H3K4 Methylation Research Articles

Characterization of Multiple Myeloma Subpopulations Associated with Treatment Resistance By Single-Cell RNA Sequencing

Multiple myeloma (MM) has extensive genomic and transcriptomic heterogeneity with identical variable-diversity-joining (VDJ) region recombination of immunoglobulin loci (called repertoire). Even though new treatment approaches have been developed, most cases of MM remain incurable. Recent evidence suggests that epigenetic cell plasticity and changes in cell state underlie the therapy resistance, but the mechanisms remain largely unknown. We attempted to understand the comprehensive architecture of the entire tumor cell populations using repertoire clonality as a fingerprint. First, we subdivided bone marrow samples of primary MM at different stages (n=8) into 11 fractions and performed bulk RNA sequencing (bulk RNA-seq). Analysis of the repertoire revealed that tumor cells were present at very low frequencies in some minor fractions other than the main fraction (Lineage marker - (Lin -)/CD19 -/CD38 +/CD138 +) as the disease stage progressed. Importantly, MM cells were consistently present in CD138 negative fraction (Lin -/CD19 -/CD38 +/CD138 -) regardless of disease stage. Flow cytometry (FCM) analysis confirmed that CD138negative MM cells were present at a rate of less than 10% across the disease stages. Compared with CD138 positive cells, they were slowly cycling and showed unique morphology with large cytoplasm (n=59). To achieve a selective and comprehensive evaluation of MM population, we performed single-cell RNA sequencing (scRNA-seq) coupled with VDJ targeted sequencing (scVDJ-seq) on Lin -/CD19 -/CD38 +(without CD138 gating) samples from 11 MM patients, including those at diagnosis and in remission with minimal residual disease (MRD). This method allowed us to discriminate MM cells (20,084 cells) clearly from others by clonal repertoire sequence even in the post-treatment samples with extremely small populations of tumor cells. Because mRNA levels of CD138 did not correspond to the differential intensity in FCM, we defined gene signatures of CD138 positive and negative cells respectively by bulk RNA-seq (n=6). As a result of integration of these gene signatures using Gene Set Enrichment Analysis (GSEA), the heterogeneity on the CD138 axis was also unraveled in scRNA-seq data. Interestingly, gene ontology (GO) analysis of bulk RNA-seq data revealed that several terms related to H3K4 methylation were positively enriched in CD138 negative fraction, which included KMT2A, KMT2B, KMT2C and KMT2D. Western blotting confirmed significant reductions in H3K4me1 and H3K4me3 levels in CD138 negative cells. Furthermore, primary MM cell culture indicated the plasticity between CD138 positive and negative cells, which was also suggested by trajectory/velocity analysis of scRNA-seq data. Collectively, it was suggested that epigenetic changes, including those in histone modifications, contribute to the heterogeneity along the CD138 axis. We next compared the size of each cluster before and after treatment in scRNA-seq data to identify the MM populations associated with treatment resistance. Remarkably, we identified unique clusters which tended to remain at high proportions against treatment in the CD138 negative population. They were characterized by low expression of MHC class-I genes and TNFRSF17/BCMA, as well as 198 significantly upregulated genes including MCL1, MALAT1 and NEAT1, which have been previously implicated in treatment resistance of MM. To systematically interrogate their contribution in MM cell survival, we performed CRISPR/Cas9 knockout screening of selected 100 genes, which were differentially up-regulated in the resistant clusters and associated with poor prognosis in a MMRF CoMMpass Study database. GO analysis of indispensable genes reproducibly identified among three MM cell lines revealed enrichment of several pathways related to mRNA splicing. These results suggested that the RNA splicing pathway is a promising therapeutic target to eliminate resistant populations. Indeed, we confirmed that inhibition of a splicing factor by CRISPR/Cas9-mediated knockout and pharmacological protein degradation suppresses MM cell proliferation effectively. Taken together, we achieved a complete view of MM diversity by using repertoire clonality and confirmed heterogeneity along the CD138 axis, which may be characterized by epigenetic plasticity. Our results highlight a novel subpopulation as a candidate therapeutic target for cure.

H3K36 methyltransferase NSD1 protects against osteoarthritis through regulating chondrocyte differentiation and cartilage homeostasis.

