Single Amino Acid Change Underlies Distinct Roles of H2A.Z Subtypes in Human Syndrome.

The developmental disorder Floating-Harbor syndrome (FHS) is caused by heterozygous truncating mutations in SRCAP, a gene encoding a chromatin remodeler mediating incorporation of histone variant H2A.Z. Here, we demonstrate that FHS-associated mutations result in loss of SRCAP nuclear localization, alter neural crest gene programs in human in vitro models and Xenopus embryos, and cause craniofacial defects. These defects are mediated by one of two H2A.Z subtypes, H2A.Z.2, whose knockdown mimics and whose overexpression rescues the FHS phenotype. Selective rescue by H2A.Z.2 is conferred by one of the three amino acid differences between the H2A.Z subtypes, S38/T38. We further show that H2A.Z.1 and H2A.Z.2 genomic occupancy patterns are qualitatively similar, but quantitatively distinct, and H2A.Z.2 incorporation at AT-rich enhancers and expression of their associated genes are both sensitized to SRCAP truncations. Altogether, our results illuminate the mechanism underlying a human syndrome and uncover selective functions of H2A.Z subtypes during development.

FACT mediates the depletion of macroH2A1.2 to expedite gene transcription.

The histone variant macroH2A is generally linked to transcriptionally inactive chromatin, but how macroH2A regulates chromatin structure and functions in the transcriptional process remains elusive. This study reveals that while the integration of human macroH2A1.2 into nucleosomes does not affect their stability or folding dynamics, it notably hinders the maintenance of facilitates chromatin transcription's (FACT's) function. We show that FACT effectively diminishes the stability of macroH2A1.2-nucleosomes and expedites their depletion subsequent to the initial unfolding process. Furthermore, we identify the residue S139 in macroH2A1.2 as a critical switch to modulate FACT's function in nucleosome maintenance. Genome-wide analyses demonstrate that FACT-mediated depletion of macroH2A-nucleosomes allows the correct localization of macroH2A, while the S139 mutation reshapes macroH2A distribution and influences stimulation-induced transcription and cellular response in macrophages. Our findings provide mechanistic insights into the intricate interplay between macroH2A and FACT at the nucleosome level and elucidate their collective role in transcriptional regulation and immune response of macrophages.

Histone variant H2BE enhances chromatin accessibility in neurons to promote synaptic gene expression and long-term memory.

Histone proteins affect gene expression through multiple mechanisms, including through exchange with histone variants. Recent findings link histone variants to neurological disorders, yet few are well studied in the brain. Most notably, widely expressed variants of H2B remain elusive. We applied recently developed antibodies, biochemical assays, and sequencing approaches to reveal broad expression of the H2B variant H2BE and defined its role in regulating chromatin structure, neuronal transcription, and mouse behavior. We find that H2BE is enriched at promoters, and a single unique amino acid allows it to dramatically enhance chromatin accessibility. Further, we show that H2BE is critical for synaptic gene expression and long-term memory. Together, these data reveal a mechanism linking histone variants to chromatin accessibility, transcriptional regulation, neuronal function, and memory. This work further identifies a widely expressed H2B variant and uncovers a single histone amino acid with profound effects on genomic structure.

Catalytic and non-catalytic mechanisms of histone H4 lysine 20 methyltransferase SUV420H1.

SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/H4K20me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation. It is dysregulated in several cancers. Many of these processes were linked to its catalytic activity. However, deletion and inhibition of SUV420H1 have shown distinct phenotypes, suggesting that the enzyme likely has uncharacterized non-catalytic activities. Our cryoelectron microscopy (cryo-EM), biochemical, biophysical, and cellular analyses reveal how SUV420H1 recognizes its nucleosome substrates, and how histone variant H2A.Z stimulates its catalytic activity. SUV420H1 binding to nucleosomes causes a dramatic detachment of nucleosomal DNA from the histone octamer, which is a non-catalytic activity. We hypothesize that this regulates the accessibility of large macromolecular complexes to chromatin. We show that SUV420H1 can promote chromatin condensation, another non-catalytic activity that we speculate is needed for its heterochromatin functions. Together, our studies uncover and characterize the catalytic and non-catalytic mechanisms of SUV420H1, a key histone methyltransferase that plays an essential role in genomic stability.

