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An ideal chemotherapeutic agent damages DNA, specifically in cancer cells, without harming normal cells. Recently, we used Box A of HMGB1 plasmid as molecular scissors to produce DNA gaps in normal cells. The DNA gap relieves DNA tension and increases DNA strength, preventing DNA double-strand breaks (DSBs). Since the formation of HMGB1-produced DNA gaps in cancers may differ from normal cells, the outcome of introducing Box A into cancer cells may be different. We demonstrated that in lung cancer cells, γH2AX foci and histone modification associating DSBs were produced by Box A. We transfected Box A plasmid into lung cancer cell lines to overexpress Box A and evaluated the expression levels of γH2AX foci and other DNA damage response (DDR) signaling cascade markers, including ATM, ATR, and p53. Then, we demonstrated the downstream effects of DSBs on lung cancer, lowering cell proliferation, decreasing cell migration, and promoting apoptosis. Thus, Box A in lung cancer promoted the opposite outcome to normal cells by breaking cancer DNA., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Sequence-dependent mechanical properties of DNA could play essential roles in nuclear processes by affecting histone-DNA interactions. Previously, we found that the DNA entry site of the first nucleosomes from the transcription start site (+ 1 nucleosome) in budding yeast enriches AA/TT steps, but not the exit site, and the biased presence of AA/TT in the entry site was associated with the transcription levels of yeast genes. Because AA/TT is a rigid dinucleotide step, we considered that AA/TT causes DNA unwrapping. However, our previous MNase-seq experiments with reconstituted nucleosomes left some doubt regarding this interpretation, owing to its high exonuclease activity. Furthermore, MNase cleavage did not provide direct evidence of its structural state. In this study, Förster resonance energy transfer (FRET) measurements were used to investigate salt-induced conformational changes in nucleosomal DNA containing AA/TT repeats at the entry site. We observed that the AA/TT region wrapped around the histone core was as likely as other DNA sequences at physiological salt concentrations. However, it unwrapped at a lower salt concentration, indicating weaker electrostatic interactions with the histone core. Ethidium-induced nucleosome disruption assay showed that the intercalator had greater access to DNA with AA/TT at the entry site. Taken together, these results suggest that AA/TT at the entry sites induces DNA unwrapping from the histone core on the promoter side, which may promote transcriptional activation in response to the approach of transcription-related proteins., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The cytotoxic mechanisms of thymidylate synthase inhibitors, such as the multitarget antifolate pemetrexed, are not yet fully understood. Emerging evidence indicates that combining pemetrexed with histone deacetylase inhibitors (HDACi) may enhance therapeutic efficacy in non-small cell lung cancer (NSCLC). To explore this further, A549 NSCLC cells were treated with various combinations of pemetrexed and the HDACi MS275 (Entinostat), and subsequently assessed for cell viability, cell cycle changes, and genotoxic markers. Proteomic alterations were analyzed using label-free shotgun and targeted LC-MS/MS. MS275 enhanced the sensitivity of A549 cells to pemetrexed, but only when administered following prior treatment with pemetrexed. Both HeLa (p53 negative) and A549 (p53 positive) showed robust activation of γH2AX upon treatment with this combination. Importantly, CRISPR/Cas9 knockout of the uracil-DNA glycosylase UNG did not affect γH2AX activation or sensitivity to pemetrexed. Proteomic analysis revealed that MS275 altered the expression of known pemetrexed targets, as well as several proteins involved in pyrimidine metabolism and DNA repair, which could potentiate pemetrexed cytotoxicity. Contrary to the conventional model of antifolate toxicity, which implicates futile cycles of uracil incorporation and excision in DNA, we propose that ribonucleotide incorporation in nuclear and mitochondrial DNA significantly contributes to the cytotoxicity of antifolates like pemetrexed, and likely also of fluorinated pyrimidine analogs. HDAC inhibition apparently exacerbates cytotoxicity of these agents by inhibiting error-free repair of misincorporated ribonucleotides in DNA. The potential of HDACis to modulate pyrimidine metabolism and DNA damage responses offers novel strategies for improving NSCLC outcomes., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world and is associated with an overall poor prognosis. The protein methyltransferase SET and MYND domain-containing 3 (SMYD3), which trimethylates H3K4, activates gene transcription and enhances several oncogenic pathways, including epithelial-mesenchymal transition and cell cycle related pathways, in various cancer types. It was also recently shown that SMYD3 is overexpressed in HPV-negative HNSCC, and represses the expression of type I IFN response genes, contributing to resistance to anti-PD-1 checkpoint blockade in this disease. In this study, we show that SMYD3 depletion using siRNA interference or CRISPR decreases cellular proliferation and clonal capacity, induces cell cycle arrest and decreases the invasive potential of HPV-negative HNSCC cell lines. Accordingly, xenografts of SMYD3 knockout tumors derived from a human HPV-negative HNSCC cell line grew significantly slower compared to control tumors in mice. Genome-wide mapping for SMYD3 and H3K4me3 in HPV-negative HNSCC cells using cleavage under targets and release using nuclease (CUT&RUN) assays identified direct downstream gene targets regulated by SMYD3, including cell cycle- and EMT-promoting genes. This study provides insights into the epigenetic role of SMYD3 as an oncogene in HPV-negative HNSCC and supports SMYD3 as a rational therapeutic target in HPV-negative HNSCC., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Histone H3 monoaminylations at Gln5 represent an important family of epigenetic marks in brain that have critical roles in permissive gene expression 1-3 . We previously demonstrated that serotonylation 4-10 and dopaminylation 9,11-13 of Gln5 of histone H3 (H3Q5ser and H3Q5dop, respectively) are catalysed by transglutaminase 2 (TG2), and alter both local and global chromatin states. Here we found that TG2 additionally functions as an eraser and exchanger of H3 monoaminylations, including H3Q5 histaminylation (H3Q5his), which displays diurnally rhythmic expression in brain and contributes to circadian gene expression and behaviour. We found that H3Q5his, in contrast to H3Q5ser, inhibits the binding of WDR5, a core member of histone H3 Lys4 (H3K4) methyltransferase complexes, thereby antagonizing methyltransferase activities on H3K4. Taken together, these data elucidate a mechanism through which a single chromatin regulatory enzyme has the ability to sense chemical microenvironments to affect the epigenetic states of cells, the dynamics of which have critical roles in the regulation of neural rhythmicity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The "epigenetics" concept was first described in 1942. Thus far, chemical modifications on histones, DNA, and RNA have emerged as three important building blocks of epigenetic modifications. Many epigenetic modifications have been intensively studied and found to be involved in most essential biological processes as well as human diseases, including cancer. Precisely and quantitatively mapping over 100 [1], 17 [2], and 160 [3] different known types of epigenetic modifications in histone, DNA, and RNA is the key to understanding the role of epigenetic modifications in gene regulation in diverse biological processes. With the rapid development of sequencing technologies, scientists are able to detect specific epigenetic modifications with various quantitative, high-resolution, whole-genome/transcriptome approaches. Here, we summarize recent advances in epigenetic modification sequencing technologies, focusing on major histone, DNA, and RNA modifications in mammalian cells., Competing Interests: Competing interests: The authors declare no competing interests., (© 2023. The Author(s).)