Osteoarthritis (OA) is one of the most common joint diseases, there are no effective disease-modifying drugs, and the pathological mechanisms of OA need further investigation. Here, we show that H3K36 methylations were decreased in senescent chondrocytes and age-related osteoarthritic cartilage. Prrx1-Cre inducible H3.3K36M transgenic mice showed articular cartilage destruction and osteophyte formation. Conditional knockout Nsd1Prrx1-Cre mice, but not Nsd2Prrx1-Cre or Setd2Prrx1-Cre mice, replicated the phenotype of K36M/+; Prrx1-Cre mice. Immunostaining results showed decreased anabolic and increased catabolic activities in Nsd1Prrx1-Cre mice, along with decreased chondrogenic differentiation. Transcriptome and ChIP-seq data revealed that Osr2 was a key factor affected by Nsd1. Intra-articular delivery of Osr2 adenovirus effectively improved the homeostasis of articular cartilage in Nsd1Prrx1-Cre mice. In human osteoarthritic cartilages, both mRNA and protein levels of NSD1 and OSR2 were decreased. Our results indicate that NSD1-induced H3K36 methylations and OSR2 expression play important roles in articular cartilage homeostasis and OA. Targeting H3K36 methylation and OSR2 would be a novel strategy for OA treatment.

2-Hydroxyglutarate modulates histone methylation at specific loci and alters gene expression via Rph1 inhibition.

2-Hydroxyglutarate (2-HG) is an oncometabolite that accumulates in certain cancers. Gain-of-function mutations in isocitrate dehydrogenase lead to 2-HG accumulation at the expense of alpha-ketoglutarate. Elevated 2-HG levels inhibit histone and DNA demethylases, causing chromatin structure and gene regulation changes with tumorigenic consequences. We investigated the effects of elevated 2-HG levels in Saccharomyces cerevisiae, a yeast devoid of DNA methylation and heterochromatin-associated histone methylation. Our results demonstrate genetic background-dependent gene expression changes and altered H3K4 and H3K36 methylation at specific loci. Analysis of histone demethylase deletion strains indicated that 2-HG inhibits Rph1 sufficiently to induce extensive gene expression changes. Rph1 is the yeast homolog of human KDM4 demethylases and, among the yeast histone demethylases, was the most sensitive to the inhibitory effect of 2-HG in vitro. Interestingly, Rph1 deficiency favors gene repression and leads to further down-regulation of already silenced genes marked by low H3K4 and H3K36 trimethylation, but abundant in H3K36 dimethylation. Our results provide novel insights into the genome-wide effects of 2-HG and highlight Rph1 as its preferential demethylase target.

DOT1L interaction partner AF10 controls patterning of H3K79 methylation and RNA polymerase II to maintain cell identity

Histone 3 lysine 79 methylation (H3K79me) is enriched on gene bodies proportional to gene expression levels and serves as a strong barrier for the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). DOT1L is the sole histone methyltransferase that deposits all three orders-mono (me1), di (me2), and tri (me3) methylation-at H3K79. Here, we leverage genetic and chemical approaches to parse the specific functions of orders of H3K79me in maintaining cell identity. DOT1L interacts with AF10 (Mllt10), which recognizes unmodified H3K27 and boosts H3K79me2/3 methylation. AF10 deletion evicts H3K79me2/3 and reorganizes H3K79me1 to the transcription start site to facilitate iPSC formation in the absence of steady-state transcriptional changes. Instead, AF10 loss redistributes RNA polymerase II to a uniquely pluripotent pattern at highly expressed, rapidly transcribed housekeeping genes. Taken together, we reveal a specific mechanism for H3K79me2/3 located at the gene body in reinforcing cell identity.

LEO1 Is Required for Efficient Entry into Quiescence, Control of H3K9 Methylation and Gene Expression in Human Fibroblasts.

(1) Background: The LEO1 (Left open reading frame 1) protein is a conserved subunit of the PAF1C complex (RNA polymerase II-associated factor 1 complex). PAF1C has well-established mechanistic functions in elongation of transcription and RNA processing. We previously showed, in fission yeast, that LEO1 controls histone H3K9 methylation levels by affecting the turnover of histone H3 in chromatin, and that it is essential for the proper regulation of gene expression during cellular quiescence. Human fibroblasts enter a reversible quiescence state upon serum deprivation in the growth media. Here we investigate the function of LEO1 in human fibroblasts. (2) Methods: We knocked out the LEO1 gene using CRISPR/Cas9 methodology in human fibroblasts and verified that the LEO1 protein was undetectable by Western blot. We characterized the phenotype of the ΔLEO1 knockout cells with FACS analysis and cell growth assays. We used RNA-sequencing using spike-in controls to measure gene expression and spike-in controlled ChIP-sequencing experiments to measure the histone modification H3K9me2 genome-wide. (3) Results: Gene expression levels are altered in quiescent cells, however factors controlling chromatin and gene expression changes in quiescent human cells are largely unknown. The ΔLEO1 knockout fibroblasts are viable but have reduced metabolic activity compared to wild-type cells. ΔLEO1 cells showed a slower entry into quiescence and a different morphology compared to wild-type cells. Gene expression was generally reduced in quiescent wild-type cells. The downregulated genes included genes involved in cell proliferation. A small number of genes were upregulated in quiescent wild-type cells including several genes involved in ERK1/ERK2 and Wnt signaling. In quiescent ΔLEO1 cells, many genes were mis-regulated compared to wild-type cells. This included genes involved in Calcium ion transport and cell morphogenesis. Finally, spike-in controlled ChIP-sequencing experiments demonstrated that the histone modification H3K9me2 levels are globally increased in quiescent ΔLEO1 cells. (4) Conclusions: Thus, LEO1 is important for proper entry into cellular quiescence, control of H3K9me2 levels, and gene expression in human fibroblasts.