The missing linker: emerging trends for H1 variant-specific functions.

Major advances in the chromatin and epigenetics fields have uncovered the importance of core histones, histone variants and their post-translational modifications (PTMs) in modulating chromatin structure. However, an acutely understudied related feature of chromatin structure is the role of linker histone H1. Previous assumptions of the functional redundancy of the 11 nonallelic H1 variants are contrasted by their strong evolutionary conservation, variability in their potential PTMs, and increased reports of their disparate functions, sub-nuclear localizations and unique expression patterns in different cell types. The commonly accepted notion that histone H1 functions solely in chromatin compaction and transcription repression is now being challenged by work from multiple groups. These studies highlight histone H1 variants as underappreciated facets of chromatin dynamics that function independently in various chromatin-based processes. In this review, we present notable findings involving the individual somatic H1 variants of which there are seven, underscoring their particular contributions to distinctly significant chromatin-related processes.

Histone Variant and Cell Context Determine H3K27M Reprogramming of the Enhancer Landscape and Oncogenic State.

Development of effective targeted cancer therapies is fundamentally limited by our molecular understanding of disease pathogenesis. Diffuse intrinsic pontine glioma (DIPG) is a fatal malignancy of the childhood pons characterized by a unique substitution to methionine in histone H3 at lysine 27 (H3K27M) that results in globally altered epigenetic marks and oncogenic transcription. Through primary DIPG tumor characterization and isogenic oncohistone expression, we show that the same H3K27M mutation displays distinct modes of oncogenic reprogramming and establishes distinct enhancer architecture depending upon both the variant of histone H3 and the cell context in which the mutation occurs. Compared with non-malignant pediatric pontine tissue, we identify and functionally validate both shared and variant-specific pathophysiology. Altogether, we provide a powerful resource of epigenomic data in 25 primary DIPG samples and 5 rare normal pediatric pontine tissue samples, revealing clinically relevant functional distinctions previously unidentified in DIPG.

Inherited mutations affecting the SRCAP complex are central in moderate-penetrance predisposition to uterine leiomyomas.

Uterine leiomyomas (ULs) are benign smooth muscle tumors that are common in premenopausal women. Somatic alterations in MED12, HMGA2, FH, genes encoding subunits of the SRCAP complex, and genes involved in Cullin 3-RING E3 ligase neddylation are mutually exclusive UL drivers. Established predisposition genes explain only partially the estimated heritability of leiomyomas. Here, we examined loss-of-function variants across 18,899 genes in a cohort of 233,614 White European women, revealing variants in four genes encoding SRCAP complex subunits (YEATS4, ZNHIT1, DMAP1, and ACTL6A) with a significant association to ULs, and YEATS4 and ZNHIT1 strikingly rank first and second, respectively. Positive mutation status was also associated with younger age at diagnosis and hysterectomy. Moderate-penetrance UL risk was largely attributed to rare non-synonymous mutations affecting the SRCAP complex. To examine this disease phenotype more closely, we set out to identify inherited mutations affecting the SRCAP complex in our in-house sample collection of Finnish individuals with ULs (n = 860). We detected one individual with an ACTL6A splice-site mutation, two individuals with a YEATS4 missense mutation, and four individuals with DMAP1 mutations: one splice-site, one nonsense, and two missense variants. These individuals had large and/or multiple ULs, were often diagnosed at an early age, and many had family history of ULs. When a somatic second hit was found, ACTL6A and DMAP1 were silenced in tumors by somatic mutation and YEATS4 by promoter hypermethylation. Decreased H2A.Z staining was observed in the tumors, providing further evidence for the pathogenic nature of the germline mutations. Our results establish inactivation of genes encoding SRCAP complex subunits as a central contributor to moderate-penetrance UL predisposition.