Facultative heterochromatin is marked by the repressive histone modification H3K27me3 in eukaryotes. Deposited by the PRC2 complex, H3K27me3 is essential for regulating gene expression during development, and chromatin bearing this mark is generally considered transcriptionally inert. The PRC2 complex has also been linked to programmed DNA elimination during development in ciliates such as Paramecium. Due to a lack of mechanistic insight, a direct involvement has been questioned as most eliminated DNA segments in Paramecium are shorter than the size of a nucleosome. Here, we identify two sets of histone methylation readers essential for PRC2-mediated DNA elimination in Paramecium: Firefly1/2 and Mayfly1-4. The chromodomain proteins Firefly1/2 act in tight association with TFIIS4, a transcription elongation factor required for noncoding RNA transcription. These noncoding transcripts act as scaffolds for sequence-specific targeting by PIWI-bound sRNAs, resulting in local nucleosome depletion and DNA elimination. Our findings elucidate the molecular mechanism underlying the role of PRC2 in PIWI-mediated DNA elimination and suggest that its role in IES elimination may be to activate rather than repress transcription., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Identification of mutations in histones in a number of human neoplasms and developmental syndromes represents the most compelling evidence to date for a causal role of epigenetic perturbations in human disease. In most cases, these mutations have gain of function properties that cause deviation from normal developmental processes leading to embryo defects and/or neoplastic transformation. These exciting discoveries represent a step-change in our understanding of the role of chromatin (dys)regulation in development and disease. However, the mechanisms of action of oncogenic histone mutations (oncohistones) remain only partially understood. Here, we critically assess existing literature on oncohistones focussing mainly on bone neoplasms. We show how it is possible to draw parallels with some of the cell-autonomous mechanisms of action described in paediatric brain cancer, although the functions of oncohistones in bone tumours remain under-investigated. In this respect, it is becoming clear that histone mutations targeting the same residues display, at least in part, tissue-specific oncogenic mechanisms. Furthermore, it is emerging that cancer cells carrying oncohistones can modify the surrounding microenvironment to support growth and/or alter differentiation trajectories. A better understanding of oncohistone function in different neoplasms provide potential for identification of signalling that could be targeted therapeutically. Finally, we discuss some of the main concepts and future directions in this research area, while also drawing possible connections and parallels with other cancer epigenetic mechanisms., Competing Interests: Competing interests: The authors declare no competing interests., (© 2023. The Author(s).)

In early mammalian embryogenesis, a shift from non-canonical histone H3 lysine 4 trimethylation (H3K4me3) linked to transcriptional repression to canonical H3K4me3 indicating active promoters occurs during zygotic genome activation (ZGA). However, the mechanisms and roles of these H3K4me3 states in embryogenesis remain poorly understood. Our research reveals that the histone methyltransferase MLL2 is responsible for installing H3K4me3 (both non-canonical and canonical) in totipotent embryos, while a transition to SETD1A/B-deposited H3K4me3 occurs in pluripotent embryos. Interestingly, MLL2-mediated H3K4me3 operates independently of transcription, fostering a relaxed chromatin state conducive to totipotency rather than directly influencing transcription. Conversely, SETD1A/B-mediated H3K4me3, which depends on transcription, is crucial for facilitating expression of genes essential for pluripotency and pre-implantation development. Our findings highlight the role of the H3K4me3 transition, mediated by an MLL2-to-SETD1A/B relay mechanism, in the regulation of transition from totipotency to pluripotency during early embryogenesis., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

In organisms ranging from vertebrates to plants, major components of centromeres are rapidly evolving repeat sequences, such as tandem repeats (TRs) and transposable elements (TEs), which harbour centromere-specific histone H3 (CENH3) 1,2 . Complete centromere structures recently determined in human and Arabidopsis suggest frequent integration and purging of retrotransposons within the TR regions of centromeres 3-5 . Despite the high impact of 'centrophilic' retrotransposons on the paradox of rapid centromere evolution, the mechanisms involved in centromere targeting remain poorly understood in any organism. Here we show that both Ty3 and Ty1 long terminal repeat retrotransposons rapidly turnover within the centromeric TRs of Arabidopsis species. We demonstrate that the Ty1/Copia element Tal1 (Transposon of Arabidopsis lyrata 1) integrates de novo into regions occupied by CENH3 in Arabidopsis thaliana, and that ectopic expansion of the CENH3 region results in spread of Tal1 integration regions. The integration spectra of chimeric TEs reveal the key structural variations responsible for contrasting chromatin-targeting specificities to centromeres versus gene-rich regions, which have recurrently converted during the evolution of these TEs. Our findings show the impact of centromeric chromatin on TE-mediated rapid centromere evolution, with relevance across eukaryotic genomes., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

H3K27M-mutant diffuse midline gliomas (DMGs) express high levels of the disialoganglioside GD2 (ref. 1 ). Chimeric antigen receptor-modified T cells targeting GD2 (GD2-CART) eradicated DMGs in preclinical models 1 . Arm A of Phase I trial no. NCT04196413 (ref.  2 ) administered one intravenous (IV) dose of autologous GD2-CART to patients with H3K27M-mutant pontine (DIPG) or spinal DMG (sDMG) at two dose levels (DL1, 1 × 10 6  kg - 1 ; DL2, 3 × 10 6  kg -1 ) following lymphodepleting chemotherapy. Patients with clinical or imaging benefit were eligible for subsequent intracerebroventricular (ICV) intracranial infusions (10-30 × 10 6 GD2-CART). Primary objectives were manufacturing feasibility, tolerability and the identification of maximally tolerated IV dose. Secondary objectives included preliminary assessments of benefit. Thirteen patients enroled, with 11 receiving IV GD2-CART on study (n = 3 DL1 (3 DIPG); n = 8 DL2 (6 DIPG, 2 sDMG)). GD2-CART manufacture was successful for all patients. No dose-limiting toxicities occurred on DL1, but three patients experienced dose-limiting cytokine release syndrome on DL2, establishing DL1 as the maximally tolerated IV dose. Nine patients received ICV infusions, with no dose-limiting toxicities. All patients exhibited tumour inflammation-associated neurotoxicity, safely managed with intensive monitoring and care. Four patients demonstrated major volumetric tumour reductions (52, 54, 91 and 100%), with a further three patients exhibiting smaller reductions. One patient exhibited a complete response ongoing for over 30 months since enrolment. Nine patients demonstrated neurological benefit, as measured by a protocol-directed clinical improvement score. Sequential IV, followed by ICV GD2-CART, induced tumour regressions and neurological improvements in patients with DIPG and those with sDMG., Competing Interests: Competing interests: M.M., R.M. and C. Mackall are coinventors on a patent for the use of GD2-CAR T cells in regard to H2K27M gliomas, coordinated through Stanford University. C. Mackall is a coinventor on patents for the use of dasatinib and other small molecules to modulate CAR function and control CAR-associated toxicity, and on several patents related to CAR T cell therapies. C. Mackall holds equity in CARGO Therapeutics, Link Cell Therapies and GBM Newco, which are developing CAR-based therapies, and consults for CARGO, Link, Immatics, Ensoma, GBM NewCo and Red Tree Capital. She receives research funding from Lyell and Tune Therapeutics. R.M. is a cofounder of, and holds equity in, Link Cell Therapies and CARGO Therapeutics, and is a consultant for Lyell Immunopharma, NKarta, Arovella Pharmaceuticals, Innervate Radiopharmaceuticals, Aptorum Group, Gadeta, FATE Therapeutics (Data and Safety Monitoring Board) and Waypoint Bio. S.A.F. holds several patents in the field of cellular immunotherapy. V.B. is an investor and Director at Umoja Biopharma and Arsenal Bio. S.P.R. holds equity in Lyell Immunopharma. M.M. holds equity in MapLight Therapeutics and CARGO Therapeutics. The other authors declare no competing interests., (© 2024. The Author(s).)