DOT1L is a barrier to histone acetylation during reprogramming to pluripotency.

Embryonic stem cells (ESCs) have transcriptionally permissive chromatin enriched for gene activation-associated histone modifications. A striking exception is DOT1L-mediated H3K79 dimethylation (H3K79me2) that is considered a positive regulator of transcription. We find that ESCs are depleted for H3K79me2 at shared locations of enrichment with somatic cells, which are highly and ubiquitously expressed housekeeping genes, and have lower RNA polymerase II (RNAPII) at the transcription start site (TSS) despite greater nascent transcription. Inhibiting DOT1L increases the efficiency of reprogramming of somatic to induced pluripotent stem cells, enables an ESC-like RNAPII pattern at the TSS, and functionally compensates for enforced RNAPII pausing. DOT1L inhibition increases H3K27 methylation and RNAPII elongation-enhancing histone acetylation without changing the expression of the causal histone-modifying enzymes. Only the maintenance of elevated histone acetylation is essential for enhanced reprogramming and occurs at loci that are depleted for H3K79me2. Thus, DOT1L inhibition promotes the hyperacetylation and hypertranscription pluripotent properties.

EPCO-33. H3K4 METHYLATION IS A PREREQUISITE OF CELL FATE CHANGE DURING IDH-MUTANT GLIOMAGENESIS

Abstract RATIONALE: Low grade gliomas (LGGs) are defined by isocitrate dehydrogenase (IDH)-mutations that mediate broad epigenetic changes. However, additional determinants of low-grade gliomagenesis remain unknown, including factors necessary for the activation of LGG gene programs. Here, we aim at identifying epigenetic prerequisites of IDH-dependent gliomagenesis by modulating key chromatin modifiers in a conditionally IDHR132H-expressing immortalized human astrocyte (IHA) model system. METHODS: Using chemical compounds, we targeted key histone modifications and DNA methylation in an inducibly IDHR132H expressing IHA cell line. Flow cytometry for the LGG-associated marker L1CAM served as a primary read-out. Compound candidates were further assessed with RNA-Seq and mass spectrometry. Phenotypic evaluation included sphere formation, clonogenicity, cell viability and invasion assays. Relevant findings were additionally assessed in established LGG cell lines TS603, NCH612 and NCH1681. RESULTS: L1CAM was significantly altered by DOT1L inhibitor EPZ00477, MLL1 inhibitor MM-102, and EZH2 inhibitor GSK-126. MM-102 treatment was associated with broad transcriptional downregulation. Intersecting data from MM-102 and GSK126 treated cells, we identified potential bivalent genes directly affected by inhibitor treatment that were enriched for Wnt signaling and developmental transcription factors implicated in gliomagenesis. Overlapping transcriptional and proteomics data, MM-102-treated cells exhibited a signature of predominantly downregulated genes associated with invasion/migration, extracellular matrix (ECM) and cholesterol metabolism. Fittingly, cells showed impaired invasion, clonal proliferation and sphere forming capacity. The negative effect on sphere formation tracked inversely with aggressiveness of established LGG cell lines. CONCLUSION: Interfering with histone H3K4 methylation through MLL1 inhibition is associated with broad downregulation of gene sets related to cell identity and ECM/migration, reflected by disruption of clonal proliferation in IDHR132H astrocytes and LGG cell lines. This points to deposition of H3K4 methylation as a prerequisite of cell fate change in LGG development. Persisting effects on established LGG cell lines warrant its investigation as a potential therapeutic target.

