Nucleosomal Arrays Research Articles

Probing voltage-dependent stability of nucleosomes using glass nanopores.

The packaging of DNA in the form of chromatin helps the cell to compact and regulate its genetic material. It is known that the nucleosomes exist in various structural and positional combinations that leads to chromatin assuming locally folded structures. This allows for its efficient packaging and also helps in the gene regulation. In this work, using resistive pulse technique with our glass nanopores, we have investigated the voltage dependent stability of mono-nucleosomes as well as the 12-mer nucleosome arrays. In experiments with our in vitro assembled mononucleosomes, we show that the octasomal nucleosomes, comprising of octameric histone complexes on DNA, systematically breakdown into its partial structures, which we reason to be hexasomes and tetrasomes, in a voltage dependent manner. We attribute these structural changes to the opposing electrical forces experienced by the positively charged histone protein complex and the negatively charged DNA. We next investigate the stability of 12-mer nucleosome arrays and find discrete multi-level events corresponding to discrete structures on the array molecules. A detailed analysis of these discrete levels revealed that these structures correspond to single, multiple as well as partial nucleosomes on the array molecule. We further show that these structures disassemble in a voltage dependent manner. Our work demonstrates, for the first time, application of the glass nanopore platform to perform stability studies on nucleosome-DNA complexes, both individually as well as on nucleosomal arrays. This opens up future studies to evaluate force dependent structural transitions in the context of epigenetic control of chromatin structure.

Nucleosome chaperone activity of LEDGF and HDGF2 characterized with single molecule force spectroscopy.

The nucleosome, two turns of DNA wrapped around a histone octamer, is the fundamental structure of DNA organization in eukaryotic cells. Lens epithelium-derived growth factor (LEDGF) and hepatoma-derived growth factor 2 (HDGF2) are recently discovered proteins that facilitate transcription without ATP hydrolysis, suggesting roles as nucleosome chaperones during the integration of lentiviral DNA, including HIV-1 DNA, into infected cell genomes. Both proteins have methyl-lysine reading PWWP domains that can recognize H3K36me2/3 marks. To better understand these activities, we used optical tweezers to stretch reconstituted DNA nucleosome arrays containing twelve Widom 601 positioning sequences with unmodified (WT) and methylated histones (mimicking the H3K36me2/3 modifications), in the presence of LEDGF and HDGF2. To test the function of these nucleosome chaperone proteins during HIV-1 DNA integration, we exposed the modified nucleosomes to HIV-1 integrase (IN) together with equal concentrations of LEDGF and HDGF2. Analysis of force-extension data showed that while some destabilization of the inner wrapping of nucleosomes occurs due to histone methylation, exposure to chaperones results in further destabilization. Notably, the outer half turn unwrapping of nucleosomes that typically occurs at lower forces disappears with chaperone binding. Kinetic analysis of histone-DNA interactions in the presence of these chaperones via survival probability and confocal fluorescence measurements indicates that LEDGF not only facilitates nucleosome unwrapping but also moderately promotes nucleosome reassembly. These results offer insight into the functions of LEDGF and HDGF2 as nucleosome chaperone proteins and their effect on IN binding to the nucleosome array.

Tuning the phase separation properties of HP1α through ligand interactions.

Heterochromatin protein 1α (HP1α) is a crucial component for gene repression and the structural integrity of heterochromatin. It is proposed that HP1α functions through a liquid-liquid phase separation (LLPS) mechanism where HP1α compacts nucleosome arrays into a liquid compartment and prevents transcription factors from accessing chromatin. HP1α can undergo LLPS via phosphorylation of the N-terminus extension (NTE) or through interactions with DNA/nucleosomes. The multidomain architecture of HP1α allows for simultaneous multivalent interactions with other HP1α proteins and multiple binding partners. These multivalent interactions are believed to be important for forming and maintaining heterochromatin structure but their exact role in HP1α LLPS is not well understood. In our study, we used experimental and computational approaches to investigate how a series of peptides from HP1α binding partners can fine tune the ability of the protein to undergo phase separation. Our results showed that in phosphorylation-driven LLPS, positively charged peptides enhance phase separation, while negatively or near neutral charged peptides disrupt the process. DNA-driven LLPS can also be inhibited by negatively or near neutral charged ligands. However, in this case, positively charged peptides compete with HP1α for DNA interactions, which also disrupts LLPS. Our results suggest that phosphorylation-driven LLPS has a larger tunability range compared to phase separation with DNA, and that the electrostatic properties of HP1α ligands play an important role in modulating this process. Our study provides insights into the potential mechanisms of heterochromatin regulation where binding partners may modulate the biophysical properties of HP1α to control access to chromatin and the genetic information in the cell.

