA and nucleosome core particles supports the direct involvement of these tails in chromatin phase separation. These results provide insight into fundamental mechanisms underlying phase separation of chromatin, which starts at the level of the nucleosome core particle, and support that long-range inter-nucleosomal interactions are sufficient to drive phase separation at nuclear concentrations. Additionally, our data have implications for understanding crosstalk between histone tails and provide a lens through which to interpret the effect of histone post-translational modifications and sequence variants. STATEMENT OF SIGNIFICANCE: Emerging models propose that chromatin organization is based in phase separation, however, mechanisms that drive this dynamic nuclear organization are only beginning to be understood. Previous focus has been on phase separation driven by chromatin-associating proteins, but this has recently shifted to recognize a direct role of chromatin in phase separation. Here, we take a fundamental approach in understanding chromatin phase separation and present new findings that the basic subunit of chromatin, the nucleosome core particle, undergoes phase separation under physiological concentrations of nucleosome and monovalent salt. Furthermore, the histone H3 and H4 tails are involved in phase separation in a manner independent of histone-associating proteins. These data suggest that H3 and H4 tail epigenetic factors may modulate chromatin phase separation.

The BAF chromatin remodeling complex is critical for genome regulation. The central ATPase of BAF is either BRM or BRG1, both of which contain a C-terminal bromodomain, known to associate with acetylated lysines. We have recently demonstrated that in addition to acetyl-lysine binding, the BRG1/BRM bromodomain can associate with DNA through a lysine/arginine rich patch that is adjacent to the acetyl-lysine binding pocket. Flanking the bromodomain is an AT-hook separated by a short, proline-rich linker. We previously found that the AT-hook and bromodomain can associate with DNA in a multivalent manner. Here, we investigate the conservation of this composite module and find that the AT-hook, linker, and lysine/arginine rich bromodomain patch are ancient, conserved over ~1 billion years. We utilize extensive mutagenesis, NMR spectroscopy, and fluorescence anisotropy to dissect the contribution of each of these conserved elements in association of this module with DNA. Our results reveal a structural and functional coupling of the AT-hook and bromodomain mediated by the linker. The lysine/arginine rich patch on the bromodomain and the conserved elements of the AT-hook are critical for robust affinity for DNA, while the conserved elements of the linker are dispensable for overall DNA affinity but critical for maintaining the relative conformation of the AT-hook and bromodomain in binding to DNA. This supports that the coupled action of the AT-hook and bromodomain are important for BAF activity.

Hexasomes and tetrasomes are intermediates in nucleosome assembly and disassembly. Their formation is promoted by histone chaperones, ATP-dependent remodelers, and RNA polymerase II. In addition, hexasomes are maintained in transcribed genes and could be an important regulatory factor. While nucleosome composition has been shown to affect the structure and accessibility of DNA, its influence on histone tails is largely unknown. Here, we investigate the conformational dynamics of the H3 tail in the hexasome and tetrasome. Using a combination of NMR spectroscopy, MD simulations, and trypsin proteolysis, we find that the conformational ensemble of the H3 tail is regulated by nucleosome composition. As has been found for the nucleosome, the H3 tails bind robustly to DNA within the hexasome and tetrasome, but upon loss of the H2A/H2B dimer, we determined that the adjacent H3 tail has an altered conformational ensemble, increase in dynamics, and increase in accessibility. Similar to observations of DNA dynamics, this is seen to be asymmetric in the hexasome. Our results indicate that nucleosome composition has the potential to regulate chromatin signaling and ultimately help shape the chromatin landscape.

Accessibility of the human genome must be dynamically modulated for proper gene control. As such, the structure of chromatin undergoes continual temporal and spatial rearrangements during which nucleosomes, the basic structural unit of chromatin, are reorganized and DNA is repositioned around the nucleosome core. The canonical nucleosome consists of two copies each of the histones H2A, H2B, H3 and H4. Recent evidence suggests that subnucleosomal species lacking the standard number of histones exist in cells, at least as transcriptional and remodeling intermediates. Altering the composition of nucleosomes may serve as a mechanism of chromatin regulation. We recently found that the H3 tails exist in a dynamic ensemble of states within the nucleosome core particle, collapsed onto the core DNA. This results in inhibition of binding by a model histone reader domain in the absence of other nuclear factors. We sought to investigate the effects of removing one or both H2A/H2B dimers on the conformational ensemble of the H3 tails. In this study, we demonstrate that the conformation of the H3 tails is sensitive to the assembly state of the nucleosome core particle (NCP). Solution NMR studies demonstrate that the H3 tails experience distinct environments between octasomes (canonical nucleosomes), hexasomes (lacking one dimer), and tetrasomes (lacking both dimers). These studies additionally demonstrate that the accessibility of the H3 tails is influenced by the assembly state of the NCP. Altogether, these studies suggest that nucleosome assembly state is another mechanism of modulating histone tail conformation and accessibility, with implications in chromatin signaling and remodeling.