BIOM-30. CELL LINE STUDY OF NUCLEOSOME-BASED BIOMARKERS IN THE DIAGNOSIS AND DETECTION OF RELAPSES IN GLIOBLASTOMA

Abstract Currently, glioblastoma (GBM) diagnosis and monitoring rely on neuroimaging and histopathological confirmation. However, overall survival has not improved in last decades due to therapeutic failure and to a lack of biomarkers for relapses’ detection. Liquid biopsies (i.e. blood or CSF) using nucleosomes-containing-histone post-translational modifications (PTMs) have the potential to become valuable biomarkers for diagnosis and monitoring GBM. Four glioblastoma cell lines (SF-126, U-87MG, U-118MG, and U-138MG) compared to a healthy microglia cell line (HMC3) and other solid cancer cell lines including pancreas (MIA-PaCa-2), liver (HepG2), and cervix/uterus (HeLa) have been analyzed to identify their epigenetic profile. Nucleosomes were extracted and analyzed with the Nu.Q® Discover immunoassays platform addressing 13 histone-PTMs. Quantitative results of PTMs expression were normalized on quantification of total H3.1-nucleosomes. The immunoassay identified three candidate biomarkers compared to control cell lines (n = 4): citrullinated-histone-H3 (H3R8Cit) (control = 4.68%; GBM = 13.88%; p< 0.05), phosphorylation of H3S10 (H3S10Ph) (control = 2.31%; GBM = 5.57%; p< 0.05), and a trend in H3K4Me2 elevation (control = 0.43%; GBM = 1.07%; p = 0.066) in GBM cell lines (n = 4). These results were confirmed by western blot. Moreover, principal component analysis revealed a segregation between GBM and control cells, mainly driven by these three PTMs. Moreover, to demonstrate the capacity of the Nu.Q® assays to quantitatively monitor PTMs, GBM cell line SF-126 (n = 3) was treated with an EZH2 inhibitor (responsible of H3K27 methylation) (iEZH2). After 24 hours and 48 hours of exposure, the expression level of H3K27Me3 –expressed as ratio of total nucleosomes– was decreased by 32% (control = 14.67%±0.58%; iEZH2= 10.00%±1.73%; p< 0.05) and 41% (control = 15.33%±1.53%; iEZH2= 9.00%±0.00%; p< 0.05), respectively. Those results were also confirmed by western blot analysis. In conclusion, we identified three specific in vitro epigenetic-based marks of GBM and demonstrated that the Nu.Q® technology is a valuable tool to monitor the degree of PTMs expression.

Histone retention preserves epigenetic marks during heat stress-induced transcriptional memory in plants.

Plants often experience recurrent stressful events, for example, during heat waves. They can be primed by heat stress (HS) to improve the survival of more severe heat stress conditions. At certain genes, sustained expression is induced for several days beyond the initial heat stress. This transcriptional memory is associated with hyper-methylation of histone H3 lysine 4 (H3K4me3), but it is unclear how this is maintained for extended periods. Here, we determined histone turnover by measuring the chromatin association of HS-induced histone H3.3. Genome-wide histone turnover was not homogenous; in particular, H3.3 was retained longer at heat stress memory genes compared to HS-induced non-memory genes during the memory phase. While low nucleosome turnover retained H3K4 methylation, methylation loss did not affect turnover, suggesting that low nucleosome turnover sustains H3K4 methylation, but not vice versa. Together, our results unveil the modulation of histone turnover as a mechanism to retain environmentally mediated epigenetic modifications.

Photoaged Polystyrene Nanoplastics Result in Transgenerational Reproductive Toxicity Associated with the Methylation of Histone H3K4 and H3K9 in Caenorhabditis elegans.

Polystyrene nanoplastics (PS-NPs) are emerging environmental contaminants that are ubiquitously detected in various environments and have toxic effects on various organisms. Nevertheless, the transgenerational reproductive toxicity and underlying mechanisms of PS-NPs remain largely unknown, especially for photoaged PS-NPs under ultraviolet irradiation. In this study, only the parental generation (P0) was exposed to virgin and aged PS-NPs at environmentally relevant concentrations (0.1-100 μg/L), and subsequent generations (F1-F4) were cultured under normal conditions. Ultraviolet irradiation induced the generation of environmentally persistent free radicals and reactive oxygen species, which altered the physical and chemical characteristics of PS-NPs. The results of toxicity testing suggested that exposure to aged PS-NPs caused a more severe decrease in brood size, egg ejection rate, number of fertilized eggs, and hatchability than did the virgin PS-NPs in the P0, F1, and F2 generations. Additionally, a single maternal exposure to aged PS-NPs resulted in transgenerational effects on fertility in the F1 and F2 generations. Increased levels of H3K4 and H3K9 methylation were observed in the F1 and F2 generations, which were concomitant with the transgenerational downregulation of the expression of associated genes, such as spr-5, set-17, and met-2. On the basis of correlation analyses, the levels of histone methylation and the expression of these genes were significantly correlated to transgenerational reproductive effects. Further research showed that transgenerational effects on fertility were not observed in spr-5(by134), met-2(n4256), and set-17(n5017) mutants. Overall, maternal exposure to aged PS-NPs induced transgenerational reproductive effects via H3K4 and H3K9 methylation, and the spr-5, met-2, and set-17 genes were involved in the regulation of transgenerational toxicity. This study provides new insights into the potential risks of photoaging PS-NPs in the environment.