Histone H3 core domain in chromatin with different DNA linker lengths studied by 1H-Detected solid-state NMR spectroscopy.

Chromatin, a dynamic protein-DNA complex that regulates eukaryotic genome accessibility and essential functions, is composed of nucleosomes connected by linker DNA with each nucleosome consisting of DNA wrapped around an octamer of histones H2A, H2B, H3 and H4. Magic angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy can yield unique insights into histone structure and dynamics in condensed nucleosomes and nucleosome arrays representative of chromatin at physiological concentrations. Recently we used J-coupling-based solid-state NMR methods to investigate with residue-specific resolution the conformational dynamics of histone H3 N-terminal tails in 16-mer nucleosome arrays containing 15, 30 or 60 bp DNA linkers. Here, we probe the H3 core domain in the 16-mer arrays as a function of DNA linker length via dipolar coupling-based 1H-detected solid-state NMR techniques. Specifically, we established nearly complete assignments of backbone chemical shifts for H3 core residues in arrays with 15-60 bp DNA linkers reconstituted with 2H,13C,15N-labeled H3. Overall, these chemical shifts were similar irrespective of the DNA linker length indicating no major changes in H3 core conformation. Notably, however, multiple residues at the H3-nucleosomal DNA interface in arrays with 15 bp DNA linkers exhibited relatively pronounced differences in chemical shifts and line broadening compared to arrays with 30 and 60 bp linkers. These findings are consistent with increased heterogeneity in nucleosome packing and structural strain within arrays containing short DNA linkers that likely leads to side-chains of these interfacial residues experiencing alternate conformations or shifts in their rotamer populations relative to arrays with the longer DNA linkers.

A pioneer factor locally opens compacted chromatin to enable targeted ATP-dependent nucleosome remodeling.

To determine how different pioneer transcription factors form a targeted, accessible nucleosome within compacted chromatin and collaborate with an ATP-dependent chromatin remodeler, we generated nucleosome arrays in vitro with a central nucleosome containing binding sites for the hematopoietic E-Twenty Six (ETS) factor PU.1 and Basic Leucine Zipper (bZIP) factors C/EBPα and C/EBPβ. Our long-read sequencing reveals that each factor can expose a targeted nucleosome on linker histone-compacted arrays, but with different nuclease sensitivity patterns. The DNA binding domain of PU.1 binds mononucleosomes, but requires an additional intrinsically disordered domain to bind and open compacted chromatin. The canonical mammalian SWI/SNF (cBAF) remodeler was unable to act upon two forms of locally open chromatin unless cBAF was enabled by a separate transactivation domain of PU.1. cBAF potentiates the PU.1 DNA binding domain to weakly open chromatin in the absence of the PU.1 disordered domain. Our findings reveal a hierarchy by which chromatin is opened and show that pioneer factors can provide specificity for action by nucleosome remodelers.

Beyond Sequence: Internucleosomal Interactions Dominate Array Assembly.

The organization of the nucleosome array is a critical component of the chromatin assembly into higher order structure as well as its function. Here, we investigated the contributions of the DNA sequence and internucleosomal interactions on the organization of the nucleosomal arrays in compact structures using atomic force microscopy. We assembled nucleosomes on DNA substrates allowing for the formation of tetranucleosomes. We found that nucleosomes are capable of close positioning with no discernible space between them, even in the case of assembled dinucleosomes. This morphology of the array is in contrast with that observed for arrays assembled with repeats of the nucleosome positioning motifs separated by uniform spacers. Simulated assembly of tetranucleosomes by random placement along the substrates revealed that nucleosome array compaction is promoted by the interaction of the nucleosomes. We developed a theoretical model to account for the role of DNA sequence and internucleosomal interactions in the formation of the nucleosome structures. These findings suggest that, in the chromatin assembly, the affinity of the nucleosomes to the DNA sequence and the strengths of the internucleosomal interactions are the two major factors defining the compactness of the chromatin.