Actin-related protein 6 facilitates proneural protein-induced gene activation for rapid neural differentiation.

Neurogenesis is initiated by basic helix-loop-helix proneural proteins. Here, we show that Actin-related protein 6 (Arp6), a core component of the H2A.Z exchange complex SWR1, interacts with proneural proteins and is crucial for efficient onset of proneural protein target gene expression. Arp6 mutants exhibit reduced transcription in sensory organ precursors (SOPs) downstream of the proneural protein patterning event. This leads to retarded differentiation and division of SOPs and smaller sensory organs. These phenotypes are also observed in proneural gene hypomorphic mutants. Proneural protein expression is not reduced in Arp6 mutants. Enhanced proneural gene expression fails to rescue retarded differentiation in Arp6 mutants, suggesting that Arp6 acts downstream of or in parallel with proneural proteins. H2A.Z mutants display Arp6-like retardation in SOPs. Transcriptomic analyses demonstrate that loss of Arp6 and H2A.Z preferentially decreases expression of proneural protein-activated genes. H2A.Z enrichment in nucleosomes around the transcription start site before neurogenesis correlates highly with greater activation of proneural protein target genes by H2A.Z. We propose that upon proneural protein binding to E-box sites, H2A.Z incorporation around the transcription start site allows rapid and efficient activation of target genes, promoting rapid neural differentiation.

Transcription-coupled H3.3 recycling: A link with chromatin states.

Histone variant H3.3 is incorporated into chromatin throughout the cell cycle and even in non-cycling cells. This histone variant marks actively transcribed chromatin regions with high nucleosome turnover, as well as silent pericentric and telomeric repetitive regions. In the past few years, significant progress has been made in our understanding of mechanisms involved in the transcription-coupled deposition of H3.3. Here we review how, during transcription, new H3.3 deposition intermingles with the fate of the old H3.3 variant and its recycling. First, we describe pathways enabling the incorporation of newly synthesized vs old H3.3 histones in the context of transcription. We then review the current knowledge concerning differences between these two H3.3 populations, focusing on their PTMs composition. Finally, we discuss the implications of H3.3 recycling for the maintenance of the transcriptional state and underline the emerging importance of H3.3 as a potent epigenetic regulator for both maintaining and switching a transcriptional state.

Structural basis of nucleosome dynamics modulation by histone variants H2A.B and H2A.Z.2.2.

Nucleosomes are dynamic entities with wide-ranging compositional variations. Human histone variants H2A.B and H2A.Z.2.2 play critical roles in multiple biological processes by forming unstable nucleosomes and open chromatin structures, but how H2A.B and H2A.Z.2.2 confer these dynamic features to nucleosomes remains unclear. Here, we report cryo-EM structures of nucleosome core particles containing human H2A.B (H2A.B-NCP) at atomic resolution, identifying large-scale structural rearrangements in the histone octamer in H2A.B-NCP. H2A.B-NCP compacts approximately 103 bp of DNA wrapping around the core histones in approximately 1.2 left-handed superhelical turns, in sharp contrast to canonical nucleosome encompassing approximately 1.7 turns of DNA. Micrococcal nuclease digestion assay reveals that nineteen H2A.B-specific residues, including a ROF ("regulating-octamer-folding") sequence of six consecutive residues, are responsible for loosening of H2A.B-NCPs. Unlike H2A.B-NCP, the H2A.Z.2.2-containing nucleosome (Z.2.2-NCP) adopts a less-extended structure and compacts around 125 bp of DNA. Further investigation uncovers a crucial role for the H2A.Z.2.2-specific ROF in both H2A.Z.2.2-NCP opening and SWR1-dependent histone replacement. Taken together, these first high-resolution structure of unstable nucleosomes induced by histone H2A variants elucidate specific functions of H2A.B and H2A.Z.2.2 in enhancing chromatin dynamics.