Testicular cell differentiation is a highly regulated process, essential for male reproductive health. The histone variant H3.5 is apparently a critical player in this intricate orchestra of cell types, but its regulation and function remains poorly understood. To elucidate its role, we fractionized testicular cells using c-Kit/CD117 as a separation marker and analyzed H3.5 expression. Further, we investigated the regulation of H3.5 expression using public data repositories. We explored DNA methylation patterns in specific regions of the H3-5 gene and assessed H3-5 copy number gain in seminoma specimens. Additionally, we examined the testicular localization of H3.5 and its histone chaperone interactions to understand its regulation at the protein level. We used qRT-PCR, MeDIP, and qPCR to study H3.5 expression and DNA methylation in various cell types. H3-5 copy number gain was analyzed using qPCR. Protein interactions were investigated through fluorescence-2-hybrid assays in baby hamster kidney cells. H3.5 is primarily enriched in spermatocytes. DNA methylation of a CpG island overlapping the H3-5 promoter appeared to be involved in the tissue-specific regulation of H3.5 expression. Elevated H3.5 expression was observed in seminoma specimens, suggesting a potential link to testicular tumors. H3-5 copy number gain was associated with elevated H3.5 expression in seminoma specimens. Furthermore, we identified physical interactions between H3.5 and histone chaperones Asf1a and Asf1b, HIRA, CAF p150 and DAXX, shedding light on the protein-level regulation of H3.5. These findings provide valuable insights into the molecular mechanisms governing testicular cell differentiation and the potential role of H3.5 in testicular pathologies., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval: This study was conducted with approval of the Witten/Herdecke University Ethics board (No’s. 90/2011 and 138/2013). In addition, for investigations involving human subjects, informed consent has been obtained from all participants. Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of our research. Research involving human research participants were performed in accordance with the Declaration of Helsinki. Informed consent: Informed written consent was obtained by all involved donors., (© 2024. The Author(s).)

Single-molecule tracking provides direct real-time measurements of protein dynamics in live cells with high spatiotemporal resolution. In the last decade, this method has been adopted by several labs to understand the dynamics of DNA replication, transcription, telomerase, chromatin remodeling, etc. in a variety of model systems (bacteria, yeast S. cerevisiae, cell lines, embryo). However, it has not been employed for the yeast Schizosaccharomyces pombe, despite being a valuable model system. Here, we present single-molecule tracking datasets for chromatin-bound histone H3 (Hht1) in live and fixed cells and freely diffusing GFP in S. pombe nuclei to benchmark the diffusion parameters (diffusion coefficient, mean squared displacement, fraction of bound molecules, residence time). These parameters will be used to differentiate chromatin-bound molecules from unbound (free) molecules of any protein. It will be a valuable resource for any lab that starts employing this method for studying protein dynamics. This dataset can also be used to validate the performance of various tracking software and as a training dataset for machine learning-based automated tracking., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The expression of uncoupling protein 1 (UCP1), which regulates energy expenditure, is limited to brown/beige adipocytes in most mammals; however, it is also detected in the skeletal muscles of cattle. We previously observed a positive relationship between Ucp1 and fast-twitch myosin heavy chain (Myh) expression in bovine skeletal muscles. In the present study, we explored the regulatory expression of Ucp1 in bovine myogenic cells using cell culture. Vitamin C and high-dose capsaicin, which induce the formation of fast-twitch myotubes in murine myogenic cells, did not stimulate myogenesis in bovine myosatellite cells. Treatment with 4-phenylbutyric acid (PBA), a histone deacetylase inhibitor that enhances histone acetylation, upregulates the expression of all myogenic regulatory factors (MRFs), except Myog, in bovine myogenic cells. Consistent with this, PBA increased the expression levels of acetylated lysine 27 of histone 3 (H3K27), the fast-twitch component MYH1/2, and Ucp1 in bovine myogenic cells. SB203580, an inhibitor of p38 MAP kinase, blocked PBA-induced myogenesis and Ucp1 upregulation. PBA is a butyric acid-related molecule, and cattle produce large amounts of volatile fatty acids (VFAs), including acetic acid, propionic acid, and butyric acid, through ruminal fermentation. Propionic acid treatment stimulated H3K27 acetylation, myogenesis, and Ucp1 induction. Thus, the upregulation of muscular Ucp1 may be related to myogenic stimulation through the modulation of histone acetylation status in cattle; we propose that the cattle-specific expression of muscular UCP1 results from VFA production through ruminal fermentation., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The tumor-suppressing p-53 binding protein is a crucial protein that is involved in the prevention of cancer via its regulatory effect on a number of cellular processes. Recent evidence indicates that it interacts with a number of other proteins involved in cancer in ways that are not fully understood. An understanding of such interactions could provide insights into novel ways p53 further exerts its tumour prevention role via its interactions with diverse proteins. Thus, this study aimed to examine the interactions of the p53 protein with other proteins (peptides and histones) using molecular simulation dynamics. We opted for a total of seven proteins, namely 2LVM, 2MWO, 2MWP, 4CRI, 4 × 34, 5Z78, and 6MYO (control), and had their PBD files retrieved from the protein database. These proteins were then docked against the p-53 protein and the resulting interactions were examined using molecular docking simulations run at 500 ns. The result of the interactions revealed the utilisation of various amino acids in the process. The peptide that interacted with the highest number of amino acids was 5Z78 and these were Lys10, Gly21, Trp24, Pro105, His106, and Arg107, indicating a stronger interaction. The RMSD and RMSF values indicate that the complexes formed were stable, with 4CRI, 6MYO, and 2G3R giving the most stable values (less than 2.5 Å). Other parameters, including the SASA, Rg, and number of hydrogen bonds, all indicated the formation of fairly stable complexes. Our study indicates that overall, the interactions of 53BP1 with p53K370me2, p53K382me2, methylated K810 Rb, p53K381acK382me2, and tudor-interacting repair regulator protein indicated interactions that were not as strong as those with the histone protein. Thus, it could be that P53 may mediate its tumour suppressing effect via interactions with amino acids and histone., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Primordial germ cells (PGCs) are the precursors of sperm and eggs. They undergo genome-wide epigenetic reprogramming to erase epigenetic memory and reset the genomic potential for totipotency. Global DNA methylation erasure is a crucial part of epigenetic resetting when DNA methylation levels decrease across the genome to <5%. However, certain localized regions exhibit slower demethylation or resistance to reprogramming. Since DNA methylation plays a crucial role in transcriptional regulation, this depletion in PGCs requires mechanisms independent of DNA methylation to regulate transcriptional control during PGC reprogramming. Histone modifications are predicted to compensate for the loss of DNA methylation in gene regulation. Different histone modifications exhibit distinct patterns in PGCs undergoing epigenetic programming at the genomic level during PGC development in conjunction with changes in DNA methylation. Together, they contribute to PGC-specific genomic regulation. Recent findings related to these processes provide a comprehensive overview of germline epigenetic reprogramming and its importance in mouse and human PGC development. Additionally, we evaluated the extent to which in vitro culture techniques have replicated the development processes of human PGCs., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Lysine-specific demethylase 1 (LSD1/KDM1A) is a pivotal epigenetic enzyme that contributes to several malignancies including malignant glioma. LSD1 is a flavin adenine dinucleotide dependent histone demethylase that specifically targets histone H3 lysine (K) 4 mono- (me1) and di-methylation (me2) and H3K9me1/2 for demethylation. Herein we report the development of an LSD inhibitor, S2172, which efficiently penetrates the blood-brain barrier. S2172 effectively suppresses LSD1 enzymatic activity, resulting in the depletion of cell growth both in vitro in glioma stem cells (GSCs) (mean half-maximal inhibitory concentration (IC 50 ) of 13.8 μM) and in vivo in a GSC orthotopic xenograft mouse model. Treatment with S2172 robustly reduced the expression of the stemness-related genes MYC and Nestin in GSC cells. Consistent with this, chromatin immunoprecipitation-sequencing revealed a significant S2172-dependent alteration in H3K4me2/H3K4me3 status. Furthermore, we identified 284 newly acquired H3K4me2 peak regions after S2172 treatment, which were encompassed within super-enhancer regions. The altered H3K4me2/H3K4me3 status induced by S2172 treatment affected the expression of genes related to tumorigenesis. Our data suggest that targeting LSD1 with S2172 could provide a promising treatment option for glioblastomas, particularly due to targeting of GSC populations., Competing Interests: Competing interests: The authors declare no competing interests. Ethical approval: The research protocol was approved by the Institutional Review Board of Nagoya University of Graduate School of Medicine (21-115). Animal study was performed under protocols approved by the Institutional Animal Care and Use Committee of Nagoya University of Graduate School of Medicine (M220315-002)., (© 2024. The Author(s).)