Nanopore Sensing of DNA-Histone Complexes on Nucleosome Arrays.

The location of nucleosomes in DNA and their structural stability are critical in regulating DNA compaction, site accessibility, and epigenetic gene regulation. Here, we combine the nanopore platform-based fast and label-free single-molecule detection technique with a voltage-dependent force rupture assay to detect distinct structures on nucleosomal arrays and then to induce breakdown of individual nucleosome complexes. Specifically, we demonstrate direct measurement of distinct nucleosome structures present on individual 12-mer arrays. A detailed event analysis showed that nucleosomes are present as a combination of complete and partial structures, during translocation through the pore. By comparing with the voltage-dependent translocation of the mononucleosomes, we find that the partial nucleosomes result from voltage-dependent structural disintegration of nucleosomes. High signal-to-noise detection of heterogeneous levels in translocation of 12-mer array molecules quantifies the heterogeneity and nucleosomal substructure sizes on the arrays. These results facilitate the understanding of electrostatic interactions responsible for the integrity of the nucleosome structure and possible mechanisms of its unraveling by chromatin remodeling enzymes. This study also has potential applications in chromatin profiling.

Chromatin Liquid-Liquid Phase Separation (LLPS) Is Regulated by Ionic Conditions and Fiber Length.

The dynamic regulation of the physical states of chromatin in the cell nucleus is crucial for maintaining cellular homeostasis. Chromatin can exist in solid- or liquid-like forms depending on the surrounding ions, binding proteins, post-translational modifications and many other factors. Several recent studies suggested that chromatin undergoes liquid–liquid phase separation (LLPS) in vitro and also in vivo; yet, controversial conclusions about the nature of chromatin LLPS were also observed from the in vitro studies. These inconsistencies are partially due to deviations in the in vitro buffer conditions that induce the condensation/aggregation of chromatin as well as to differences in chromatin (nucleosome array) constructs used in the studies. In this work, we present a detailed characterization of the effects of K+, Mg2+ and nucleosome fiber length on the physical state and property of reconstituted nucleosome arrays. LLPS was generally observed for shorter nucleosome arrays (15-197-601, reconstituted from 15 repeats of the Widom 601 DNA with 197 bp nucleosome repeat length) at physiological ion concentrations. In contrast, gel- or solid-like condensates were detected for the considerably longer 62-202-601 and lambda DNA (~48.5 kbp) nucleosome arrays under the same conditions. In addition, we demonstrated that the presence of reduced BSA and acetate buffer is not essential for the chromatin LLPS process. Overall, this study provides a comprehensive understanding of several factors regarding chromatin physical states and sheds light on the mechanism and biological relevance of chromatin phase separation in vivo.

Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies.

Atomic Force Microscopy (AFM) is widely used for topographic imaging of DNA and protein-DNA complexes in ambient conditions with nanometer resolution. In AFM studies of protein-DNA complexes, identifying the protein’s location on the DNA substrate is one of the major goals. Such studies require distinguishing between the DNA ends, which can be accomplished by end-specific labeling of the DNA substrate. We selected as labels three-way DNA junctions (3WJ) assembled from synthetic DNA oligonucleotides with two arms of 39–40 bp each. The third arm has a three-nucleotide overhang, GCT, which is paired with the sticky end of the DNA substrate generated by the SapI enzyme. Ligation of the 3WJ results in the formation of a Y-type structure at the end of the linear DNA mole cule, which is routinely identified in the AFM images. The yield of labeling is 69%. The relative orientation of arms in the Y-end varies, such dynamics were directly visualized with time-lapse AFM studies using high-speed AFM (HS-AFM). This labeling approach was applied to the characterization of the nucleosome arrays assembled on different DNA templates. HS-AFM experiments revealed a high dynamic of nucleosomes resulting in a spontaneous unraveling followed by disassembly of nucleosomes.