Abstract 17329: Histone Modifications Regulate Hypertrophy Pathways in Obstructive Hypertrophic Cardiomyopathy

Introduction: Recently, we demonstrated transcriptional down-regulation of hypertrophy pathways in myectomy tissue derived from patients with obstructive hypertrophic cardiomyopathy (HCM) despite translational activation of associated hypertrophy pathways. Previous studies of other forms of cardiac hypertrophy demonstrate hypertrophy pathways are activated via histone acetylation and histone deacetylase (HDAC) inhibitors prevented development of hypertrophy. Whether histone modifications regulate hypertrophy pathways in HCM remains unexplored. Methods: Chromatin immunoprecipitation sequencing (ChIP-seq) was performed on myectomy tissues from 40 patients with HCM and 23 controls to characterize differential histone marks for H3K4me3 (active promoter), H3K9ac (active promoter), H3K9me3 (heterochromatin), H3K27ac (active enhancer), H3K27me3 (repression) across the genome. The differential histone marks were integrated with RNA-sequencing (RNA-seq) data derived from the same patients. Results: Overall, we identified 2,912 (7%) differential H3K4me3 peaks impacting 5 pathways, 23,339 (21%) differential H3K9ac peaks impacting 87 pathways, 33 (0.05%) differential H3K9me3 peaks, and 58,837 (42%) differential H3K27ac peaks impacting 50 pathways. There was increased H3K27ac (q-value = 0.002) and H3K9ac (q-value = 0.009) at RAS-MAPK loci. Univariate analysis of concordance between H3K9ac with RNA-seq data showed activation of cardiac hypertrophy signaling (z-score=4.5; -log [BH p-value] = 3.1), while H3K27me showed down-regulation of cardiac hypertrophy signaling (z-score=-2.4; -log [BH p-value] = 2.4). Multivariate integration of the ChIP-seq data with RNA-seq data identified 3,189 transcripts concordant with all five histone marks impacting 254 pathways, 18 (7%) of which are involved in cardiac hypertrophy. Conclusions: Myectomy tissue from patients with obstructive HCM shows enrichment of histone modifications at cardiac hypertrophy loci with activation via H3K27 and H3K9 acetylation and repression via H3K27 methylation. Further studies are needed to understand the balance and temporal relationship between these competing regulators in HCM pathobiology and the potential for epigenetic therapies.