DAXX safeguards heterochromatin formation in embryonic stem cells.

Genomes comprise a large fraction of repetitive sequences folded into constitutive heterochromatin, which protect genome integrity and cell identity. De novo formation of heterochromatin during preimplantation development is an essential step for preserving the ground-state of pluripotency and the self-renewal capacity of embryonic stem cells (ESCs). However, the molecular mechanisms responsible for the remodeling of constitutive heterochromatin are largely unknown. Here, we identify that DAXX, an H3.3 chaperone essential for the maintenance of mouse ESCs in the ground state, accumulates in pericentromeric regions independently of DNA methylation. DAXX recruits PML and SETDB1 to promote the formation of heterochromatin, forming foci that are hallmarks of ground-state ESCs. In the absence of DAXX or PML, the three-dimensional (3D) architecture and physical properties of pericentric and peripheral heterochromatin are disrupted, resulting in de-repression of major satellite DNA, transposable elements and genes associated with the nuclear lamina. Using epigenome editing tools, we observe that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX is crucial for the maintenance and 3D organization of the heterochromatin compartment and protects ESC viability.

A Dual Inhibitory Mechanism Sufficient to Maintain Cell-Cycle-Restricted CENP-A Assembly.

Chromatin featuring the H3 variant CENP-A at the centromere is critical for its mitotic function and epigenetic maintenance. Assembly of centromeric chromatin is restricted to G1 phase through inhibitory action of Cdk1/2 kinases in other phases of the cell cycle. Here, we identify the two key targets sufficient to maintain cell-cycle control of CENP-A assembly. We uncovered a single phosphorylation site in the licensing factor M18BP1 and a cyclin A binding site in the CENP-A chaperone, HJURP, that mediated specific inhibitory phosphorylation. Simultaneous expression of mutant proteins lacking these residues results in complete uncoupling from the cell cycle. Consequently, CENP-A assembly is fully recapitulated under high Cdk activities, indistinguishable from G1 assembly. We find that Cdk-mediated inhibition is exerted by sequestering active factors away from the centromere. Finally, we show that displacement of M18BP1 from the centromere is critical for the assembly mechanism of CENP-A.

Identification of a novel DNA oxidative damage repair pathway, requiring the ubiquitination of the histone variant macroH2A1.1.

BACKGROUND: The histone variant macroH2A (mH2A), the most deviant variant, is about threefold larger than the conventional histone H2A and consists of a histone H2A-like domain fused to a large Non-Histone Region responsible for recruiting PARP-1 to chromatin. The available data suggest that the histone variant mH2A participates in the regulation of transcription, maintenance of heterochromatin, NAD+ metabolism, and double-strand DNA repair. RESULTS: Here, we describe a novel function of mH2A, namely its implication in DNA oxidative damage repair through PARP-1. The depletion of mH2A affected both repair and cell survival after the induction of oxidative lesions in DNA. PARP-1 formed a specific complex with mH2A nucleosomes in vivo. The mH2A nucleosome-associated PARP-1 is inactive. Upon oxidative damage, mH2A is ubiquitinated, PARP-1 is released from the mH2A nucleosomal complex, and is activated. The in vivo-induced ubiquitination of mH2A, in the absence of any oxidative damage, was sufficient for the release of PARP-1. However, no release of PARP-1 was observed upon treatment of the cells with either the DNA alkylating agent MMS or doxorubicin. CONCLUSIONS: Our data identify a novel pathway for the repair of DNA oxidative lesions, requiring the ubiquitination of mH2A for the release of PARP-1 from chromatin and its activation.

Histone Variant H3.3 Controls Arabidopsis Fertility by Regulating Male Gamete Development.