Regulation of gene expression is a complex process that involves the modification of histone proteins by epigenetic regulators and the formation of subnucleosomal particles such as hexasomes and tetrasomes. Moreover, the disordered tail region of histones harbor several post-translational modification sites for key epigenetic signals, and their conformation is central in determining their accessibility to histone-modifying enzymes. Despite several studies, the conformation and dynamics of tails in canonical and subnucleosomal particles remain elusive due to their intrinsically disordered nature. Using molecular dynamics simulations, we have studied the H3 tail dynamics in canonical and subnucleosomal particles. Results obtained from our study show that H3 tails are more compact in canonical than subnucleosomal particles due to DNA unwrapping. Although tails consistently collapsed onto nucleosomal DNA in simulations with two different water models, they were more extended and solvent accessible in OPC waters relative to TIP3P. Selection of appropriate water models for studying disordered tails in all-atom simulations is important to get detailed insights into the conformation of histone tails in intra and inter-nucleosome interactions and to elucidate the structural basis of chromatin condensation and epigenetic controls in the genome. Furthermore, we are combining our study with experiments to validate our computational findings on tail conformations in the context of canonical and subnucleosomal particles.

Chromatin is the form in which the eukaryotic genome is packaged into the cell nucleus, which regulates accessibility for all DNA templated processes. The nucleosome, which is the repeating subunit of chromatin, is composed of DNA wrapped around a histone octameric protein complex. It is at the level of the nucleosome that chromatin structure is modulated by chromatin regulators. Interactions between these regulators and the nucleosome are mediated by reader domains found in the chromatin regulatory complexes. Reader domain association is driven by a variety of covalent post-translational modifications (PTMs) found on histone proteins. The unstructured histone tails are known to be enriched in these PTMs. Histone tail/reader domain interactions have been widely investigated. However, studies have largely been conducted using peptide fragments to represent the histone tails, leaving a large gap in our knowledge of how reader domains recognize their substrate in the proper context of the nucleosome. Previously, our lab demonstrated that the association of the PHD finger of BPTF is inhibited by histone tail accessibility in the context of the nucleosome. Here, utilizing a novel nucleosome screen approach and NMR spectroscopy, we investigate further with the BPTF tandem domains, consisting of the PHD finger and a bromodomain. We find that, in addition to regulating the affinity of reader domains, the nucleosomal context also dictates the specificity of the reader domains. These studies provide a fundamental insight into the importance of investigating histone reader domains in the context of the nucleosome. Further, this will help us understand mechanisms of chromatin signaling and genome regulation, while providing us insight into a variety of epigenetic disease mechanisms.

BACKGROUND:Prostate adenocarcinoma (AdPC) progression to treatment-induced neuroendocrine prostate cancer (t-NEPC) is associated with poor patient survival. While AdPC and t-NEPC share similar genomes, they possess distinct transcriptomes, suggesting that RNA splicing and epigenetic mechanisms may regulate t-NEPC development. OBJECTIVE:To characterize the role of alternative RNA splicing of the histone demethylase BHC80 during t-NEPC progression. DESIGN, SETTING, AND PARTICIPANTS:The expression of BHC80 splice variants (BHC80-1 and BHC80-2) were compared between AdPC and t-NEPC patient tumors. Regulatory mechanisms of RNA splicing of the BHC80 gene were studied, and the signal pathways mediated by BHC80 splice variants were investigated in t-NEPC cell and xenograft models. RESULTS:Global transcriptome analyses identified that the BHC80-2 variant is highly expressed in t-NEPC. Compared with the known histone demethylation activities of the BHC80 gene, we discovered a novel nonepigenetic action of BHC80-2, whereby BHC80-2 is localized in the cytoplasm to trigger the MyD88-p38-TTP pathway, which results in increased RNA stability of multiple tumor-promoting cytokines. While BHC80-2 does not induce neuroendocrine differentiation of cancer cells, it stimulates cell proliferation and tumor progression independent of androgen receptor signaling. Blockade of BHC80-2-regulated MyD88 signaling suppresses growth of several t-NEPC cell spheroid and xenograft models. CONCLUSIONS:Gain of function of BHC80-2 through alternative RNA splicing activates immune responses of cancer cells to promote t-NEPC development. PATIENT SUMMARY:The main obstacle to develop effective therapies for patients with t-NEPC is the lack of understanding on how t-NEPC is developed. Our study not only identifies a previously unknown BHC80-2-MyD88 signaling pathway that plays an important role during t-NEPC development, but also provides a proof of principle that targeting this signal pathway may offer an avenue to treat t-NEPC.

Histone tails harbor a plethora of post-translational modifications (PTMs) that direct the function of chromatin regulators. Recognition of histone PTMs by these regulatory complexes is mediated through the action of reader sub-domains. The interaction of reader domains with modified histone tails has been extensively studied using peptide fragments of the tails. However, we have very little knowledge of how these domains associate with the full nucleosome. We are using NMR spectroscopy and complimentary orthogonal techniques to investigate this, and have found that the conformation of the histone tails in the context of the nucleosome has a dramatic effect on reader domain binding. As a model system, we are investigating the interaction of the BPTF PHD finger with its known cognate modification, methylated lysine 4 on histone H3 (H3K4me3). Here, we show that the conformation adopted by the histone H3 tails within the context of the nucleosome is inhibitory to binding of the BPTF PHD finger to H3K4me3, as compared to histone peptides. Using NMR spectroscopy and MD simulations, we find that the H3 tails interact robustly but dynamically with nucleosomal DNA, and demonstrate that this inhibits PHD finger association. Modifications and mutations of the H3 tail outside the binding region increase the accessibility to PHD finger binding, indicating that PTM crosstalk can regulate reader domain binding by altering the nucleosome conformation. Together, our results demonstrate that the nucleosome context has a dramatic impact on signaling events at the histone tails, and highlights the importance of studying histone binding in the context of the nucleosome.