EZH2 as a major histone methyltransferase in PDGF-BB-activated orbital fibroblast in the pathogenesis of Graves' ophthalmopathy.

Graves' ophthalmopathy (GO) is an extra-thyroidal complication of Graves' disease which can lead to vision loss in severe cases. Currently, treatments of GO are not sufficiently effective, so novel therapeutic strategies are needed. As platelet-derived growth factor (PDGF)-BB induces several effector mechanisms in GO orbital fibroblasts including cytokine production and myofibroblast activation, this study aims to investigate the roles of histone lysine methyltransferases (HKMTs) in PDGF-BB-activated GO orbital fibroblasts by screening with HKMTs inhibitors library. From the total of twelve selective HKMT inhibitors in the library, EZH2, G9a and DOT1L inhibitors, DZNeP, BIX01294 and Pinometostat, respectively, prevented PDGF-BB-induced proliferation and hyaluronan production by GO orbital fibroblasts. However, only EZH2 inhibitor, DZNeP, significantly blocked pro-inflammatory cytokine production. For the HKMTs expression in GO orbital fibroblasts, PDGF-BB significantly and time-dependently induced EZH2, G9a and DOT1L mRNA expression. To confirm the role of EZH2 in PDGF-BB-induced orbital fibroblast activation, EZH2 silencing experiments revealed suppression of PDGF-BB-induced collagen type I and α-SMA expression along with decreasing histone H3 lysine 27 trimethylation (H3K27me3) level. In a more clinically relevant model than orbital fibroblast culture experiments, DZNeP treated GO orbital tissues significantly reduced pro-inflammatory cytokine production while slightly reduced ACTA2 mRNA expression. Our data is the first to demonstrate that among all HKMTs EZH2 dominantly involved in the expression of myofibroblast markers in PDGF-BB-activated orbital fibroblast from GO presumably via H3K27me3. Thus, EZH2 may represent a novel therapeutics target for GO.

MLL1 histone methyltransferase and UTX histone demethylase functionally cooperate to regulate the expression of NRF2 in response to ROS-induced oxidative stress.

The transcription factor NRF2 plays a pivotal role in maintaining redox and metabolic homeostasis by orchestrating oxidative stress-dependent transcription programs. Despite growing evidence implicating various cellular components in the regulation of NRF2 activity at the posttranslational stage, relatively less is known about the factors dictating the transcriptional activation of NRF2 in response to oxidative stress. In this study, we report the crucial roles of MLL1, an H3K4-specific methyltransferase, and UTX, an H3K27-specific histone demethylase, in the NRF2-dependent transcription program under oxidative stress. We find that the depletion of MLL1 or UTX results in increased susceptibility to oxidative stress, accompanied by higher intracellular ROS and the failed activation of antioxidant genes, including NRF2. In addition, MLL1 and UTX selectively target the NRF2 promoter, and exogenous FLAG-NRF2 expression increases the viability of MLL1-or UTX-depleted cells upon exposure to hydrogen peroxide. RNA-seq analysis demonstrates that depletion of MLL1 or UTX affects the changes in NRF2-dependent transcriptome in response to oxidative stress. Furthermore, ChIP and ChIP-seq analyses find that MLL1 and UTX functionally cooperate to establish a chromatin environment that favors active transcription at the H3K4me3/H3K27me3 bivalent NRF2 promoter in response to ROS-induced oxidative stress. Collectively, these findings provide a molecular mechanism underlying the cellular response to oxidative stress and highlight the importance of the chromatin structure and function in maintaining redox homeostasis.

Selfish conflict underlies RNA-mediated parent-of-origin effects.