Reprograming of chromatin structures and changes in gene expression are critical for plant male gamete development, and epigenetic marks play an important role in these processes. Histone variant H3.3 is abundant in euchromatin and is largely associated with transcriptional activation. The precise function of H3.3 in gamete development remains unclear in plants. Here, we report that H3.3 is abundantly expressed in Arabidopsis anthers and its knockout mutant h3.3-1 is sterile due to male sterility. Transcriptome analysis of young inflorescence has identified 2348 genes downregulated in h3.3-1 mutant, among which 1087 target genes are directly bound by H3.3, especially at their 3' ends. As a group, this set of H3.3 targets is enriched in the reproduction-associated processes including male gamete generation, pollen sperm cell differentiation and pollen tube growth. The function of H3.3 in male gamete development is dependent on the Anti-Silencing Factor 1A/1B (ASF1A/1B)-Histone regulator A (HIRA)-mediated pathway. Our results suggest that ASF1A/1B-HIRA-mediated H3.3 deposition at its direct targets for transcription activation forms the regulatory networks responsible for male gamete development.

SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin.

Chromatin integrity is essential for cellular homeostasis. Polycomb group proteins modulate chromatin states and transcriptionally repress developmental genes to maintain cell identity. They also repress repetitive sequences such as major satellites and constitute an alternative state of pericentromeric constitutive heterochromatin at paternal chromosomes (pat-PCH) in mouse pre-implantation embryos. Remarkably, pat-PCH contains the histone H3.3 variant, which is absent from canonical PCH at maternal chromosomes, which is marked by histone H3 lysine 9 trimethylation (H3K9me3), HP1, and ATRX proteins. Here, we show that SUMO2-modified CBX2-containing Polycomb Repressive Complex 1 (PRC1) recruits the H3.3-specific chaperone DAXX to pat-PCH, enabling H3.3 incorporation at these loci. Deficiency of Daxx or PRC1 components Ring1 and Rnf2 abrogates H3.3 incorporation, induces chromatin decompaction and breakage at PCH of exclusively paternal chromosomes, and causes their mis-segregation. Complementation assays show that DAXX-mediated H3.3 deposition is required for chromosome stability in early embryos. DAXX also regulates repression of PRC1 target genes during oogenesis and early embryogenesis. The study identifies a novel critical role for Polycomb in ensuring heterochromatin integrity and chromosome stability in mouse early development.

Chromatin sensing: integration of environmental signals to reprogram plant development through chromatin regulators.

Chromatin regulation in eukaryotes plays pivotal roles in controlling the developmental regulatory gene network. This review explores the intricate interplay between chromatin regulators and environmental signals, elucidating their roles in shaping plant development. As sessile organisms, plants have evolved sophisticated mechanisms to perceive and respond to environmental cues, orchestrating developmental programs that ensure adaptability and survival. A central aspect of this dynamic response lies in the modulation of versatile gene regulatory networks, mediated in part by various chromatin regulators. Here, we summarized current understanding of the molecular mechanisms through which chromatin regulators integrate environmental signals, influencing key aspects of plant development.

Histone variant H3.3 promotes metastasis in alveolar rhabdomyosarcoma.

The relatively quiet mutational landscape of rhabdomyosarcoma (RMS) suggests that epigenetic deregulation could be central to oncogenesis and tumour aggressiveness. Histone variants have long been recognised as important epigenetic regulators of gene expression. However, the role of histone variants in RMS has not been studied hitherto. In this study, we show that histone variant H3.3 is overexpressed in alveolar RMS (ARMS), an aggressive subtype of RMS. Functionally, knockdown of H3F3A, which encodes for H3.3, significantly impairs the ability of ARMS cells to undertake migration and invasion and reduces Rho activation. In addition, a striking reduction in metastatic tumour burden and improved survival is apparent in vivo. Using RNA-sequencing and ChIP-sequencing analyses, we identified melanoma cell adhesion molecule (MCAM/CD146) as a direct downstream target of H3.3. Loss of H3.3 resulted in a reduction in the presence of active marks and an increase in the occupancy of H1 at the MCAM promoter. Cell migration and invasion were rescued in H3F3A-depleted cells through MCAM overexpression. Moreover, we identified G9a, a lysine methyltransferase encoded by EHMT2, as an upstream regulator of H3F3A. Therefore, this study identifies a novel H3.3 dependent axis involved in ARMS metastasis. These findings establish the potential of MCAM as a therapeutic target for high-risk ARMS patients. © 2022 The Pathological Society of Great Britain and Ireland.