Chromatin structure meets cryo-EM: Dynamic building blocks of the functional architecture

Chromatin is a dynamic molecular complex composed of DNA and proteins that package the DNA in the nucleus of eukaryotic cells. The basic structural unit of chromatin is the nucleosome core particle, composed of ~150 base pairs of genomic DNA wrapped around a histone octamer containing two copies each of four histones, H2A, H2B, H3, and H4. Individual nucleosome core particles are connected by short linker DNAs, forming a nucleosome array known as a beads-on-a-string fiber. Higher-order structures of chromatin are closely linked to nuclear events such as replication, transcription, recombination, and repair. Recently, a variety of chromatin structures have been determined by single-particle cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), and their structural details have provided clues about the chromatin architecture functions in the cell. In this review, we highlight recent cryo-EM structural studies of a fundamental chromatin unit to clarify the functions of chromatin.

Molecular organization of the early stages of nucleosome phase separation visualized by cryo-electron tomography

It has been proposed that the intrinsic property of nucleosome arrays to undergo liquid-liquid phase separation (LLPS) invitro is responsible for chromatin domain organization invivo. However, understanding nucleosomal LLPS has been hindered by the challenge to characterize the structure of the resulting heterogeneous condensates. We used cryo-electron tomography and deep-learning-based 3D reconstruction/segmentation to determine the molecular organization of condensates at various stages of LLPS. We show that nucleosomal LLPS involves a two-step process: a spinodal decomposition process yielding irregular condensates, followed by their unfavorable conversion into more compact, spherical nuclei that grow into larger spherical aggregates through accretion of spinodal materials or by fusion with other spherical condensates. Histone H1 catalyzes more than 10-fold the spinodal-to-spherical conversion. We propose that this transition involves exposure of nucleosome hydrophobic surfaces causing modified inter-nucleosome interactions. These results suggest a physical mechanism by which chromatin may transition from interphase to metaphase structures.

Phosphorylation-dependent association of human chromatin protein PC4 to linker histone H1 regulates genome organization and transcription.

Human Positive Coactivator 4 (PC4) is a multifaceted chromatin protein involved in diverse cellular processes including genome organization, transcription regulation, replication, DNA repair and autophagy. PC4 exists as a phospho-protein in cells which impinges on its acetylation by p300 and thereby affects its transcriptional co-activator functions via double-stranded DNA binding. Despite the inhibitory effects, the abundance of phosphorylated PC4 in cells intrigued us to investigate its role in chromatin functions in a basal state of the cell. We found that casein kinase-II (CKII)-mediated phosphorylation of PC4 is critical for its interaction with linker histone H1. By employing analytical ultracentrifugation and electron microscopy imaging of in vitro reconstituted nucleosomal array, we observed that phospho-mimic (PM) PC4 displays a superior chromatin condensation potential in conjunction with linker histone H1. ATAC-sequencing further unveiled the role of PC4 phosphorylation to be critical in inducing chromatin compaction of a wide array of coding and non-coding genes in vivo. Concordantly, phospho-PC4 mediated changes in chromatin accessibility led to gene repression and affected global histone modifications. We propose that the abundance of PC4 in its phosphorylated state contributes to genome compaction contrary to its co-activator function in driving several cellular processes like gene transcription and autophagy.

Identification and Analysis of Six Phosphorylation Sites Within the Xenopus laevis Linker Histone H1.0 C-Terminal Domain Indicate Distinct Effects on Nucleosome Structure