Genomic imprinting-the non-equivalence of maternal and paternal genomes-is a critical process that has evolved independently in many plant and mammalian species1,2. According to kinship theory, imprinting is the inevitable consequence of conflictive selective forces acting on differentially expressed parental alleles3,4. Yet, how these epigenetic differences evolve in the first place is poorly understood3,5,6. Here we report the identification and molecular dissection of a parent-of-origin effect on gene expression that might help to clarify this fundamental question. Toxin-antidote elements (TAs) are selfish elements that spread in populations by poisoning non-carrier individuals7-9. In reciprocal crosses between two Caenorhabditis tropicalis wild isolates, we found that the slow-1/grow-1 TA is specifically inactive when paternally inherited. This parent-of-origin effect stems from transcriptional repression of the slow-1 toxin by the PIWI-interacting RNA (piRNA) host defence pathway. The repression requires PIWI Argonaute and SET-32 histone methyltransferase activities and is transgenerationally inherited via small RNAs. Remarkably, when slow-1/grow-1 is maternally inherited, slow-1 repression is halted by a translation-independent role of its maternal mRNA. That is, slow-1 transcripts loaded into eggs-but not SLOW-1 protein-are necessary and sufficient to counteract piRNA-mediated repression. Our findings show that parent-of-origin effects can evolve by co-option of the piRNA pathway and hinder the spread of selfish genes that require sex for their propagation.

Controllers of histone methylation-modifying enzymes in gastrointestinal cancers.

Gastrointestinal (GI) cancers have been considered primarily genetic malignancies, caused by a series of progressive genetic alterations. Accumulating evidence shows that histone methylation, an epigenetic modification program, plays an essential role in the different pathological stages of GI cancer progression, such as precancerous lesions, tumorigenesis, and tumor metastasis. Histone methylation-modifying enzymes, including histone methyltransferases (HMTs) and demethylases (HDMs), are the main executor of post-transcriptional modification. The abnormal expression of histone methylation-modifying enzymes characterizes GI cancers with complex pathogenesis and progression. Interactions between upstream controllers and histone methylation-modifying enzymes have recently been revealed, and have provided numerous opportunities to elucidate the pathogenesis of GI cancers in depth and clearly. Here we focus on the association between histone methylation-modifying enzymes and their controllers, aiming to provide a new perspective on the molecular research and clinical management of GI cancers.

Characterization of the Pristionchus pacificus "epigenetic toolkit" reveals the evolutionary loss of the histone methyltransferase complex PRC2.

Comparative approaches have revealed both divergent and convergent paths to achieving shared developmental outcomes. Thus, only through assembling multiple case studies can we understand biological principles. Yet, despite appreciating the conservation-or lack thereof-of developmental networks, the conservation of epigenetic mechanisms regulating these networks is poorly understood. The nematode Pristionchus pacificus has emerged as a model system of plasticity and epigenetic regulation as it exhibits a bacterivorous or omnivorous morph depending on its environment. Here, we determined the "epigenetic toolkit" available to P. pacificus as a resource for future functional work on plasticity, and as a comparison with Caenorhabditis elegans to investigate the conservation of epigenetic mechanisms. Broadly, we observed a similar cast of genes with putative epigenetic function between C. elegans and P. pacificus. However, we also found striking differences. Most notably, the histone methyltransferase complex PRC2 appears to be missing in P. pacificus. We described the deletion/pseudogenization of the PRC2 genes mes-2 and mes-6 and concluded that both were lost in the last common ancestor of P. pacificus and a related species P. arcanus. Interestingly, we observed the enzymatic product of PRC2 (H3K27me3) by mass spectrometry and immunofluorescence, suggesting that a currently unknown methyltransferase has been co-opted for heterochromatin silencing. Altogether, we have provided an inventory of epigenetic genes in P. pacificus to compare with C. elegans. This inventory will enable reverse-genetic experiments related to plasticity and has revealed the first loss of PRC2 in a multicellular organism.

Histone methyltransferase SETD2 is required for proper hippocampal lamination and neuronal maturation.

Proper formation of the hippocampus is crucial for the brain to execute memory and learning functions. However, many questions remain regarding how pyramidal neurons (PNs) of the hippocampus mature and precisely position. Here we revealed that Setd2, the methyltransferase for histone 3 lysine 36 trimethylation (H3K36me3), is essential for the precise localization and maturation of PNs in the hippocampal CA1. The ablation of Setd2 in neural progenitors leads to irregular lamination of the CA1 and increased numbers of PNs in the stratum oriens. Setd2 deletion in postmitotic neurons causes mislocalization and immaturity of CA1 PNs. Transcriptome analyses revealed that SETD2 maintains the expressions of clustered protocadherin (cPcdh) genes. Together, Setd2 is required for proper hippocampal lamination and maturation of CA1 PNs.

