HIRA complex deposition of histone H3.3 is driven by histone tetramerization and histone-DNA binding.

The HIRA histone chaperone complex is comprised of four protein subunits: HIRA, UBN1, CABIN1, and transiently associated ASF1a. All four subunits have been demonstrated to play a role in deposition of the histone variant H3.3 onto areas of actively transcribed euchromatin in cells. The mechanism by which these subunits function together to drive histone deposition has remained poorly understood. Here we present biochemical and biophysical data supporting a model whereby ASF1a delivers histone H3.3/H4 dimers to the HIRA complex, H3.3/H4 tetramerization drives the association of two HIRA/UBN1 complexes, and the affinity of the histones for DNA drives release of ASF1a and subsequent histone deposition. These findings have implications for understanding how other histone chaperone complexes may mediate histone deposition.

Histone H3.3 chaperone HIRA renders stress-responsive genes poised for prospective lethal stresses in acquired tolerance.

Appropriate responses to environmental challenges are imperative for the survival of all living organisms. Exposure to low-dose stresses is recognized to yield increased cellular fitness, a phenomenon termed hormesis. However, our molecular understanding of how cells respond to low-dose stress remains profoundly limited. Here we report that histone variant H3.3-specific chaperone, HIRA, is required for acquired tolerance, where low-dose heat stress exposure confers resistance to subsequent lethal heat stress. We found that human HIRA activates stress-responsive genes, including HSP70, by depositing histone H3.3 following low-dose stresses. These genes are also marked with histone H3 Lys-4 trimethylation and H3 Lys-9 acetylation, both active chromatin markers. Moreover, depletion of HIRA greatly diminished acquired tolerance, both in normal diploid fibroblasts and in HeLa cells. Collectively, our study revealed that HIRA is required for eliciting adaptive stress responses under environmental fluctuations and is a master regulator of stress tolerance.

Haspin mediates H3.3S31 phosphorylation downstream of Aurora B in mouse embryonic stem cells.

Histone phosphorylation is instrumental in regulating diverse cellular processes across eukaryotes. Unraveling the kinases that target specific histone sites is key to deciphering the underlying mechanisms. Among the various sites on histone tails that can undergo phosphorylation, the kinase responsible for H3.3S31 phosphorylation remained elusive. Since both H3.3S31ph and H3T3ph occur specifically during mitosis, and Haspin is the known kinase for H3T3 phosphorylation, we investigated its potential role in H3.3S31 phosphorylation. We employed CRISPR/Cas9, RNA interference, and specific small molecule inhibitors to eliminate Haspin function in various cell types. Our data consistently revealed a link between Haspin and H3.3S31ph. Furthermore, in vitro kinase assays provided evidence supporting Haspin's contribution to H3.3S31ph. Loss- and gain-of-function experiments targeting Haspin and Aurora B further suggested a hierarchical relationship. Haspin acts as a downstream kinase of Aurora B, specifically orchestrating H3.3S31 phosphorylation in mESCs. This study unveils a novel role for Haspin as a kinase in regulating H3.3S31 phosphorylation during mitosis. This discovery holds promise for expanding our understanding of the functional significance of Haspin and H3.3S31ph in mammals.

Structural and Biochemical Characterization of the Nucleosome Containing Variants H3.3 and H2A.Z.

Variant H3.3, along with H2A.Z, is notably enriched at promoter regions and is commonly associated with transcriptional activation. However, the specific molecular mechanisms through which H3.3 influences chromatin dynamics at transcription start sites, and its role in gene regulation, remain elusive. Using a combination of biochemistry and cryo-electron microscopy (cryo-EM), we show that the inclusion of H3.3 alone does not induce discernible changes in nucleosome DNA dynamics. Conversely, the presence of both H3.3 and H2A.Z enhances DNA's flexibility similarly to H2A.Z alone. Interestingly, our findings suggest that the presence of H3.3 in the H2A.Z nucleosome provides slightly increased protection to DNA at internal sites within the nucleosome. These results imply that while H2A.Z at active promoters promotes the formation of more accessible nucleosomes with increased DNA accessibility to facilitate transcription, the simultaneous presence of H3.3 offers an additional mechanism to fine-tune nucleosome accessibility and the chromatin environment.