An essential role for the Ino80 chromatin remodeling complex in regulation of gene expression during cellular quiescence.

Cellular quiescence is an important physiological state both in unicellular and multicellular eukaryotes. Quiescent cells are halted for proliferation and stop the cell cycle at the G0 stage. Using fission yeast as a model organism, we have previously found that several subunits of a conserved chromatin remodeling complex, Ino80C (INOsitol requiring nucleosome remodeling factor), are required for survival in quiescence. Here, we demonstrate that Ino80C has a key function in the regulation of gene expression in G0 cells. We show that null mutants for two Ino80C subunits, Iec1 and Ies2, a putative subunit Arp42, a null mutant for the histone variant H2A.Z, and a null mutant for the Inositol kinase Asp1 have very similar phenotypes in quiescence. These mutants show reduced transcription genome-wide and specifically fail to activate 149 quiescence genes, of which many are localized to the subtelomeric regions. Using spike in normalized ChIP-seq experiments, we show that there is a global reduction of H2A.Z levels in quiescent wild-type cells but not in iec1∆ cells and that a subtelomeric chromosome boundary element is strongly affected by Ino80C. Based on these observations, we propose a model in which Ino80C is evicting H2A.Z from chromatin in quiescent cells, thereby inactivating the subtelomeric boundary element, leading to a reorganization of the chromosome structure and activation of genes required to survive in quiescence.

High-Speed Atomic Force Microscopy Reveals Spontaneous Nucleosome Sliding of H2A.Z at the Subsecond Time Scale.

Nucleosome dynamics, such as nucleosome sliding and DNA unwrapping, are important for gene regulation in eukaryotic chromatin. H2A.Z, a variant of histone H2A that is highly evolutionarily conserved, participates in gene regulation by forming unstable multipositioned nucleosomes in vivo and in vitro. However, the subsecond dynamics of this unstable nucleosome have not been directly visualized under physiological conditions. Here, we used high-speed atomic force microscopy (HS-AFM) to directly visualize the subsecond dynamics of human H2A.Z.1-nucleosomes. HS-AFM videos show nucleosome sliding along 4 nm of DNA within 0.3 s in any direction. This sliding was also visualized in an H2A.Z.1 mutant, in which the C-terminal half was replaced by the corresponding canonical H2A amino acids, indicating that the interaction between the N-terminal region of H2A.Z.1 and the DNA is responsible for nucleosome sliding. These results may reveal the relationship between nucleosome dynamics and gene regulation by histone H2A.Z.

The Histone Variant H3.3 Is Required for Plant Growth and Fertility in Arabidopsis.

Histones are the core components of the eukaryote chromosome, and have been implicated in transcriptional gene regulation. There are three major isoforms of histone H3 in Arabidopsis. Studies have shown that the H3.3 variant is pivotal in modulating nucleosome structure and gene transcription. However, the function of H3.3 during development remains to be further investigated in plants. In this study, we disrupted all three H3.3 genes in Arabidopsis. Two triple mutants, h3.3cr-4 and h3.3cr-5, were created by the CRISPR/Cas9 system. The mutant plants displayed smaller rosettes and decreased fertility. The stunted growth of h3.3cr-4 may result from reduced expression of cell cycle regulators. The shorter stamen filaments, but not the fertile ability of the gametophytes, resulted in reduced fertility of h3.3cr-4. The transcriptome analysis suggested that the reduced filament elongation of h3.3cr-4 was probably caused by the ectopic expression of several JASMONATE-ZIM DOMAIN (JAZ) genes, which are the key repressors of the signaling pathway of the phytohormone jasmonic acid (JA). These observations suggest that the histone variant H3.3 promotes plant growth, including rosette growth and filament elongation.