Regulation of adipogenesis by histone methyltransferases.

Epigenetic regulation is a critical component of lineage determination. Adipogenesis is the process through which uncommitted stem cells or adipogenic precursor cells differentiate into adipocytes, the most abundant cell type of the adipose tissue. Studies examining chromatin modification during adipogenesis have provided further understanding of the molecular blueprint that controls the onset of adipogenic differentiation. Unlike histone acetylation, histone methylation has context dependent effects on the activity of a transcribed region of DNA, with individual or combined marks on different histone residues providing distinct signals for gene expression. Over half of the 42 histone methyltransferases identified in mammalian cells have been investigated in their role during adipogenesis, but across the large body of literature available, there is a lack of clarity over potential correlations or emerging patterns among the different players. In this review, we will summarize important findings from studies published in the past 15 years that have investigated the role of histone methyltransferases during adipogenesis, including both protein arginine methyltransferases (PRMTs) and lysine methyltransferases (KMTs). We further reveal that PRMT1/4/5, H3K4 KMTs (MLL1, MLL3, MLL4, SMYD2 and SET7/9) and H3K27 KMTs (EZH2) all play positive roles during adipogenesis, while PRMT6/7 and H3K9 KMTs (G9a, SUV39H1, SUV39H2, and SETDB1) play negative roles during adipogenesis.

The dePARylase NUDT16 promotes radiation resistance of cancer cells by blocking SETD3 for degradation via reversing its ADP-ribosylation.

Poly(ADP-ribosyl)ation (PARylation) is a critical posttranslational modification that plays a vital role in maintaining genomic stability via a variety of molecular mechanisms, including activation of replication stress and the DNA damage response. The nudix hydrolase NUDT16 was recently identified as a phosphodiesterase that is responsible for removing ADP-ribose units and that plays an important role in DNA repair. However, the roles of NUDT16 in coordinating replication stress and cell cycle progression remain elusive. Here, we report that SETD3, which is a member of the SET-domain containing protein (SETD) family, is a novel substrate for NUDT16, that its protein levels fluctuate during cell cycle progression, and that its stability is strictly regulated by NUDT16-mediated dePARylation. Moreover, our data indicated that the E3 ligase CHFR is responsible for the recognition and degradation of endogenous SETD3 in a PARP1-mediated PARylation-dependent manner. Mechanistically, we revealed that SETD3 associates with BRCA2 and promotes its recruitment to stalled replication fork and DNA damage sites upon replication stress or DNA double-strand breaks, respectively. Importantly, depletion of SETD3 in NUDT16-deficient cells did not further exacerbate DNA breaks or enhance the sensitivity of cancer cells to IR exposure, suggesting that the NUDT16-SETD3 pathway may play critical roles in the induction of tolerance to radiotherapy. Collectively, these data showed that NUDT16 functions as a key upstream regulator of SETD3 protein stability by reversing the ADP-ribosylation of SETD3, and NUDT16 participates in the resolution of replication stress and facilitates HR repair.

Histone methyltransferase SUV39H2 regulates apoptosis and chemosensitivity in prostate cancer through AKT/FOXO signaling pathway.

Prostate cancer (PCa) is one of the most common malignant tumors that exhibit both chemoresistance and recurrence. SUV39H2 is highly expressed in many types of human tumors, but its role in the development and progression of PCa has never been clarified. The aim of this study is to elucidate the role of SUV39H2 in the development and progression of PCa, its association with the AKT/FOXO signaling pathway, and its potential implications for PCa diagnosis and treatment. SUV39H2 expression was analyzed in The Cancer Genome Atlas (TCGA) and genotype tissue expression pan-cancer data. The TCGA database was evaluated for SUV39H2 enrichment and its correlation to immune cell infiltration. SUV39H2 levels in PCa tissues and control tissues were determined in 30 patients using qPCR and IHC. Clinical relevance was assessed via The Cancer Genome Atlas (TCGA). In vitro assessments including colony formation assays, Western Blot analysis, CCK-8 assays, and flow cytometry were utilized to establish SUV39H2's contribution to PCa cell growth. The influence of SUV39H2 on PC3 and DU145 cell proliferation was assessed through a cell line-derived xenograft model. Sphere formation assays and qPCR were employed to delineate SUV39H2's role in PCa stemness and chemosensitivity. In vitro macrophage polarization assays provided insights into SUV39H2's association with M2 macrophages, while enrichment analysis shed light on its role in FOXO signaling. PCa tissues expressed higher levels of SUV39H2 than normal tissues. By knocking down SUV39H2, PCa cells were made more chemosensitive to docetaxel and cell proliferation and stemness were inhibited. Additionally, SUV39H2 knockdown significantly inhibited in vivo PCa cell growth and inhibited the polarization of macrophages. Furthermore, SUV39H2 was found to regulate AKT/FOXO signaling by increasing Akt and FOXO3a phosphorylation. Our findings highlight SUV39H2's role in PCa cell apoptosis and chemosensitivity mainly by regulating the AKT/FOXO signaling pathway and suggest that SUV39H2 could be a potential target for PCa diagnosis and treatment.