Bone cancer pain (BCP) affects ~70% of patients in advanced stages, primarily due to bone metastasis, presenting a substantial therapeutic challenge. Here, we profile orphan G protein-coupled receptors in the dorsal root ganglia (DRG) following tumor infiltration, and observe a notable increase in GPR160 expression. Elevated Gpr160 mRNA and protein levels persist from postoperative day 6 for over 18 days in the affected DRG, predominantly in small-diameter C-fiber type neurons specific to the tibia. Targeted interventions, including DRG microinjection of siRNA or AAV delivery, mitigate mechanical allodynia, cold, and heat hyperalgesia induced by the tumor. Tumor infiltration increases DRG neuron excitability in wild-type mice, but not in Gpr160 gene knockout mice. Tumor infiltration results in reduced H3K27me3 and increased H3K27ac modifications, enhanced binding of the transcription activator Sp1 to the Gpr160 gene promoter region, and induction of GPR160 expression. Modulating histone-modifying enzymes effectively alleviated pain behavior. Our study delineates a novel mechanism wherein elevated Sp1 levels facilitate Gpr160 gene transcription in nociceptive DRG neurons during BCP in rodents., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

DNA methyltransferase 3A (DNMT3A) plays a critical role in establishing and maintaining DNA methylation patterns in vertebrates. Here we structurally and biochemically explore the interaction of DNMT3A1 with diverse modified nucleosomes indicative of different chromatin environments. A cryo-EM structure of the full-length DNMT3A1-DNMT3L complex with a H2AK119ub nucleosome reveals that the DNMT3A1 ubiquitin-dependent recruitment (UDR) motif interacts specifically with H2AK119ub and makes extensive contacts with the core nucleosome histone surface. This interaction facilitates robust DNMT3A1 binding to nucleosomes, and previously unexplained DNMT3A disease-associated mutations disrupt this interface. Furthermore, the UDR-nucleosome interaction synergises with other DNMT3A chromatin reading elements in the absence of histone ubiquitylation. H2AK119ub does not stimulate DNMT3A DNA methylation activity, as observed for the previously described H3K36me2 mark, which may explain low levels of DNA methylation on H2AK119ub marked facultative heterochromatin. This study highlights the importance of multivalent binding of DNMT3A to histone modifications and the nucleosome surface and increases our understanding of how DNMT3A1 chromatin recruitment occurs., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

The yeast SWR1 complex catalyses the exchange of histone H2A-H2B dimers in nucleosomes, with Htz1-H2B dimers 1-3 . Here we used single-molecule analysis to demonstrate two-step double exchange of the two H2A-H2B dimers in a canonical yeast nucleosome with Htz1-H2B dimers, and showed that double exchange can be processive without release of the nucleosome from the SWR1 complex. Further analysis showed that bound nucleosomes flip between two states, with each presenting a different face, and hence histone dimer, to SWR1. The bound dwell time is longer when an H2A-H2B dimer is presented for exchange than when presented with an Htz1-H2B dimer. A hexasome intermediate in the reaction is bound to the SWR1 complex in a single orientation with the 'empty' site presented for dimer insertion. Cryo-electron microscopy analysis revealed different populations of complexes showing nucleosomes caught 'flipping' between different conformations without release, each placing a different dimer into position for exchange, with the Swc2 subunit having a key role in this process. Together, the data reveal a processive mechanism for double dimer exchange that explains how SWR1 can 'proofread' the dimer identities within nucleosomes., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Lysine-specific histone demethylase 1 (LSD1) is a histone demethylase that plays a critical role in epigenetic regulation by removing the methyl group from mono- and di-methylated lysine 4 on histone H3 (H3K4me1/2), acting as a repressor of gene expression. Recently, catalytically independent functions of LSD1, serving as a scaffold for assembling chromatin-regulator and transcription factor complexes, have been identified. Herein, we show for the first time that LSD1 interacts with chromodomain-helicase-DNA-binding protein 7 (CHD7) in mouse embryonic stem cells (ESCs). To further investigate the CHD7-LSD1 crosstalk, we engineered Chd7 and Chd7/Lsd1 knockout (KO) mouse ESCs. We show that CHD7 is dispensable for ESC self-renewal and survival, while Chd7 KO ESCs can differentiate towards embryoid bodies (EBs) with defective expression of ectodermal markers. Intriguingly, Chd7/Lsd1 double KO mouse ESCs exhibit proliferation defects similar to Lsd1 KO ESCs and have lost the capacity to differentiate properly. Furthermore, the increased co-occupancy of H3K4me1 and CHD7 on chromatin following Lsd1 deletion suggests that LSD1 is required for facilitating the proper binding of CHD7 to chromatin and regulating differentiation. Collectively, our results suggest that LSD1 and CHD7 work in concert to modulate gene expression and influence proper cell fate determination., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

DNA damage occurs in all living cells. γ-H2AX imaging by fluorescent microscopy is widely used across disciplines in the analysis of double-strand break (DSB) DNA damage. Here we demonstrate a method for the quantitative analysis of such DBSs. Ionising radiation, well known to induce DSBs, is used in this demonstration, and additional DBSs are induced if high-Z nanoparticles are present during irradiation. As a deliberate test of the methodology, cells are exposed to a spatially fractionated ionising radiation field, characterised by regions of high and low absorbed radiation dose that are only ever qualitatively verified biologically via γ-H2AX imaging. Here we validate our bio-dosimetric quantification method using γ-H2AX assays in the assessment of DSB enhancement. Our method reliably quantifies DSB enhancement in cells when exposed to either a spatially contiguous or fractionated irradiation fields. Using the γ-H2AX assay, we deduce the biological dose response, and for the first time, demonstrate equivalence to the independently measured physical absorbed dose. Using our novel method, we are also able quantify the nanoparticle DSB enhancement at the cellular level, which is not possible using physical dose measurement techniques. Our method therefore provides a new paradigm in γ-H2AX image quantification of DSBs, as well as an independently validated bio-dosimetry technique., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Chromatin structure is a key regulator of DNA transcription, replication and repair 1 . In humans, the TIP60-EP400 complex (TIP60-C) is a 20-subunit assembly that affects chromatin structure through two enzymatic activities: ATP-dependent exchange of histone H2A-H2B for H2A.Z-H2B, and histone acetylation. In yeast, however, these activities are performed by two independent complexes-SWR1 and NuA4, respectively 2,3 . How the activities of the two complexes are merged into one supercomplex in humans, and what this association entails for the structure and mechanism of the proteins and their recruitment to chromatin, are unknown. Here we describe the structure of the endogenous human TIP60-C. We find a three-lobed architecture composed of SWR1-like (SWR1L) and NuA4-like (NuA4L) parts, which associate with a TRRAP activator-binding module. The huge EP400 subunit contains the ATPase motor, traverses the junction between SWR1L and NuA4L twice and constitutes the scaffold of the three-lobed architecture. NuA4L is completely rearranged compared with its yeast counterpart. TRRAP is flexibly tethered to NuA4L-in stark contrast to its robust connection to the completely opposite side of NuA4 in yeast 4-7 . A modelled nucleosome bound to SWR1L, supported by tests of TIP60-C activity, suggests that some aspects of the histone exchange mechanism diverge from what is seen in yeast 8,9 . Furthermore, a fixed actin module (as opposed to the mobile actin subcomplex in SWR1; ref. 8 ), the flexibility of TRRAP and the weak effect of extranucleosomal DNA on exchange activity lead to a different, activator-based mode of enlisting TIP60-C to chromatin., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The count of mitotic figures (MFs) observed in hematoxylin and eosin (H&E)-stained slides is an important prognostic marker, as it is a measure for tumor cell proliferation. However, the identification of MFs has a known low inter-rater agreement. In a computer-aided setting, deep learning algorithms can help to mitigate this, but they require large amounts of annotated data for training and validation. Furthermore, label noise introduced during the annotation process may impede the algorithms' performance. Unlike H&E, where identification of MFs is based mainly on morphological features, the mitosis-specific antibody phospho-histone H3 (PHH3) specifically highlights MFs. Counting MFs on slides stained against PHH3 leads to higher agreement among raters and has therefore recently been used as a ground truth for the annotation of MFs in H&E. However, as PHH3 facilitates the recognition of cells indistinguishable from H&E staining alone, the use of this ground truth could potentially introduce an interpretation shift and even label noise into the H&E-related dataset, impacting model performance. This study analyzes the impact of PHH3-assisted MF annotation on inter-rater reliability and object level agreement through an extensive multi-rater experiment. Subsequently, MF detectors, including a novel dual-stain detector, were evaluated on the resulting datasets to investigate the influence of PHH3-assisted labeling on the models' performance. We found that the annotators' object-level agreement significantly increased when using PHH3-assisted labeling (F1: 0.53 to 0.74). However, this enhancement in label consistency did not translate to improved performance for H&E-based detectors, neither during the training phase nor the evaluation phase. Conversely, the dual-stain detector was able to benefit from the higher consistency. This reveals an information mismatch between the H&E and PHH3-stained images as the cause of this effect, which renders PHH3-assisted annotations not well-aligned for use with H&E-based detectors. Based on our findings, we propose an improved PHH3-assisted labeling procedure., (© 2024. The Author(s).)