Set2 and H3K36 regulate the Drosophila male X chromosome in a context-specific manner, independent from MSL complex spreading.

Dosage compensation in Drosophila involves upregulating male X-genes two-fold. This process is carried out by the MSL (male-specific lethal) complex, which binds high-affinity sites and spreads to surrounding genes. Current models of MSL spreading focus on interactions of MSL3 (male-specific lethal 3) with histone marks; in particular, Set2-dependent H3 lysine-36 trimethylation (H3K36me3). However, Set2 might affect DC via another target, or there could be redundancy between canonical H3.2 and variant H3.3 histones. Further, it is difficult to parse male-specific effects from those that are simply X-specific. To discriminate among these possibilities, we employed genomic approaches in H3K36 (residue) and Set2 (writer) mutants. The results confirm a role for Set2 in X-gene regulation, but show that expression trends in males are often mirrored in females. Instead of global male-specific reduction of X-genes in Set2/H3K36 mutants, the effects were heterogeneous. We identified cohorts of genes whose expression was significantly altered following loss of H3K36 or Set2, but the changes were in opposite directions, suggesting that H3K36me states have reciprocal functions. In contrast to H4K16R controls, analysis of combined H3.2K36R/H3.3K36R mutants neither showed consistent reduction in X-gene expression, nor any correlation with MSL3 binding. Examination of other developmental stages/tissues revealed additional layers of context-dependence. Our studies implicate BEAF-32 and other insulator proteins in Set2/H3K36-dependent regulation. Overall, the data are inconsistent with the prevailing model wherein H3K36me3 directly recruits the MSL complex. We propose that Set2 and H3K36 support DC indirectly, via processes that are utilized by MSL but common to both sexes.

Targeting the reprogrammed metabolism in H3.3K27M pediatric high-grade gliomas.

Recent studies in Diffuse Midline Gliomas (DMG) demonstrated a strong connection between epigenome dysregulation and metabolic rewiring. Here, we evaluated the value of targeting a glycolytic protein named Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3 (PFKFB3) in H3.3K27M DMG. We observed that the viability of H3.3K27M cells is dramatically reduced by PFK15, a potent inhibitor of PFKFB3. Furthermore, PFKFB3 inhibition induced apoptosis and G2/M arrest. Interestingly, CRISPR-Knockout of the K27M mutant allele has a synergistic effect on the observed phenotype. Altogether, we identified PFKFB3 as a new target for H3.3K27M DMG, making PFK15 a potential candidate for future animal studies and clinical trials.

Treatment of Clival Giant Cell Tumor: A Case Report and Literature Review.

Giant cell tumors (GCTs) are locally aggressive primary bone tumors of osteoclast-like cells. Most GCTs occur within the long bones, and primary GCTs involving the clivus are extremely rare. We present the case of an 18-year-old boy with binocular horizontal diplopia with an insidious onset who was found to have a hypointense enhancing mass involving the clivus and left side dorsum sellae on magnetic resonance images. The tumor was completely resected via an endoscopic endonasal transclival approach, and histopathologic examination via immunohistochemistry indicated a GCT. The patient's left abducens nerve palsy improved slightly after surgery. Because of the rarity of GCTs, there is no consensus about the definitive treatment protocol. However, we suggest that gross total resection is the treatment of choice, and denosumab plays a critical role in patients with subtotal resection.

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.

The role of Smarcad1 in retroviral repression in mouse embryonic stem cells.