ULTRAPETALAs in action: Unraveling their role in root development.

The epigenetic complex Trithorax (TrxG) regulates gene transcription through post-translational histone modifications and is involved in a wide range of developmental processes. ULTRAPETALA1 (ULT1) is a SAND domain plant-exclusive TrxG protein that regulates the H3K4me3 active mark to counteract PcG repression. ULT1 has been identified to be involved in multiple tissue-specific processes. In the Arabidopsis root, ULT1 is required to maintain the stem cell niche, a role that is independent of the histone methyltransferase ATX1. Here we show the contribution of ULT2 in the maintenance of root stem cell niche. We also analyzed the gene expression in the ult1, ult2, and ult1ult2 mutants, evidencing three ways in which ULT1 and ULT2 regulate gene expression, one of them, where ULT1 or ULT2 regulate specific genes each, another where ULT1 and ULT2 act redundantly, as well as a regulation that requires of ULT1 and ULT2 together, supporting a coregulation, never reported. Furthermore, we also evidenced the participation of ULT1 in transcriptional repression synergically with CLF, a key histone methyltransferase of PcG.

Role of histone H3K4 methyltransferase in regulating Monascus pigments production by red light-coupled magnetic field.

Light, magnetic field, and methylation affected the growth and secondary metabolism of fungi. The regulation effect of the three factors on the growth and Monascus pigments (MPs) synthesis of Monascus purpureus was investigated in this study. 5-azacytidine (5-AzaC), DNA methylation inhibitor, was used to treat M. purpureus (wild-type, WT). Twenty micromolar 5-AzaC significantly promoted the growth, development, and MPs yield. Moreover, 250 lux red light and red light coupled magnetic field (RLCMF) significantly promoted the biomass. For WT, red light, and RLCMF significantly promoted MPs yield. But compared with red light treatment, only 0.2 mT RLCMF promoted the alcohol-soluble MPs yield. For histone H3K4 methyltransferase complex subunit Ash2 gene knockout strain (ΔAsh2), only 0.2 mT RLCMF significantly promoted water-soluble MPs yield. Yet red light, 1.0 and 0.2 mT RLCMF significantly promoted alcohol-soluble MPs yield. This indicated that methylation affected the MPs biosynthesis. Red light and weaker MF had a synergistic effect on the growth and MPs synthesis of ΔAsh2. This result was further confirmed by the expression of related genes. Therefore, histone H3K4 methyltransferase was involved in the regulation of the growth, development, and MPs synthesis of M. purpureus by the RLCMF.

Histone methyltransferase SUV420H1/KMT5B contributes to poor prognosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a high rate of recurrence and poor prognosis, even after curative surgery. Multikinase inhibitors have been applied for HCC patients, but their effect has been restricted. This study aims to clarify the clinical impact of SUV420H1/KMT5B, one of the methyltransferases for histone H4 at lysine 20, and elucidate the novel mechanisms of HCC progression. We retrospectively investigated SUV420H1 expression using HCC clinical tissue samples employing immunohistochemical analysis (n = 350). We then performed loss-of-function analysis of SUV420H1 with cell cycle analysis, migration assay, invasion assay and RNA sequence for Gene Ontology (GO) pathway analysis in vitro, and animal experiments with xenograft mice in vivo. The SUV420H1-high-score group (n = 154) had significantly poorer prognosis for both 5-year overall and 2-year/5-year disease-free survival than the SUV420H1-low-score group (n = 196) (p < 0.001 and p < 0.05, respectively). The SUV420H1-high-score group had pathologically larger tumor size, more tumors, poorer differentiation, and more positive vascular invasion than the SUV420H1-low-score group. Multivariate analysis demonstrated that SUV420H1 high score was the poorest independent factor for overall survival. SUV420H1 knockdown could suppress cell cycle from G1 to S phase and cell invasion. GO pathway analysis showed that SUV420H1 contributed to cell proliferation, cell invasion, and/or metastasis. Overexpression of SUV420H1 clinically contributed to poor prognosis in HCC, and the inhibition of SUV420H1 could repress tumor progression and invasion both in vitro and in vivo; thus, further analyses of SUV420H1 are necessary for the discovery of future molecularly targeted drugs.