Epigenetic modifiers (miRNAs, histone methyltransferases (HMTs)/demethylases, and DNA methyltransferases/demethylases) are associated with cancer proliferation, metastasis, angiogenesis, and drug resistance. Among these modifiers, HMTs are frequently overexpressed in various cancers, and recent studies have increasingly identified these proteins as potential therapeutic targets. In this review, we discuss members of the SET and MYND domain-containing protein (SMYD) family that are topics of extensive research on the histone methylation and nonhistone methylation of cancer-related genes. Various members of the SMYD family play significant roles in cancer proliferation, metastasis, and drug resistance by regulating cancer-specific histone methylation and nonhistone methylation. Thus, the development of specific inhibitors that target SMYD family members may lead to the development of cancer treatments, and combination therapy with various anticancer therapeutic agents may increase treatment efficacy., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

To maintain protein homeostasis in changing nutrient environments, cells must precisely control the amount of their proteins, despite the accompanying changes in cell growth and biosynthetic capacity. As nutrients are major regulators of cell cycle length and progression, a particular challenge arises for the nutrient-dependent regulation of 'cell cycle genes', which are periodically expressed during the cell cycle. One important example are histones, which are needed at a constant histone-to-DNA stoichiometry. Here we show that budding yeast achieves histone homeostasis in different nutrients through a decoupling of transcript and protein abundance. We find that cells downregulate histone transcripts in poor nutrients to avoid toxic histone overexpression, but produce constant amounts of histone proteins through nutrient-specific regulation of translation efficiency. Our findings suggest that this allows cells to balance the need for rapid histone production under fast growth conditions with the tight regulation required to avoid toxic overexpression in poor nutrients., (© 2024. The Author(s).)

NASH is characterized by hepatic lipid accumulation and inflammation; and JMJD2B-a histone demethylase-upregulation has been linked to its progression. Pirfenidone (PFD) is an antifibrotic agent with anti-inflammatory and antioxidant effects recognized to decrease NASH symptoms. Herein, our aim was to investigate PFD-induced epigenetics mechanisms involving JMJD2B and histone modifications in experimental NASH. Male C57BL/6J mice were fed with normo-diet, or high fat/carbohydrate diet (HF) for 16 weeks. A HF-subgroup was treated with PFD 300 mg/kg/d from week 8th to the end of protocol. Insulin tolerance test and liver and fat histological and biochemical analyses were carried out. Hepatic transcriptome was examined. Liver proteins were studied by western blot (WB) and Chromatin immunoprecipitation. In vitro, lipotoxicity was induced in HepG2 cells and proteins were evaluated using WB. Molecular docking was used to explore binding of PFD to JMJD2B. Mice treated with PFD reduced weight gain, epididymal fat and inflammatory nodules, and steatosis in liver tissue, as well as, improved biochemical test. PFD modified the expression of Jmjd2b, Pparg, Fasn and Srebp1, and restored JMJD2B protein and H3K9me3 repressive mark, both in animal and cell models. PFD increased hepatic enrichment of H3K9me2 and H3K9me3 at the promoter region of Fasn and Srebp1, and Pparg. In HepG2 cells, PFD reduced lipid vacuole accumulation. In silico, PFD interacted with JMJD2B catalytic site. PFD is an epigenetic regulator modifying JMJD2B activity, resulting in reduced NASH traits., (© 2024. The Author(s).)

Various techniques using fluorescent reporter probes have been developed, such as GFP transgenic mouse lines that are used to detect spatial-temporal expression levels of genes. Although GFP expression is largely considered non-toxic, recent reports have indicated that under certain conditions GFP can display cellular toxicity. We hereby report the nuclear toxicity of H2B-GFP using a K14 specific Tet-on reporter mouse system. Using this system, GFP accumulates in the nucleus of all K14 expressing cells, such as the ocular surface epithelia and ocular adnexa. Expression of high levels of nuclear GFP during embryonic stages led to an eye open-at-birth (EOB) phenotype and abnormal ocular adnexa development and during adult and aging stages showed notable toxicity to ocular tissues. Other tissues, such as skin, also presented multiple defects associated with H2B-GFP expression. This toxicity was found to be concentration dependent, with homozygous mice presenting extremely high toxicity, while heterozygous mice presented limited toxicity. Upon induction, the accumulation of H2B-GFP in the nucleus of homozygous mice led to apoptosis within 2 weeks. This study therefore shows that although the use of nuclear GFP reporter mice is a valuable tool, at high levels, nuclear GFP can be toxic, leading to cell death and affecting tissue function., (© 2024. The Author(s).)

The differentiation of developmental cell lineages is associated with genome-wide modifications in histone H3 methylation. However, the causal role of histone H3 methylation in transcriptional regulation and cell differentiation has been difficult to test in mammals. The experimental overexpression of histone H3 mutants carrying lysine-to-methionine (K-to-M) substitutions has emerged as an alternative tool for inhibiting the endogenous levels of histone H3 methylation at specific lysine residues. Here, we leverage the use of histone K-to-M mutants by creating Enhanced Episomal Vectors that enable the simultaneous depletion of multiple levels of histone H3 lysine 4 (H3K4) or lysine 9 (H3K9) methylation in projection neurons of the mouse cerebral cortex. Our approach also facilitates the simultaneous depletion of H3K9 and H3K27 trimethylation (H3K9me3 and H3K27me3, respectively) in cortical neurons. In addition, we report a tamoxifen-inducible Cre-FLEX system that allows the activation of mutant histones at specific developmental time points or in the adult cortex, leading to the depletion of specific histone marks. The tools presented here can be implemented in other experimental systems, such as human in vitro models, to test the combinatorial role of histone methylations in developmental fate decisions and the maintenance of cell identity., (© 2024. The Author(s).)

Although the role of low-density granulocytes (LDGs), neutrophils in the peripheral blood mononuclear cell (PBMC) fraction, and neutrophil extracellular traps (NETs) in assessing lupus disease severity is acknowledged, data specific to childhood-onset lupus remains scarce. This study analyzed 46 patients with childhood-onset systemic lupus erythematosus (82.6% females, mean age 14.5 ± 0.3 years), including 26 cases with normal complement levels and 20 with low complement levels, along with 20 healthy adult volunteers. Key parameters that distinguished healthy volunteers from lupus patients and differentiated between lupus patients with low and normal complement were serum interferon (IFN)-α, serum citrullinated histone 3 (CitH3), and extracellular traps (ETs) in LDGs. However, NETs (assessed by nuclear staining morphology), LDG abundance, and other parameters (such as endotoxemia, cytokines, and double-stranded (ds) DNA) did not show such differentiation. When lipopolysaccharide (LPS) was administered to LDGs in the PBMC fraction, it induced ETs in both low and normal complement groups, indicating the inducible nature of ETs. In adult healthy volunteers, activation by recombinant IFN-α or dsDNA in isolated neutrophils induced LDGs and NETs (identified using immunofluorescent staining for CitH3, myeloperoxidase, and neutrophil elastase) at 45 min and 3 h post-stimulation, respectively. Additionally, approximately half of the LDGs underwent late apoptosis at 3 h post-stimulation, as determined by flow cytometry analysis. Activation by IFN-α or dsDNA in LDGs also led to a more pronounced expression of CD66b, an adhesion molecule, compared to regular-density neutrophils, suggesting higher activity in LDGs. In conclusion, IFN-α and/or dsDNA in serum may transform regular-density neutrophils into LDGs before progressing to NETosis and apoptosis, potentially exacerbating lupus severity through cell death-induced self-antigens. Therefore, LDGs and ETs in LDGs could provide deeper insights into the pathophysiology of childhood-onset lupus., (© 2024. The Author(s).)