As a key structural component of the chromatin of higher eukaryotes, linker histones (H1s) are involved in stabilizing the folding of extended nucleosome arrays into higher-order chromatin structures and function as a gene-specific regulator of transcription in vivo. The H1 C-terminal domain (CTD) is essential for high-affinity binding of linker histones to chromatin and stabilization of higher-order chromatin structure. Importantly, the H1 CTD is an intrinsically disordered domain that undergoes a drastic condensation upon binding to nucleosomes. Moreover, although phosphorylation is a prevalent post-translational modification within the H1 CTD, exactly where this modification is installed and how phosphorylation influences the structure of the H1 CTD remains unclear for many H1s. Using novel mass spectrometry techniques, we identified six phosphorylation sites within the CTD of the archetypal linker histone Xenopus H1.0. We then analyzed nucleosome-dependent CTD condensation and H1-dependent linker DNA organization for H1.0 in which the phosphorylated serine residues were replaced by glutamic acid residues (phosphomimics) in six independent mutants. We find that phosphomimetics at residues S117E, S155E, S181E, S188E, and S192E resulted in a significant reduction in nucleosome-bound H1.0 CTD condensation compared with unphosphorylated H1.0, whereas S130E did not alter CTD structure. Furthermore, we found distinct effects among the phosphomimetics on H1-dependent linker DNA trajectory, indicating unique mechanisms by which this modification can influence H1 CTD condensation. These results bring to light a novel role for linker histone phosphorylation in directly altering the structure of nucleosome-bound H1 and a potential novel mechanism for its effects on chromatin structure and function.

A biochemistry platform to incorporate replication forks, DNA lesions, and nucleosome arrays to single‐molecule experiments

Fundamental biological processes such as DNA replication, DNA repair, and chromatin organization are precisely regulated in space and time by sophisticated multiprotein molecular machinery. The high spatiotemporal resolution of optical tweezers combined with single molecule fluorescence microscopy enables real‐time visualization of these processes while simultaneously measuring their enzymatic activity via detection of mechanical manipulation of the DNA substrates. Our biochemistry platform provides a set of tools to incorporate replication forks, DNA lesions, and specific sequences (e.g. nucleosome arrays) in DNA molecules tethered between two beads of the optical tweezers. Importantly, our tools also allow labeling of exact positions within these DNA substrates with fluorophores. This enables precise localization of the imaged proteins in the DNA sequence during processes including DNA scanning, as well as recognition and enzymatic manipulation of specific DNA structures or lesions. Our next developments aim to combine multiple DNA features in a single DNA molecule, such as a replication fork in the presence of DNA lesions or in the context of a nucleosome array. These advances will allow more complex experimental set ups and increase the capability to mimic cellular biological processes at a single molecule level.

Histone H1 binding to nucleosome arrays depends on linker DNA length and trajectory

Throughout the genome, nucleosomes often form regular arrays that differ in nucleosome repeat length (NRL), occupancy of linker histone H1 and transcriptional activity. Here, we report cryo-EM structures of human H1-containing tetranucleosome arrays with four physiologically relevant NRLs. The structures show a zig-zag arrangement of nucleosomes, with nucleosomes 1 and 3 forming a stack. H1 binding to stacked nucleosomes depends on the NRL, whereas H1 always binds to the non-stacked nucleosomes 2 and 4. Short NRLs lead to altered trajectories of linker DNA, and these altered trajectories sterically impair H1 binding to the stacked nucleosomes in our structures. As the NRL increases, linker DNA trajectories relax, enabling H1 contacts and binding. Our results provide an explanation for why arrays with short NRLs are depleted of H1 and suited for transcription, whereas arrays with long NRLs show full H1 occupancy and can form transcriptionally silent heterochromatin regions.

CENP-N promotes the compaction of centromeric chromatin

The histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. How nucleosomes at the centromere arrange into higher order structures is unknown. Here we demonstrate that the human CENP-A-interacting protein CENP-N promotes the stacking of CENP-A-containing mononucleosomes and nucleosomal arrays through a previously undefined interaction between the α6 helix of CENP-N with the DNA of a neighboring nucleosome. We describe the cryo-EM structures and biophysical characterization of such CENP-N-mediated nucleosome stacks and nucleosomal arrays and demonstrate that this interaction is responsible for the formation of densely packed chromatin at the centromere in the cell. Our results provide first evidence that CENP-A, together with CENP-N, promotes specific chromatin higher order structure at the centromere.