Elevation of H3K27me3 level contributes to the radioresistance of nasopharyngeal carcinoma by inhibiting OAS1 expression.

Radiotherapy has long been a main treatment option for nasopharyngeal carcinoma (NPC). However, during clinical treatment, NPC is prone to developing radioresistance, resulting in treatment failure. This study aims to examine the role of histone methylation in the induction of radioresistance. It was found that the radioresistance of NPC cells was related to the increase of the level of histone H3 lysine 27 trimethylation (H3K27me3). Treatment of cells with histone methyltransferase inhibitor GSK126 increased the radiosensitivity of NPC cells by triggering Bcl2 apoptosis regulator/BCL2-associated X, apoptosis regulator (Bcl2/BAX) signaling pathway. Bioinformatics analysis indicated that the expression of 2'-5'-oligoadenylate synthetase 1 (OAS1) was reduced in the radioresistant cells but increased in the GSK126-treated cells. Chromatin immunoprecipitation assay confirmed that the decrease of OAS1 expression in radioresistant cells was mainly due to the enrichment of H3K27me3 in its promoter region. Furthermore, downregulation of OAS1 reduced apoptosis due to the inhibition of Bcl2/BAX pathway after irradiation, while OAS1 overexpression increased radiosensitivity. Our findings revealed for the first time that the increase of H3K27me3 level was associated with the decrease of OAS1 expression, leading to the inhibition of apoptosis and ultimately contributing to the radioresistance of NPC cells. Moreover, the histone methyltransferase inhibitor GSK126 could overcome the radioresistance and thus might be a potential therapeutic strategy for NPC.NEW & NOTEWORTHY Our findings revealed for the first time that the increase of H3K27me3 level was associated with the decrease of OAS1 expression, leading to the inhibition of apoptosis and ultimately contributing to the radioresistance of NPC cells. Moreover, we demonstrated that the histone methyltransferase inhibitor GSK126 could be a promising therapeutic strategy for NPC by overcoming radioresistance, providing valuable insights into the clinical treatment of NPC.

YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia†.

Genomic integrity is critical for sexual reproduction, ensuring correct transmission of parental genetic information to the descendant. To preserve genomic integrity, germ cells have evolved multiple DNA repair mechanisms, together termed as DNA damage response. The RNA N6-methyladenosine is the most abundant mRNA modification in eukaryotic cells, which plays important roles in DNA damage response, and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) is a well-acknowledged N6-methyladenosine reader protein regulating the mRNA decay and stress response. Despite this, the correlation between YTHDF2 and DNA damage response in germ cells, if any, remains enigmatic. Here, by employing a Ythdf2-conditional knockout mouse model as well as a Ythdf2-null GC-1 mouse spermatogonial cell line, we explored the role and the underlying mechanism for YTHDF2 in spermatogonial DNA damage response. We identified that, despite no evident testicular morphological abnormalities under the normal circumstance, conditional mutation of Ythdf2 in adult male mice sensitized germ cells, including spermatogonia, to etoposide-induced DNA damage. Consistently, Ythdf2-KO GC-1 cells displayed increased sensitivity and apoptosis in response to DNA damage, accompanied by the decreased SET domain bifurcated 1 (SETDB1, a histone methyltransferase) and H3K9me3 levels. The Setdb1 knockdown in GC-1 cells generated a similar phenotype, but its overexpression in Ythdf2-null GC-1 cells alleviated the sensitivity and apoptosis in response to DNA damage. Taken together, these results demonstrate that the N6-methyladenosine reader YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia, which provides novel insights into the mechanisms underlying spermatogonial genome integrity maintenance and therefore contributes to safe reproduction.