BACKGROUND: Moloney murine leukemia virus (MLV) replication is suppressed in mouse embryonic stem cells (ESCs) by the Trim28-SETDB1 complex. The chromatin remodeler Smarcad1 interacts with Trim28 and was suggested to allow the deposition of the histone variant H3.3. However, the role of Trim28, H3.3, and Smarcad1 in MLV repression in ESCs still needs to be fully understood. RESULTS: In this study, we used MLV to explore the role of Smarcad1 in retroviral silencing in ESCs. We show that Smarcad1 is immediately recruited to the MLV provirus. Based on the repression dynamics of a GFP-reporter MLV, our findings suggest that Smarcad1 plays a critical role in the establishment and maintenance of MLV repression, as well as other Trim28-targeted genomic loci. Furthermore, Smarcad1 is important for stabilizing and strengthening Trim28 binding to the provirus over time, and its presence around the provirus is needed for proper deposition of H3.3 on the provirus. Surprisingly, the combined depletion of Smarcad1 and Trim28 results in enhanced MLV derepression, suggesting that these two proteins may also function independently to maintain repressive chromatin states. CONCLUSIONS: Overall, the results of this study provide evidence for the crucial role of Smarcad1 in the silencing of retroviral elements in embryonic stem cells. Further research is needed to fully understand how Smarcad1 and Trim28 cooperate and their implications for gene expression and genomic stability.

Chromatin Modifier EP400 Regulates Oocyte Quality and Zygotic Genome Activation in Mice.

Epigenetic modifiers that accumulate in oocytes, play a crucial role in steering the developmental program of cleavage embryos and initiating life. However, the identification of key maternal epigenetic regulators remains elusive. In the findings, the essential role of maternal Ep400, a chaperone for H3.3, in oocyte quality and early embryo development in mice is highlighted. Depletion of Ep400 in oocytes resulted in a decline in oocyte quality and abnormalities in fertilization. Preimplantation embryos lacking maternal Ep400 exhibited reduced major zygotic genome activation (ZGA) and experienced developmental arrest at the 2-to-4-cell stage. The study shows that EP400 forms protein complex with NFYA, occupies promoters of major ZGA genes, modulates H3.3 distribution between euchromatin and heterochromatin, promotes transcription elongation, activates the expression of genes regulating mitochondrial functions, and facilitates the expression of rate-limiting enzymes of the TCA cycle. This intricate process driven by Ep400 ensures the proper execution of the developmental program, emphasizing its critical role in maternal-to-embryonic transition.

Diffuse hemispheric glioma with H3 p.K28M (K27M) mutation: Unusual non-midline presentation of diffuse midline glioma, H3 K27M-altered?

Diffuse midline glioma, H3 K27-altered (DMG-H3 K27) is an aggressive group of diffuse gliomas that predominantly occurs in pediatric patients, involves midline structures, and displays loss of H3 p.K28me3 (K27me3) expression by immunohistochemistry and characteristic genetic/epigenetic profile. Rare examples of a diffuse glioma with an H3 p.K28M (K27M) mutation and without involvement of the midline structures, so-called "diffuse hemispheric glioma with H3 p.K28M (K27M) mutation" (DHG-H3 K27), have been reported. Herein, we describe 2 additional cases of radiologically confirmed DHG-H3 K27 and summarize previously reported cases. We performed histological, immunohistochemical, molecular, and DNA methylation analysis and provided clinical follow-up in both cases. Overall, DHG-H3 K27 is an unusual group of diffuse gliomas that shows similar clinical, histopathological, genomic, and epigenetic features to DMG-H3 K27 as well as enrichment for activating alterations in MAPK pathway genes. These findings suggest that DHG-H3 K27 is closely related to DMG-H3 K27 and may represent an unusual presentation of DMG-H3 K27 without apparent midline involvement and with frequent MAPK pathway activation. Detailed reports of additional cases with clinical follow-up will be important to expand our understanding of this unusual group of diffuse gliomas and to better define the clinical outcome and how to classify DHG-H3 K27.

H2A.Z histone variants facilitate HDACi-dependent removal of H3.3K27M mutant protein in pediatric high-grade glioma cells.