Reduced Polycomb Repressor Complex 2 (PRC2) Activity and Increased TP53 Activity Mediate Hematopoietic Stem Cell Dysfunction in RPS19-Mutated Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia caused by heterozygous loss-of-function mutations in one of 23 ribosomal protein (RP) genes, with RPS19 being mutated in approximately 25% of patients. While DBA typically manifests in infancy as isolated anemia with reticulocytopenia, older patients can develop multilineage cytopenias and bone marrow (BM) hypocellularity suggesting impaired hematopoietic stem cell (HSC) function. Defects in HSCs have been observed in Rps19-depleted mice but studies on human DBA HSCs have been inconclusive, largely because patient BM cells are difficult to obtain for research. To address this gap, we developed a new, robust, and scalable cellular model using CRISPR/Cas9 to create heterozygous RPS19 loss-of-function mutations in healthy donor CD34 + hematopoietic stem and progenitor cells (HSPCs). Transplantation of RPS19+/- HSPCs into NBSGW immunodeficient mice revealed a defect in BM repopulation that was partially rescued by co-disruption of the TP53 gene (Bhoopalan et al, JCI Insight, 2023). Here we provide further mechanistic insight into this HSC defect by showing that RP imbalance caused by RPS19 deficiency leads to the activation of TP53 and inhibition of Polycomb Repressor Complex 2 (PRC2), which has been previously shown to be required for HSC maintenance. Healthy donor CD34 + HSPC were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 safe harbor locus as a negative control. RPS19 disruption was verified by next-generation sequencing of the targeted region and by Western blot analysis showing reduction of RPS19 protein. RNA-Seq at 3 days after electroporation showed enrichment of TP53 pathway genes in RPS19-edited HSPCs compared to control cells, confirming our previous observations. Additionally, we noted an enrichment of HSPC genes that were previously shown to harbor the repressive histone mark H3K27me3 in normal HSPCs. Methylation of H3K27 is catalyzed by EZH2, the catalytic component of PRC2, which maintains HSCs by inhibiting transcription of genes that promote differentiation. Gene set enrichment analysis (GSEA) revealed significant induction of PRC2 target genes in RPS19-disrupted HSPCs and in induced pluripotent stem cells (iPSCs) generated from two different DBA patients with heterozygous RPS19 mutations. The “Cleavage under targets and release using nuclease (CUT&RUN)” assay indicated globally reduced EZH2 binding to chromatin in RPS19-disrupted HSPCs. These findings suggest that PRC2 activity is inhibited by RPS19 haploinsufficiency. Previous studies have shown that RPS19 haploinsufficiency can cause accumulation of large RP subunits, RPL5 and RPL11, which bind 5S ribosomal RNA to form 5S RNP. This complex can sequester the ubiquitin ligase MDM2 to inhibit its function. MDM2 ubiquitinates TP53, targeting it for proteasomal degradation. Additionally, MDM2 binds PRC2 to augment its activity. Thus, we theorized that RPS19 haploinsufficiency leads to the accumulation of 5S RNP complex which sequesters MDM2, causing increased TP53 activity and suppression of PRC2 activity, both of which can inhibit HSC maintenance. To test this, we inhibited formation of 5S RNP complex by co-disrupting RPL5 or RPL11 along with RPS19 in CD34 + HSPCs, followed by RNA-Seq analysis or transplantation into NBSGW mice. Remarkably, haploinsufficiency of either large RP gene rescued the BM repopulation defect of RPS19 +/- HSPC (p<0.05) ( Fig. 1A), reduced TP53 activity and improved PRC2 activity (p<0.001 and FDR q<0.01) ( Fig. 1B). In summary, our data support the model that RPS19 haploinsufficiency leads to increased TP53 activity and reduced PRC2 activity through 5S RNP-mediated sequestration of MDM2, causing impairment of HSC function. These studies provide insights into the molecular mechanisms of DBA and could potentially inform new therapies.

Histone Lysine Methyltransferase MMSET : An Essential Regulator of Human Erythropoiesis.

Erythropoiesis is the process by which hematopoietic stem cells proliferate, differentiate, and eventually form mature erythrocytes. Steady-state erythropoiesis is the result of the coordinated efforts of transcription factors and epigenetic regulators during differentiation. Histone methyltransferases (HMTs) are critical regulators that drive activation and repression of histone marks during erythroid lineage progression. MMSET (multiple myeloma SET domain) protein is aH3K36 methyltransferase (HKMT) required for normal hematopoietic development and essential for B cell differentiation and mature B cell function. Knockout of MMSET induces Wolf-Hirschhorn syndrome, where lymphocytes show abnormal functions, including a deficiency in antibody production. Studies have highlighted that MMSET acts as an oncogenic driver, classically associated with solid tumours, with a special focus on Multiple myeloma. However, the biological role and function of MMSET in human erythropoiesis is untouched and needs to be explored to address its role in the erythroid cascade. To this end, we examined the role of MMSET in human erythropoiesis by studying the genotypic and phenotypic modulatory effects observed upon MMSET knockdown (KD) in erythroid cells. Erythroid cells were generated from human G-CSF mobilized peripheral blood, hematopoietic stem and progenitor cells (HSPCs) as described by (Palii et al.,2011). Cultured cells were transduced with a lentiviral vector (pLKO.1) expressing MMSET andKD was observed after 72 hours of induction. 70-80% KD of MMSET in HSPCs resulted in reduced BFU-Es and CFU-Es in the semi-solid assay (Methocult TM) as compared to the control (pLKO.1 carrying scrambled sequence) (Fig. A), which was co-validated by FACS on days 2 and 4 (Phase I). Taken together, these results indicate that MMSET is required for early erythroid differentiation. Further, to access the role of MMSET during terminal stages of erythroid differentiation, lentiviral-mediated KD of late erythroblasts (days 8-12) (phase II) was done. MMSET KD erythroblasts displayed a block in terminally differentiating (Polychromatic and Orthochromatic) erythroblasts with an increase in Proerythroblasts and Basophilic erythroblasts. Thus, our results indicate that MMSET KD at later stages is associated with disordered terminal erythropoiesis. H3K36 methylation marks are involved in the regulation of DNA replication, recombination, and repair. Specifically, these marks keep DNA replication in check; hence, we noted that loss of MMSET correlates with increased transition of G2M phase cells from S phase cells with respect to control. In lieu of the above context, we noted increased proliferation (via Click-It Edu) in both phases (I and II). Studies have shown that MMSET is associated with DNA damage and repair (Evans et al., 2016). DNA damage and fragmentation mitigates apoptosis as a result of the signalling cascade downstream of effector caspases. We, therefore, observed a significant increase in apoptotic cells (via Annexin/PI staining) in both early and late erythroblasts upon KD of MMSET. To further gain insight, we carried out whole transcriptome analysis upon KD of MMSET and observed differentially expressed erythroid genes at three time points (days 4, 8, and 12) which revealed that GATA1, ALAS2, TAL1, KLF1, and FECH were significantly altered at the specified days (Fig. B). We further observed that erythrocyte development and differentiation, followed by regulation of the G2M transition of the cell cycle, apoptosis (programmed cell death), and cell proliferation, were the most enriched processes. Thus, in the present study, we systematically characterised erythroid impairment developing upon MMSET KD, leading to global downregulation of erythroid-specific genes, which are key players in normal erythroid development. An in-depth assessment of the MMSET -mediated erythroid gene regulatory network needs elucidation for further validation of MMSET as a direct player or mediator in human erythroid development. Our findings uncovered a previously unknown role for MMSET in human erythropoiesis.