Mutation of tet methylcytosine dioxygenase 2 (encoded by TET2) drives myeloid malignancy initiation and progression 1-3 . TET2 deficiency is known to cause a globally opened chromatin state and activation of genes contributing to aberrant haematopoietic stem cell self-renewal 4,5 . However, the open chromatin observed in TET2-deficient mouse embryonic stem cells, leukaemic cells and haematopoietic stem and progenitor cells 5 is inconsistent with the designated role of DNA 5-methylcytosine oxidation of TET2. Here we show that chromatin-associated retrotransposon RNA 5-methylcytosine (m 5 C) can be recognized by the methyl-CpG-binding-domain protein MBD6, which guides deubiquitination of nearby monoubiquitinated Lys119 of histone H2A (H2AK119ub) to promote an open chromatin state. TET2 oxidizes m 5 C and antagonizes this MBD6-dependent H2AK119ub deubiquitination. TET2 depletion thereby leads to globally decreased H2AK119ub, more open chromatin and increased transcription in stem cells. TET2-mutant human leukaemia becomes dependent on this gene activation pathway, with MBD6 depletion selectively blocking proliferation of TET2-mutant leukaemic cells and largely reversing the haematopoiesis defects caused by Tet2 loss in mouse models. Together, our findings reveal a chromatin regulation pathway by TET2 through retrotransposon RNA m 5 C oxidation and identify the downstream MBD6 protein as a feasible target for developing therapies specific against TET2 mutant malignancies., (© 2024. The Author(s).)

The hierarchical packaging of chromatin fibers plays a critical role in gene regulation. The 30-nm chromatin fibers, a central-level structure bridging nucleosomal arrays to higher-order organizations, function as the first level of transcriptional dormant chromatin. The dynamics of 30-nm chromatin fiber play a crucial role in biological processes related to DNA. Here, we report a 3.6-angstrom resolution cryogenic electron microscopy structure of H5-bound dodecanucleosome, i.e., the chromatin fiber reconstituted in the presence of linker histone H5, which shows a two-start left-handed double helical structure twisted by tetranucleosomal units. An atomic structural model of the H5-bound chromatin fiber, including an intact chromatosome, is built, which provides structural details of the full-length linker histone H5, including its N-terminal domain and an HMG-motif-like C-terminal domain. The chromatosome structure shows that H5 binds the nucleosome off-dyad through a three-contact mode in the chromatin fiber. More importantly, the H5-chromatin structure provides a fine molecular basis for the intra-tetranucleosomal and inter-tetranucleosomal interactions. In addition, we systematically validated the physiological functions and structural characteristics of the tetranucleosomal unit through a series of genetic and genomic studies in Saccharomyces cerevisiae and in vitro biophysical experiments. Furthermore, our structure reveals that multiple structural asymmetries of histone tails confer a polarity to the chromatin fiber. These findings provide structural and mechanistic insights into how a nucleosomal array folds into a higher-order chromatin fiber with a polarity in vitro and in vivo., (© 2024. The Author(s).)

In mammals, the transition from mitosis to meiosis facilitates the successful production of gametes. However, the regulatory mechanisms that control meiotic initiation remain unclear, particularly in the context of complex histone modifications. Herein, we show that KDM2A, acting as a lysine demethylase targeting H3K36me3 in male germ cells, plays an essential role in modulating meiotic entry and progression. Conditional deletion of Kdm2a in mouse pre-meiotic germ cells results in complete male sterility, with spermatogenesis ultimately arrested at the zygotene stage of meiosis. KDM2A deficiency disrupts H3K36me2/3 deposition in c-KIT + germ cells, characterized by a reduction in H3K36me2 but a dramatic increase in H3K36me3. Furthermore, KDM2A recruits the transcription factor E2F1 and its co-factor HCFC1 to the promoters of key genes required for meiosis entry and progression, such as Stra8, Meiosin, Spo11, and Sycp1. Collectively, our study unveils an essential role for KDM2A in mediating H3K36me2/3 deposition and controlling the programmed gene expression necessary for the transition from mitosis to meiosis during spermatogenesis., (© 2024. The Author(s).)

The PIWI-interacting RNA (piRNA) pathway guides the DNA methylation of young, active transposons during germline development in male mice 1 . piRNAs tether the PIWI protein MIWI2 (PIWIL4) to the nascent transposon transcript, resulting in DNA methylation through SPOCD1 (refs. 2-5 ). Transposon methylation requires great precision: every copy needs to be methylated but off-target methylation must be avoided. However, the underlying mechanisms that ensure this precision remain unknown. Here, we show that SPOCD1 interacts directly with SPIN1 (SPINDLIN1), a chromatin reader that primarily binds to H3K4me3-K9me3 (ref. 6 ). The prevailing assumption is that all the molecular events required for piRNA-directed DNA methylation occur after the engagement of MIWI2. We find that SPIN1 expression precedes that of both SPOCD1 and MIWI2. Furthermore, we demonstrate that young LINE1 copies, but not old ones, are marked by H3K4me3, H3K9me3 and SPIN1 before the initiation of piRNA-directed DNA methylation. We generated a Spocd1 separation-of-function allele in the mouse that encodes a SPOCD1 variant that no longer interacts with SPIN1. We found that the interaction between SPOCD1 and SPIN1 is essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 elements. We propose that piRNA-directed LINE1 DNA methylation requires a developmentally timed two-factor authentication process. The first authentication is the recruitment of SPIN1-SPOCD1 to the young LINE1 promoter, and the second is MIWI2 engagement with the nascent transcript. In summary, independent authentication events underpin the precision of piRNA-directed LINE1 DNA methylation., (© 2024. The Author(s).)

Histone lysine methylation is pivotal in shaping the epigenetic landscape and is linked to cell physiology. Coordination of the activities of multiple histone lysine methylation modifiers, namely, methyltransferases and demethylases, modulates chromatin structure and dynamically alters the epigenetic landscape, orchestrating almost all DNA-templated processes, such as transcription, DNA replication, and DNA repair. The stability of modifier proteins, which is regulated by protein degradation, is crucial for their activity. Here, we review the current knowledge of modifier-protein degradation via specific pathways and its subsequent impact on cell physiology through epigenetic changes. By summarizing the functional links between the aberrant stability of modifier proteins and human diseases and highlighting efforts to target protein stability for therapeutic purposes, we aim to promote interest in defining novel pathways that regulate the degradation of modifiers and ultimately increase the potential for the development of novel therapeutic strategies., (© 2024. The Author(s).)

Diffuse midline glioma, H3 K27-altered (DMG) are highly aggressive malignancies of the central nervous system (CNS) that primarily affect the pediatric population. Large scale spatial transcriptomic studies have implicated that tumor microenvironmental landscape plays an important role in determining the phenotypic differences in tumor presentation and clinical course, however, data connecting overall transcriptomic changes to the protein level is lacking. The NanoString GeoMx ™  Digital Spatial Profiler platform was used to determine the spatial transcriptomic and proteomic landscape in a cohort of both pediatric and adult H3 K27-altered DMG biopsy samples. Three fluorescently labeled antibodies targeting immune cells (CD45), epithelial cells (PanCK), tumor cells (H3 K27M) and a nucleic acid stain (SYTO-13) were used to establish regions of interest (ROI) for genomic and proteomic analysis. We found genetic alterations within the tumor which can be delineated across patient age and spatial location. We show that the H3 K27M mutation itself has a profound impact on tumor cells transcriptomics and interestingly we found limited fidelity between overall transcriptome and proteome. Our data also validate a previously described genomic signature at the proteomic level and reveal a special shift in the signature based on the local TME composition., (© 2024. The Author(s).)