Diffuse intrinsic pontine gliomas (DIPGs) are deadly pediatric brain tumors, non-resectable due to brainstem localization and diffusive growth. Over 80% of DIPGs harbor a mutation in histone 3 (H3.3 or H3.1) resulting in a lysine-to-methionine substitution (H3K27M). Patients with DIPG have a dismal prognosis with no effective therapy. We show that histone deacetylase (HDAC) inhibitors lead to a significant reduction in the H3.3K27M protein (up to 80%) in multiple glioma cell lines. We discover that the SB939-mediated H3.3K27M loss is partially blocked by a lysosomal inhibitor, chloroquine. The H3.3K27M loss is facilitated by co-occurrence of H2A.Z, as evidenced by the knockdown of H2A.Z isoforms. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis confirms the occupancy of H3.3K27M and H2A.Z at the same SB939-inducible genes. We discover a mechanism showing that HDAC inhibition in DIPG leads to pharmacological modulation of the oncogenic H3.3K27M protein levels. These findings show the possibility of directly targeting the H3.3K27M oncohistone.

Deposition of onco-histone H3.3-G34W leads to DNA repair deficiency and activates cGAS/STING-mediated immune responses.

Mutations in histone H3.3-encoding genes causing mutant histone tails are associated with specific cancers such as pediatric glioblastomas (H3.3-G34R/V) and giant cell tumor of the bone (H3.3-G34W). The mechanisms by which these mutations promote malignancy are not completely understood. Here we show that cells expressing H3.3-G34W exhibit DNA double-strand breaks (DSBs) repair defects and increased cellular sensitivity to ionizing radiation (IR). Mechanistically, H3.3-G34W can be deposited to damaged chromatin, but in contrast to wild-type H3.3, does not interact with non-homologous end-joining (NHEJ) key effectors KU70/80 and XRCC4 leading to NHEJ deficiency. Together with defective cell cycle checkpoints reported previously, this DNA repair deficiency in H3.3-G34W cells led to accumulation of micronuclei and cytosolic DNA following IR, which subsequently led to activation of the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, thereby inducing release of immune-stimulatory cytokines. These findings suggest a potential for radiotherapy for tumors expressing H3.3-G34W, which can be further improved by combination with STING agonists to induce immune-mediated therapeutic efficacy.

Periosteal chondroblastoma of the femoral neck: two cases and a review of the literature.

Chondroblastoma is a rare benign cartilaginous tumor mostly confined to the epiphyses and apophyses. Cases outside the epiphyseal region are exceedingly rare. Extramedullary chondroblastomas are exceptional; to our knowledge, only two cases qualified as "periosteal chondroblastoma" have been described in the literature. We report two cases of metaphyseal periosteal chondroblastoma both located on the inferior surface of the femoral neck. Both cases were paucicellular with an unusual dense sclerotic reaction. The diagnosis of chondroblastoma was supported by the expression of histone 3.3, K36M mutant in tumor cells.

Altered histone abundance as a mode of ovotoxicity during 7,12-dimethylbenz[a]anthracene exposure with additive influence of obesity†.