Genome-wide identification and expression analysis reveal the role of histone methyltransferase and demethylase genes in heat stress response in potato (Solanum tuberosum L.).

BACKGROUND: Potato (Solanum tuberosum L.), the third most important non-cereal crop, is sensitive to high temperature. Histone modifications have been known to regulate various abiotic stress responses. However, the role of histone methyltransferases and demethylases remain unexplored in potato under heat stress. METHODS: Potato genome database was used for genome-wide analysis of StPRMT and StHDMA gene families, which were further characterized by analyzing gene structure, conserved motif, domain organization, sub-cellular localization, promoter region and phylogenetic relationships. Additionally, expression profiling under high-temperature stress in leaf and stolon tissue of heat contrasting potato genotypes was done to study their role in response to high temperature stress. RESULTS: The genome-wide analysis led to identification of nine StPRMT and eleven StHDMA genes. Structural analysis, including conserved motifs, exon/intron structure and phylogenetic relationships classified StPRMT and StHDMA gene families into two classes viz. Class I and Class II. A variety of cis-regulatory elements were explored in the promoter region associated with light, developmental, hormonal and stress responses. Prediction of sub-cellular localization of StPRMT proteins revealed their occurrence in nucleus and cytoplasm, whereas StHDMA proteins were observed in different sub-cellular compartments. Furthermore, expression profiling of StPRMT and StHDMA gene family members revealed genes responding to heat stress. Heat-inducible expression of StPRMT1, StPRMT3, StPRMT4 and StPRMT5 in leaf and stolon tissues of HS and HT cultivar indicated them as probable candidates for enhancing thermotolerance in potato. However, StHDMAs responded dynamically in leaf and stolon tissue of heat contrasting genotypes under high temperature. CONCLUSION: The current study presents a detailed analysis of histone modifiers in potato and indicates their role as an important epigenetic regulators modulating heat tolerance. GENERAL SIGNIFICANCE: Understanding epigenetic mechanisms underlying heat tolerance in potato will contribute towards breeding of thermotolerant potato varieties.

[Histone methyltransferases and regenerative myogenesis: A focus on SETDB1]. / Histones méthyltransférases et myogenèse régénérative - Un focus sur SETDB1.

Adult skeletal muscle is composed of thousands of fibers (also called myofibers) that contract thus allowing voluntary movements. Following an injury, muscle stem cells, surrounding the myofibers, activate, proliferate, and differentiate to form de novo myofibers. These steps constitute a process called adult (or regenerative) myogenesis. This process is possible thanks to various transcription factors sequentially expressed and regulated by epigenetic factors that modulate the chromatin and therefore lead to the regulation of gene expression. Gene expression changes consequently affect the fate of muscle stem cells. Histone Lysine Methyltransferases methylate some histones involved in the repression of gene expression. We describe here the role of SETDB1 during adult myogenesis, notably its role during muscle stem cell differentiation.

Title: Histones méthyltransférases et myogenèse régénérative - Un focus sur SETDB1. Abstract: Le muscle strié squelettique adulte est composé de milliers de fibres (myofibres) capables de se contracter, permettant ainsi les mouvements volontaires. Après une lésion musculaire, les cellules souches musculaires localisées autour des myofibres s'activent, prolifèrent et se différencient pour former de nouvelles myofibres. Ces différentes étapes forment un processus appelé myogenèse. Cette dernière est rendue possible grâce à l'expression séquentielle de facteurs de transcription régulés par des facteurs épigénétiques qui agissent sur la chromatine afin de moduler l'expression génique et ainsi, le devenir de la cellule souche. Les histones méthyltransférases déposent des marques dites méthyl sur certaines histones afin d'induire la répression génique de régions spécifiques. Nous décrivons ici le rôle de SETDB1 au cours de la myogenèse adulte, et plus spécifiquement pendant la différenciation des cellules souches musculaires.

Evidence of a synthetic lethality interaction between SETDB1 histone methyltransferase and CHD4 chromatin remodeling protein in a triple negative breast cancer cell line.