Histone post-translational modifications (HPTMs), as one of the core mechanisms of epigenetic regulation, are garnering increasing attention due to their close association with the onset and progression of diseases and their potential as targeted therapeutic agents. Advances in high-throughput molecular tools and the abundance of bioinformatics data have led to the discovery of novel HPTMs which similarly affect gene expression, metabolism, and chromatin structure. Furthermore, a growing body of research has demonstrated that novel histone modifications also play crucial roles in the development and progression of various diseases, including various cancers, cardiovascular diseases, infectious diseases, psychiatric disorders, and reproductive system diseases. This review defines nine novel histone modifications: lactylation, citrullination, crotonylation, succinylation, SUMOylation, propionylation, butyrylation, 2-hydroxyisobutyrylation, and 2-hydroxybutyrylation. It comprehensively introduces the modification processes of these nine novel HPTMs, their roles in transcription, replication, DNA repair and recombination, metabolism, and chromatin structure, as well as their involvement in promoting the occurrence and development of various diseases and their clinical applications as therapeutic targets and potential biomarkers. Moreover, this review provides a detailed overview of novel HPTM inhibitors targeting various targets and their emerging strategies in the treatment of multiple diseases while offering insights into their future development prospects and challenges. Additionally, we briefly introduce novel epigenetic research techniques and their applications in the field of novel HPTM research., (© 2024. The Author(s).)

Dynamic changes in maternal‒zygotic transition (MZT) require complex regulation of zygote formation, maternal transcript decay, embryonic genome activation (EGA), and cell cycle progression. Although these changes are well described, some key regulatory factors are still elusive. Sirtuin-1 (SIRT1), an NAD + -dependent histone deacetylase, is a versatile driver of MZT via its epigenetic and nonepigenetic substrates. This study focused on the dynamics of SIRT1 in early embryos and its contribution to MZT. A conditional SIRT1-deficient knockout mouse model was used, accompanied by porcine and human embryos. Embryos across mammalian species showed the prominent localization of SIRT1 in the nucleus throughout early embryonic development. Accordingly, SIRT1 interacts with histone H4 on lysine K16 (H4K16) in both mouse and human blastocysts. While maternal SIRT1 is dispensable for MZT, at least one allele of embryonic Sirt1 is required for early embryonic development around the time of EGA. This role of SIRT1 is surprisingly mediated via a transcription-independent mode of action., (© 2024. The Author(s).)

During the development of multicellular organisms and cell differentiation, the chromatin structure in the cell nucleus undergoes extensive changes, and the nucleosome structure is formed by a combination of various histone variants. Histone variants with diverse posttranslational modifications are known to play crucial roles in different regulatory functions. We have previously reported that H3t, a testis-specific histone variant, is essential for spermatogenesis. To elucidate the function of this chromatin molecule in vivo, we generated knock-in mice with a FLAG tag attached to the carboxyl terminus of H3t. In the present study, we evaluated the utility of the generated knock-in mice and comprehensively analyzed posttranslational modifications of canonical H3 and H3t using mass spectrometry. Herein, we found that H3t-FLAG was incorporated into spermatogonia and meiotic cells in the testes, as evidenced by immunostaining of testicular tissue. According to the mass spectrometry analysis, the overall pattern of H3t-FLAG posttranslational modification was comparable to that of the control H3, while the relative abundances of certain specific modifications differed between H3t-FLAG and the control bulk H3. The generated knock-in mice could be valuable for analyzing the function of histone variants in vivo., (© 2024. The Author(s).)

Skeletal muscle is a highly heterogeneous tissue, and its contractile proteins are composed of different isoforms, forming various types of muscle fiber, each of which has its own metabolic characteristics. It has been demonstrated that endurance exercise induces the transition of muscle fibers from fast-twitch to slow-twitch muscle fiber type. Herein, we discover a novel epigenetic mechanism for muscle contractile property tightly coupled to its metabolic capacity during muscle fiber type transition with exercise training. Our results show that an 8-week endurance exercise induces histone methylation remodeling of PGC-1α and myosin heavy chain (MHC) isoforms in the rat gastrocnemius muscle, accompanied by increased mitochondrial biogenesis and an elevated ratio of slow-twitch to fast-twitch fibers. Furthermore, to verify the roles of reactive oxygen species (ROS) and AMPK in exercise-regulated epigenetic modifications and muscle fiber type transitions, mouse C2C12 myotubes were used. It was shown that rotenone activates ROS/AMPK pathway and histone methylation enzymes, which then promote mitochondrial biogenesis and MHC slow isoform expression. Mitoquinone (MitoQ) partially blocking rotenone-treated model confirms the role of ROS in coupling mitochondrial biogenesis with muscle fiber type. In conclusion, endurance exercise couples mitochondrial biogenesis with MHC slow isoform by remodeling histone methylation, which in turn promotes the transition of fast-twitch to slow-twitch muscle fibers. The ROS/AMPK pathway may be involved in the regulation of histone methylation enzymes by endurance exercise., (© 2024. The Author(s).)

Diabetes mellitus (DM) is a well-documented risk factor of intervertebral disc degeneration (IVDD). The current study was aimed to clarify the effects and mechanisms of NADH: ubiquinone oxidoreductase subunit A3 (NDUFA3) in human nucleus pulposus cells (HNPCs) exposed to high glucose. NDUFA3 was overexpressed in HNPCs via lenti-virus transduction, which were co-treated with high glucose and rotenone (a mitochondrial complex I inhibitor) for 48 h. Cell activities were assessed for cell viability, cell apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) ratio, oxygen consumption rate (OCR) and mitochondrial complexes I activities. High glucose decreased cell viability, increased apoptotic cells, increased ROS production, decreased MMP levels and OCR values in HNPCs in a dose-dependent manner. Rotenone co-treatment augmented the high glucose-induced injuries on cell viability, apoptosis, ROS production and mitochondrial function. NDUFA3 overexpression counteracted the high glucose-induced injuries in HNPCs. HDAC/H3K27ac mechanism was involved in regulating NDUFA3 transcription. NDUFA3 knockdown decreased cell viability and increased apoptotic cells, which were reversed by ROS scavenger N-acetylcysteine. HDAC/H3K27ac-mediated transcription of NDUFA3 protects HNPCs against high glucose-induced injuries through suppressing cell apoptosis, eliminating ROS, improving mitochondrial function and oxidative phosphorylation. This study sheds light on candidate therapeutic targets and deepens the understanding of molecular mechanisms behind DM-induced IVDD., (© 2024. The Author(s).)

G9a is a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9), which is involved in the regulation of gene expression. We had previously reported that G9a is expressed in developing tendons in vivo and in vitro and that G9a-deficient tenocytes show impaired proliferation and differentiation in vitro. In this study, we investigated the functions of G9a in tendon development in vivo by using G9a conditional knockout (G9a cKO) mice. We crossed Sox9 Cre/+ mice with G9a fl/fl mice to generate G9a fl/fl ; Sox9 Cre/+ mice. The G9a cKO mice showed hypoplastic tendon formation at 3 weeks of age. Bromodeoxyuridine labeling on embryonic day 16.5 (E16.5) revealed decreased cell proliferation in the tenocytes of G9a cKO mice. Immunohistochemical analysis revealed decreased expression levels of G9a and its substrate, H3K9me2, in the vertebral tendons of G9a cKO mice. The tendon tissue of the vertebrae and limbs of G9a cKO mice showed reduced expression of a tendon marker, tenomodulin (Tnmd), and col1a1 genes, suggesting that tenocyte differentiation was suppressed. Overexpression of G9a resulted in enhancement of Tnmd and col1a1 expression in tenocytes in vitro. These results suggest that G9a regulates the proliferation and differentiation of tendon progenitor cells during tendon development. Thus, our results suggest that G9a plays an essential role in tendon development., (© 2024. The Author(s).)