Histones are slowly evolving chromatin components and chromatin remodeling can incorporate histone variants differing from canonical histones as an epigenetic modification. Several identified histone variants are involved with the environmental stress-induced DNA damage response (DDR). Mechanisms of DDR in transcriptionally inactive, prophase-arrested oocytes and epigenetic regulation are under-explored in ovarian toxicology. The study objective was to identify ovarian proteomic and histone modifications induced by DMBA exposure and an influence of obesity. Post-pubertal wildtype (KK.Cg-a/a; lean) and agouti (KK.Cg-Ay/J; obese) female mice, were exposed to either corn oil (control; CT) or DMBA (1 mg/kg) for 7d via intraperitoneal injection (n = 10/treatment). Ovarian proteome analysis (LC-MS/MS) determined that obesity altered 225 proteins (P < 0.05) with histone 3 being the second least abundant (FC = -5.98, P < 0.05). Histone 4 decreased by 3.33-fold, histone variant H3.3 decreased by 3.05-fold, and H1.2, H1.4 and H1.1(alpha) variants increased by 1.59, 1.90 and 2.01-fold, respectively (P < 0.05). DMBA exposure altered 48 proteins in lean mice with no observed alterations in histones or histone variants. In obese mice, DMBA exposure altered 120 proteins and histone 2B abundance increased by 0.30-fold (P < 0.05). In DMBA-exposed mice, obesity altered the abundance of 634 proteins. Histones 4, 3 and 2A type 1-F decreased by 4.03, 3.71, 0.43-fold, respectively, whereas histone variant H1.2 and linker histone, H15 increased by 2.72- and 3.07-fold, respectively (P < 0.05). Thus, DMBA exposure alters histones and histone variants, and responsivity is more pronounced during obesity, potentially altering ovarian transcriptional regulation.

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.

Daxx knockdown promoted rDNA transcription without impairing H3.3 expression in mouse preimplantation embryos.

During fertilization, DAXX (death domain-associated protein) mediates histone variant H3.3 incorporation into heterochromatin, which plays an important role in the maintenance of genomic integrity. rDNA, the ribosomal gene, is included in the first wave of gene activation after fertilization. Our and other studies indicated that loss of Daxx disturbs rDNA heterochromatinization and promotes rDNA transcription without change in protein expression of H3.3. However, maternal and zygotic deletion of Daxx impairs blastocyst development. Whether Daxx knockdown affects H3.3 expression and improves the rDNA transcription in preimplantation development has not been reported. In the present study, we injected HA-labelled H3.3 (H3.3-HA) into oocytes during ICSI procedure, and detected H3.3 and DAXX by immunofluorescent staining. Then, we knockdowned Daxx and detected the gene expression levels of Daxx, H3.3, 18s and 47s rRNA. We also performed immunofluorescent staining of B23, γH2A and EdU incorporation to demonstrate nuclear structure, DNA damage and replication. We found injection of H3.3-HA did not impair preimplantation development. Daxx siRNA did not change expression of H3.3 mRNA, and the development of two-cell embryos and blastocysts, but the overall replication and expression levels of rRNA were increased compared with that in the control group. Finally, knockdown of DAXX did not aggravate the DNA damage but loosened the nucleolus. We concluded that Daxx knockdown promoted DNA replication and rDNA transcription, but did not affect H3.3 expression and subsequent preimplantation development.

Giant Cell Tumor of the Acromion: Case Report and Literature Review.

BACKGROUND/AIM: Giant cell tumor of bone (GCTB) is a locally aggressive neoplasm that typically occurs in the ends (epiphyses) of long bones of young adults. Flat bones are uncommon sites of involvement. Herein, we describe an unusual case of pathologically proven GCT of the acromion. CASE REPORT: The patient was a 39-year-old woman with no history of trauma who presented with a 3-month history of right posterior shoulder pain. Physical examination revealed mild swelling and tenderness in the posterior aspect of the right shoulder. Plain radiograph showed a purely lytic lesion, suggestive of a bone tumor. Computed tomography demonstrated an intraosseous lytic lesion with associated cortical thinning and lack of periosteal reaction. On magnetic resonance imaging, the lesion exhibited slightly higher signal intensity compared to skeletal muscle on T1-weighted sequences and heterogeneous high signal intensity on T2-weighted sequences. Strong enhancement was observed following gadolinium administration. The lesion was treated by extensive curettage with adjuvant therapy comprising ethanol and the remaining cavity was filled with polymethylmethacrylate bone cement. Histologically, the lesion was composed of round or spindle-shaped mononuclear cells admixed with numerous osteoclast-like giant cells. Immunohistochemically, the mononuclear neoplastic cells were diffusely positive for H3.3 G34W. The patient was asymptomatic and there was no evidence of local recurrence or distant metastasis 5 months after surgery. CONCLUSION: Although rare, acromial GCTB should be considered in the differential diagnosis of posterior shoulder pain, especially in young and early middle-aged adults.