Vitamin B12 is a limiting factor for induced cellular plasticity and tissue repair

Transient reprogramming by the expression of OCT4, SOX2, KLF4 and MYC (OSKM) is a therapeutic strategy for tissue regeneration and rejuvenation, but little is known about its metabolic requirements. Here we show that OSKM reprogramming in mice causes a global depletion of vitamin B12 and molecular hallmarks of methionine starvation. Supplementation with vitamin B12 increases the efficiency of reprogramming both in mice and in cultured cells, the latter indicating a cell-intrinsic effect. We show that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters (cryptic transcription), is sensitive to vitamin B12 levels, providing evidence for a link between B12 levels, H3K36 methylation, transcriptional fidelity and efficient reprogramming. Vitamin B12 supplementation also accelerates tissue repair in a model of ulcerative colitis. We conclude that vitamin B12, through its key role in one-carbon metabolism and epigenetic dynamics, improves the efficiency of in vivo reprogramming and tissue repair.

Uncoupling the distinct functions of HP1 proteins during heterochromatin establishment and maintenance

H3K9 methylation (H3K9me) marks transcriptionally silent genomic regions called heterochromatin. HP1 proteins are required to establish and maintain heterochromatin. HP1 proteins bind to H3K9me, recruit factors that promote heterochromatin formation, and oligomerize to form phase-separated condensates. We do not understand how these different HP1 properties are involved in establishing and maintaining transcriptional silencing. Here, we demonstrate that the S.pombe HP1 homolog, Swi6, can be completely bypassed to establish silencing at ectopic and endogenous loci when an H3K4 methyltransferase, Set1, and an H3K14 acetyltransferase, Mst2, are deleted. Deleting Set1 and Mst2 enhances Clr4 enzymatic activity, leading to higher H3K9me levels and spreading. In contrast, Swi6 and its capacity to oligomerize were indispensable during epigenetic maintenance. Our results demonstrate the role of HP1 proteins in regulating histone modification crosstalk during establishment and identify a genetically separable function in maintaining epigenetic memory.

TOR inactivation triggers heterochromatin formation in rDNA during glucose starvation

In response to environmental cues, such as nutrient starvation, living organisms modulate gene expression through mechanisms involving histone modifications. Specifically, nutrient depletion inactivates the TOR (target of rapamycin) pathway, leading to reduced expression of ribosomal genes. While these regulatory mechanisms are well elucidated in budding yeast Saccharomyces cerevisiae, their conservation across diverse organisms remains unclear. In this study, we demonstrate that fission yeast Schizosaccharomyces pombe cells repress ribosomal gene transcription through a different mechanism. TORC1, which accumulates in the rDNA region, dissociates upon starvation, resulting in enhanced methylation of H3K9 and heterochromatin formation, facilitated by dissociation of the stress-responsive transcription factor Atf1 and accumulation of the histone chaperone FACT. We propose that this mechanism might be adapted in mammals that possess Suv39H1 and HP1, which are absent in budding yeast.