The emergence of bacterial resistance to antibiotics poses a global health threat, necessitating innovative solutions. The contemporary challenge lies in bacterial resistance, impacting morbidity, mortality, and global economies. Antimicrobial peptides (AMPs) offer a promising avenue for addressing antibiotic resistance. The Antimicrobial Peptide Database catalogs 3569 peptides from various organisms, representing a rich resource for drug development. Histones, traditionally recognized for their role in nucleosome structures, have gained attention for their extracellular functions, including antimicrobial and immunomodulatory properties. This review aims to thoroughly investigate antimicrobial peptides derived from histones in various organisms, elucidating their mechanisms. In addition, it gives us clues about how extracellular histones might be used in drug delivery systems to fight bacterial infections. This comprehensive analysis emphasizes the importance of histone-derived peptides in developing innovative therapeutic strategies for evolving bacterial challenges., (© 2024. The Author(s).)

The tandem Tudor-like domain-containing protein Spindlin1 (SPIN1) is a transcriptional coactivator with critical functions in embryonic development and emerging roles in cancer. However, the involvement of SPIN1 in DNA damage repair has remained unclear. Our study shows that SPIN1 is recruited to DNA lesions through its N-terminal disordered region that binds to Poly-ADP-ribose (PAR), and facilitates homologous recombination (HR)-mediated DNA damage repair. SPIN1 promotes H3K9me3 accumulation at DNA damage sites and enhances the interaction between H3K9me3 and Tip60, thereby promoting the activation of ATM and HR repair. We also show that SPIN1 increases chemoresistance. These findings reveal a novel role for SPIN1 in the activation of H3K9me3-dependent DNA repair pathways, and suggest that SPIN1 may contribute to cancer chemoresistance by modulating the efficiency of double-strand break (DSB) repair., (© 2024. The Author(s).)

The genome is constantly exposed to DNA damage from endogenous and exogenous sources. Fine modulation of DNA repair, chromatin remodeling, and transcription factors is necessary for protecting genome integrity, but the precise mechanisms are still largely unclear. We found that after ionizing radiation (IR), global trimethylation of histone H3 at lysine 4 (H3K4me3) was decreased at an early (5 min) post-IR phase but increased at an intermediate (180 min) post-IR phase in both human and mouse hematopoietic cells. We demonstrated that PTIP, a component of the MLL histone methyltransferase complex, is required for H3K4me3 upregulation in the intermediate post-IR phase and promotes cell cycle arrest by epigenetically inducing a cell cycle inhibitor, PRDM1. In addition, we found that PTIP expression is specifically downregulated in acute myeloid leukemia patients. These findings collectively suggest that the PTIP-PRDM1 axis plays an essential role in proper DNA damage response and its deregulation contributes to leukemogenesis., (© 2024. The Author(s).)

While perinatal medicine advancements have bolstered survival outcomes for premature infants, bronchopulmonary dysplasia (BPD) continues to threaten their long-term health. Gene-environment interactions, mediated by epigenetic modifications such as DNA methylation, histone modification, and non-coding RNA regulation, take center stage in BPD pathogenesis. Recent discoveries link methylation variations across biological pathways with BPD. Also, the potential reversibility of histone modifications fuels new treatment avenues. The review also highlights the promise of utilizing mesenchymal stem cells and their exosomes as BPD therapies, given their ability to modulate non-coding RNA, opening novel research and intervention possibilities. IMPACT: The complexity and universality of epigenetic modifications in the occurrence and development of bronchopulmonary dysplasia were thoroughly discussed. Both molecular and cellular mechanisms contribute to the diverse nature of epigenetic changes, suggesting the need for deeper biochemical techniques to explore these molecular alterations. The utilization of innovative cell-specific drug delivery methods like exosomes and extracellular vesicles holds promise in achieving precise epigenetic regulation., (© 2024. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Bidirectional communication between tumours and neurons has emerged as a key facet of the tumour microenvironment that drives malignancy 1,2 . Another hallmark feature of cancer is epigenomic dysregulation, in which alterations in gene expression influence cell states and interactions with the tumour microenvironment 3 . Ependymoma (EPN) is a paediatric brain tumour that relies on epigenomic remodelling to engender malignancy 4,5 ; however, how these epigenetic mechanisms intersect with extrinsic neuronal signalling during EPN tumour progression is unknown. Here we show that the activity of serotonergic neurons regulates EPN tumorigenesis, and that serotonin itself also serves as an activating modification on histones. We found that inhibiting histone serotonylation blocks EPN tumorigenesis and regulates the expression of a core set of developmental transcription factors. High-throughput, in vivo screening of these transcription factors revealed that ETV5 promotes EPN tumorigenesis and functions by enhancing repressive chromatin states. Neuropeptide Y (NPY) is one of the genes repressed by ETV5, and its overexpression suppresses EPN tumour progression and tumour-associated network hyperactivity through synaptic remodelling. Collectively, this study identifies histone serotonylation as a key driver of EPN tumorigenesis, and also reveals how neuronal signalling, neuro-epigenomics and developmental programs are intertwined to drive malignancy in brain cancer., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Heterochromatin stability is crucial for progenitor proliferation during early neurogenesis. It relays on the maintenance of local hubs of H3K9me. However, understanding the formation of efficient localized levels of H3K9me remains limited. To address this question, we used neural stem cells to analyze the function of the H3K9me2 demethylase PHF2, which is crucial for progenitor proliferation. Through mass-spectroscopy and genome-wide assays, we show that PHF2 interacts with heterochromatin components and is enriched at pericentromeric heterochromatin (PcH) boundaries where it maintains transcriptional activity. This binding is essential for silencing the satellite repeats, preventing DNA damage and genome instability. PHF2's depletion increases the transcription of heterochromatic repeats, accompanied by a decrease in H3K9me3 levels and alterations in PcH organization. We further show that PHF2's PHD and catalytic domains are crucial for maintaining PcH stability, thereby safeguarding genome integrity. These results highlight the multifaceted nature of PHF2's functions in maintaining heterochromatin stability and regulating gene expression during neural development. Our study unravels the intricate relationship between heterochromatin stability and progenitor proliferation during mammalian neurogenesis., (© 2024. The Author(s).)

Uterine luminal epithelia (LE), the first layer contacting with the blastocyst, acquire receptivity for normal embryo implantation. Besides the well-accepted transcriptional regulation dominated by ovarian estrogen and progesterone for receptivity establishment, the involvement of epigenetic mechanisms remains elusive. This study systematically profiles the transcriptome and genome-wide H3K27me3 distribution in the LE throughout the preimplantation. Combining genetic and pharmacological approaches targeting the PRC2 core enzyme Ezh1/2, we demonstrate that the defective remodeling of H3K27me3 in the preimplantation stage disrupts the differentiation of LE, and derails uterine receptivity, resulting in implantation failure. Specifically, crucial epithelial genes, Pgr, Gata2, and Sgk1, are transcriptionally silenced through de novo deposition of H3K27me3 for LE transformation, and their sustained expression in the absence of H3K27me3 synergistically confines the nuclear translocation of FOXO1. Further functional studies identify several actin-associated genes, including Arpin, Tmod1, and Pdlim2, as novel direct targets of H3K27me3. Their aberrantly elevated expression impedes the morphological remodeling of LE, a hindrance alleviated by treatment with cytochalasin D which depolymerizes F-actin. Collectively, this study uncovers a previously unappreciated epigenetic regulatory mechanism for the transcriptional silencing of key LE genes via H3K27me3, essential for LE differentiation and thus embryo implantation., (© 2024. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)