Diagnostic value of H3.3G34W,p63 and SATB2 immunohistochemical staining combined in giant cell tumor of bone / 西安交通大学学报(医学版)

【Objective】 To investigate the expressions of H3.3G34W, p63 and SATB2 in giant cell tumor of bone (GCTB) and the effect and value of their combined application in the diagnosis of GCTB. 【Methods】 We collected the samples and medical records of 54 cases of GCTB and 83 cases of non-giant cell tumor of bone (14 cases of aneurysmal bone cyst, 16 cases of chondroblastoma and 53 cases of non-ossifying fibroma) diagnosed between 2020 and 2022 in the Department of Pathology of Honghui Hospital Affiliated to Xi’an Jiaotong University. The expressions of H3.3G34W, p63 and SATB2 were detected by EliVision immunohistochemical method. χ2 test was used to determine whether there are significant differences in the positive rates of H3.3G34W, p63 and SATB2 among all the groups. The combined diagnostic model including H3.3G34W, p63 and SATB2 was established by Logistic regression analysis, and the diagnostic value of the model was evaluated by ROC curve analysis. 【Results】 The positive rates of H3.3G34W, p63 and SATB2 in GCTB group were 81.5%, 90.7% and 92.6%, respectively; the positive rates in NGCTB group were 2.4%, 28.9% and 62.7%. Compared with NGCTB group, the age of GCTB group was significantly older [(41.222±14.849) vs. (16.566±9.439) , P<0.001] , and the prevalence was higher in women than in men (51.9% vs. 48.1%, P<0.001). In addition, compared with the NGCTB group, the positive rates of H3.3G34W (81.5% vs. 2.4%, P<0.001), p63 (90.7% vs. 28.9%, P<0.001) and SATB2 (92.6% vs. 62.7%, P<0.001) were significantly higher in the GCTB group. Univariate regression analysis built a univariate prediction model and ROC curve analysis showed that age (AUC=92.9%, P<0.001), sex (AUC=64.5%, P=0.004), H3.3G34W positive rate (AUC=89.5%, P<0.001), p63 positive rate (AUC=80.9%, P<0.001) and SATB2 positive rate (AUC=65.0%, P=0.003) were independent predictors of diagnosis of giant cell tumor of bone. Multivariate regression analysis (Logistic) constructed a hybrid prediction model. ROC curve analysis suggested that the hybrid model showed better prediction value than the single factor model (AUC=98.4%, P<0.001). 【Conclusion】 H3.3G34W, p63 and SATB2 are effective molecular markers for the diagnosis of GCTB, and their combined application can improve the prediction efficiency of the diagnosis of GCTB.

Pediatric glioma histone H3.3 K27M/G34R mutations drive abnormalities in PML nuclear bodies.

BACKGROUND: Point mutations in histone variant H3.3 (H3.3K27M, H3.3G34R) and the H3.3-specific ATRX/DAXX chaperone complex are frequent events in pediatric gliomas. These H3.3 point mutations affect many chromatin modifications but the exact oncogenic mechanisms are currently unclear. Histone H3.3 is known to localize to nuclear compartments known as promyelocytic leukemia (PML) nuclear bodies, which are frequently mutated and confirmed as oncogenic drivers in acute promyelocytic leukemia. RESULTS: We find that the pediatric glioma-associated H3.3 point mutations disrupt the formation of PML nuclear bodies and this prevents differentiation down glial lineages. Similar to leukemias driven by PML mutations, H3.3-mutated glioma cells are sensitive to drugs that target PML bodies. We also find that point mutations in IDH1/2-which are common events in adult gliomas and myeloid leukemias-also disrupt the formation of PML bodies. CONCLUSIONS: We identify PML as a contributor to oncogenesis in a subset of gliomas and show that targeting PML bodies is effective in treating these H3.3-mutated pediatric gliomas.