Disruption of SUV39H1-Mediated H3K9 Methylation Sustains CAR T-cell Function.

T cells engineered with CD28-based CARs possess robust effector function and antigen sensitivity but are hampered by limited persistence, which may result in tumor relapse. We report an epigenetic strategy involving disruption of the SUV39H1-mediated histone-silencing program that promotes the functional persistence of CD28-based CAR T cells. See related article by López-Cobo et al., p. 120. This article is featured in Selected Articles from This Issue, p. 5.

SETD4 inhibits prostate cancer development by promoting H3K27me3-mediated NUPR1 transcriptional repression and cell cycle arrest

The suppressor of variegation enhancer of zeste-trithorax (SET) domain methyltransferases have been reported to function as key regulators in multiple tumor types by catalyzing histone lysine methylation. Nevertheless, our understanding on the role of these lysine methyltransferases, including SETD4, in prostate cancer (PCa) remains limited. Hence, the specific role of SETD4 in PCa was investigated in this study. The expression of SETD4 in PCa cells and tissue samples was downregulated in PCa cells and tissue specimens, and decreased SETD4 expression led to inferior clinicopathological characteristics in patients with PCa. knockdown of SETD4 facilitated the proliferation of PCa cells and accelerated cell cycle progression. Mechanistically, SETD4 repressed NUPR1 transcription by methylating H3K27 to generate H3K27me3, subsequently inactivated Akt pathway and impeded the tumorigenesis of PCa. Our results highlight that SETD4 prevents the development of PCa by catalyzing the methylation of H3K27 and suppressing NUPR1 transcription, subsequently inactivating the Akt signaling pathway. The findings suggest the potential application of SETD4 in PCa prognosis and therapeutics.

ATM, KAP1and the Epstein-Barr virus polymerase processivity factor direct traffic at the intersection of transcription and replication.

The timing of transcription and replication must be carefully regulated for heavily-transcribed genomes of double-stranded DNA viruses: transcription of immediate early/early genes must decline as replication ramps up from the same genome-ensuring efficient and timely replication of viral genomes followed by their packaging by structural proteins. To understand how the prototypic DNA virus Epstein-Barr virus tackles the logistical challenge of switching from transcription to DNA replication, we examined the proteome at viral replication forks. Specifically, to transition from transcription, the viral DNA polymerase-processivity factor EA-D is SUMOylated by the epigenetic regulator and E3 SUMO-ligase KAP1/TRIM28. KAP1's SUMO2-ligase function is triggered by phosphorylation via the PI3K-related kinase ATM and the RNA polymerase II-associated helicase RECQ5 at the transcription machinery. SUMO2-EA-D then recruits the histone loader CAF1 and the methyltransferase SETDB1 to silence the parental genome via H3K9 methylation, prioritizing replication. Thus, a key viral protein and host DNA repair, epigeneticand transcription-replication interference pathways orchestrate the handover from transcription-to-replication, a fundamental feature of DNA viruses.

BRCA1-BARD1 combines multiple chromatin recognition modules to bridge nascent nucleosomes.

Chromatin association of the BRCA1-BARD1 heterodimer is critical to promote homologous recombination repair of DNA double-strand breaks (DSBs) in S/G2. How the BRCA1-BARD1 complex interacts with chromatin that contains both damage induced histone H2A ubiquitin and inhibitory H4K20 methylation is not fully understood. We characterised BRCA1-BARD1 binding and enzymatic activity to an array of mono- and di-nucleosome substrates using biochemical, structuraland single molecule imaging approaches. We found that the BRCA1-BARD1 complex preferentially interacts and modifies di-nucleosomes over mono-nucleosomes, allowing integration of H2A Lys-15 ubiquitylation signals with other chromatin modifications and features. Using high speed- atomic force microscopy (HS-AFM) to monitor how the BRCA1-BARD1 complex recognises chromatin in real time, we saw a highly dynamic complex that bridges two nucleosomes and associates with the DNA linker region. Bridging is aided by multivalent cross-nucleosome interactions that enhance BRCA1-BARD1 E3 ubiquitin ligase catalytic activity. Multivalent interactions across nucleosomes explain how BRCA1-BARD1 can recognise chromatin that retains partial di-methylation at H4 Lys-20 (H4K20me2), a parental histone mark that blocks BRCA1-BARD1 interaction with nucleosomes, to promote its enzymatic and DNA repair activities.