DNP-enhanced solid-state NMR spectroscopy of chromatin polymers

Chromatin is a DNA-protein polymer that represents the functional form of the genome. The main building block of chromatin is the nucleosome, a structure that contains 147 base pairs of DNA and two copies each of the histone proteins H2A, H2B, H3 and H4. Previous work has shown that magic angle spinning (MAS) NMR spectroscopy can capture the nucleosome at high resolution although studies have been challenging due to low sensitivity, the presence of dynamic and rigid components, and the complex interaction networks of nucleosomes within the chromatin polymer. Here, we use dynamic nuclear polarization (DNP) to enhance the sensitivity of MAS NMR experiments of nucleosome arrays at 100 K and show that well-resolved 13C-13C MAS NMR correlations can be obtained much more efficiently. We evaluate the effect of temperature on the chemical shifts and linewidths in the spectra and demonstrate that changes are relatively minimal and clustered in regions of histone-DNA or histone-histone contacts. We also compare samples prepared with and without DNA and show that the low temperature 13C-13C correlations exhibit sufficient resolution to detect chemical shift changes and line broadening for residues that form the DNA-histone interface. On the other hand, we show that the measurement of DNP-enhanced 15N-13C histone-histone interactions within the nucleosome core is complicated by the natural 13C abundance network in the sample. Nevertheless, the enhanced sensitivity afforded by DNP can be used to detect long-range correlations between histone residues and DNA. Overall, our experiments demonstrate that DNP-enhanced MAS NMR spectroscopy of chromatin samples yields spectra with high resolution and sensitivity and can be used to capture functionally relevant protein-DNA interactions that have implications for gene regulation and genome organization.

GC content, but not nucleosome positioning, directly contributes to intron splicing efficiency in Paramecium.

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the more than 90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur cotranscriptionally, and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We show that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, among the smallest periodicities reported. Our analysis has revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint favoring introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for nonsense-mediated decay effectors, we show that introns located at the edge of nucleosomes display higher splicing efficiency than those at the center. However, multiple regression analysis indicates that the low GC content of introns, rather than nucleosome positioning, is associated with high splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning, and intron evolution in Paramecium.

Chemical Biology Approaches to Identify and Profile Interactors of Chromatin Modifications.

In eukaryotes, DNA is packaged with histone proteins in a complex known as chromatin. Both the DNA and histone components of chromatin can be chemically modified in a wide variety of ways, resulting in a complex landscape often referred to as the "epigenetic code". These modifications are recognized by effector proteins that remodel chromatin and modulate transcription, translation, and repair of the underlying DNA. In this Review, we examine the development of methods for characterizing proteins that interact with these histone and DNA modifications. "Mark first" approaches utilize chemical, peptide, nucleosome, or oligonucleotide probes to discover interactors of a specific modification. "Reader first" approaches employ arrays of peptides, nucleosomes, or oligonucleotides to profile the binding preferences of interactors. These complementary strategies have greatly enhanced our understanding of how chromatin modifications effect changes in genomic regulation, bringing us ever closer to deciphering this complex language.

Recurrent de novo missense variants across multiple histone H4 genes underlie a neurodevelopmental syndrome

SummaryChromatin is essentially an array of nucleosomes, each of which consists of the DNA double-stranded fiber wrapped around a histone octamer. This organization supports cellular processes such as DNA replication, DNA transcription, and DNA repair in all eukaryotes. Human histone H4 is encoded by fourteen canonical histone H4 genes, all differing at the nucleotide level but encoding an invariant protein. Here, we present a cohort of 29 subjects with de novo missense variants in six H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11) identified by whole-exome sequencing and matchmaking. All individuals present with neurodevelopmental features of intellectual disability and motor and/or gross developmental delay, while non-neurological features are more variable. Ten amino acids are affected, six recurrently, and are all located within the H4 core or C-terminal tail. These variants cluster to specific regions of the core H4 globular domain, where protein-protein interactions occur with either other histone subunits or histone chaperones. Functional consequences of the identified variants were evaluated in zebrafish embryos, which displayed abnormal general development, defective head organs, and reduced body axis length, providing compelling evidence for the causality of the reported disorder(s). While multiple developmental syndromes have been linked to chromatin-associated factors, missense-bearing histone variants (e.g., H3 oncohistones) are only recently emerging as a major cause of pathogenicity. Our findings establish a broader involvement of H4 variants in developmental syndromes.