During the tumorigenic process, cancer cells may become overly dependent on the activity of backup cellular pathways for their survival, representing vulnerabilities that could be exploited as therapeutic targets. Certain molecular vulnerabilities manifest as a synthetic lethality relationship, and the identification and characterization of new synthetic lethal interactions may pave the way for the development of new therapeutic approaches for human cancer. Our goal was to investigate a possible synthetic lethal interaction between a member of the Chromodomain Helicase DNA binding proteins family (CHD4) and a member of the histone methyltransferases family (SETDB1) in the molecular context of a cell line (Hs578T) representing the triple negative breast cancer (TNBC), a subtype of breast cancer lacking validated molecular targets for treatment. Therefore, we employed the CRISPR-Cas9 gene editing tool to individually or simultaneously introduce indels in the genomic loci corresponding to the catalytic domains of SETDB1 and CHD4 in the Hs578T cell line. Our main findings included: a) introduction of indels in exon 22 of SETDB1 sensitized Hs578T to the action of the genotoxic chemotherapy doxorubicin; b) by sequentially introducing indels in exon 22 of SETDB1 and exon 23 of CHD4 and tracking the percentage of the remaining wild-type sequences in the mixed cell populations generated, we obtained evidence of the existence of a synthetic lethality interaction between these genes. Considering the lack of molecular targets in TNBC, our findings provided valuable insights for development of new therapeutic approaches not only for TNBC but also for other cancer types.

Histone Methyltransferases G9a/Ehmt2 and GLP/Ehmt1 Are Associated with Cell Viability and Poorer Prognosis in Neuroblastoma and Ewing Sarcoma.

Changes in epigenetic programming have been proposed as being key events in the initiation and progression of childhood cancers. HMT euchromatic histone lysine methyltransferase 2 (G9a, EHMT2), which is encoded by the G9a (Ehmt2) gene, as well as its related protein GLP, which is encoded by the GLP/Ehmt1 gene, participate in epigenetic regulation by contributing to a transcriptionally repressed chromatin state. G9a/GLP activation has been reported in several cancer types. Herein, we evaluated the role of G9a in two solid pediatric tumors: neuroblastoma (NB) and Ewing sarcoma (ES). Our results show that G9a/Ehmt2 and GLP/Ehmt1 expression is higher in tumors with poorer prognosis, including St4 International Neuroblastoma Staging System (INSS) stage, MYCN amplified NB, and metastatic ES. Importantly, higher G9a and GLP levels were associated with shorter patient overall survival (OS) in both NB and ES. Moreover, pharmacological inhibition of G9a/GLP reduced cell viability in NB and ES cells. These findings suggest that G9a and GLP are associated with more aggressive NB and ES tumors and should be further investigated as being epigenetic targets in pediatric solid cancers.

Metabolic sinkholes: Histones as methyl repositories.

Perez and Sarkies uncover histones as methyl group repositories in normal and cancer human cells, shedding light on an intriguing function of histone methylation in optimizing the cellular methylation potential independently of gene regulation.

Non-canonical function of histone methyltransferase G9a in the translational regulation of chronic inflammation.

We report a novel translation-regulatory function of G9a, a histone methyltransferase and well-understood transcriptional repressor, in promoting hyperinflammation and lymphopenia; two hallmarks of endotoxin tolerance (ET)-associated chronic inflammatory complications. Using multiple approaches, we demonstrate that G9a interacts with multiple translation regulators during ET, particularly the N6-methyladenosine (m6A) RNA methyltransferase METTL3, to co-upregulate expression of certain m6A-modified mRNAs that encode immune-checkpoint and anti-inflammatory proteins. Mechanistically, G9a promotes m6A methyltransferase activity of METTL3 at translational/post-translational level by regulating its expression, its methylation, and its cytosolic localization during ET. Additionally, from a broader view extended from the G9a-METTL3-m6A translation regulatory axis, our translatome proteomics approach identified numerous "G9a-translated" proteins that unite the networks associated with inflammation dysregulation, T cell dysfunction, and systemic cytokine response. In sum, we identified a previously unrecognized function of G9a in protein-specific translation that can be leveraged to treat ET-related chronic inflammatory diseases.