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Methylation of histone H3 lysine 27 (H3K27) is widely recognized as a transcriptionally repressive chromatin modification but the mechanism of repression remains unclear. We devised and implemented a forward genetic scheme to identify factors required for H3K27 methylation-mediated silencing in the filamentous fungus Neurospora crassa and identified a bromo-adjacent homology (BAH)-plant homeodomain (PHD)-containing protein, EPR-1 (effector of polycomb repression 1; NCU07505). EPR-1 associates with H3K27-methylated chromatin, and loss of EPR-1 de-represses H3K27-methylated genes without loss of H3K27 methylation. EPR-1 is not fungal-specific; orthologs of EPR-1 are present in a diverse array of eukaryotic lineages, suggesting an ancestral EPR-1 was a component of a primitive Polycomb repression pathway.

Insect development is cooperatively orchestrated by the steroid hormone ecdysone and juvenile hormone (JH). The polycomb repressive complex 2 (PRC2)–mediated histone H3K27 trimethylation (H3K27me3) epigenetically silences gene transcription and is essential for a range of biological processes, but the functions of H3K27 methylation in insect hormone action are poorly understood. Here, we demonstrate that H3K27 methylation–mediated repression of Hairy transcription in the larval prothoracic gland (PG) is required for ecdysone biosynthesis in Bombyx and Drosophila. H3K27me3 levels in the PG are dynamically increased during the last larval instar. H3K27me3 reduction induced by the down-regulation of PRC2 activity via inhibitor treatment in Bombyx or PG-specific knockdown of the PRC2 component Su(z)12 in Drosophila diminishes ecdysone biosynthesis and disturbs the larval–pupal transition. Mechanistically, H3K27 methylation targets the JH signal transducer Hairy to repress its transcription in the PG; PG-specific knockdown or overexpression of the Hairy gene disrupts ecdysone biosynthesis and developmental transition; and developmental defects caused by PG-specific Su(z)12 knockdown can be partially rescued by Hairy down-regulation. The application of JH mimic to the PG decreases both H3K27me3 levels and Su(z)12 expression. Altogether, our study reveals that PRC2-mediated H3K27 methylation at Hairy in the PG during the larval period is required for ecdysone biosynthesis and the larval–pupal transition and provides insights into epigenetic regulation of the crosstalk between JH and ecdysone during insect development.

Abstract Neurospora crassa contains a minimal Polycomb repression system, which provides rich opportunities to explore Polycomb-mediated repression across eukaryotes and enables genetic studies that can be difficult in plant and animal systems. Polycomb Repressive Complex 2 is a multi-subunit complex that deposits mono-, di-, and trimethyl groups on lysine 27 of histone H3, and trimethyl H3K27 is a molecular marker of transcriptionally repressed facultative heterochromatin. In mouse embryonic stem cells and multiple plant species, H2A.Z has been found to be colocalized with H3K27 methylation. H2A.Z is required for normal H3K27 methylation in these experimental systems, though the regulatory mechanisms are not well understood. We report here that Neurospora crassa mutants lacking H2A.Z or SWR-1, the ATP-dependent histone variant exchanger, exhibit a striking reduction in levels of H3K27 methylation. RNA-sequencing revealed downregulation of eed, encoding a subunit of PRC2, in an hH2Az mutant compared to wild type, and overexpression of EED in a ΔhH2Az;Δeed background restored most H3K27 methylation. Reduced eed expression leads to region-specific losses of H3K27 methylation, suggesting that differential dependence on EED concentration is critical for normal H3K27 methylation at certain regions in the genome.

Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), mediates histone H3 lysine 27 trimethylation (H3K27me3), an epigenetic modification associated with transcriptional repression. EZH2 inhibitors (EZH2is) gained attention after the first-in-class drug Tazemetostat received FDA approval for treating epithelioid sarcoma. Preclinical studies suggest that EZH2is could be effective against melanoma, but their general inability to cross the blood–brain barrier (BBB), among others, limits the treatment of secondary brain metastases. Based on these limitations, we designed SG-8, a novel compound derived from TDI-6118 (a known brain-penetrant EZH2i). In silico docking predicted that SG-8 may exhibit high affinity for EZH2 as well as for another PRC2 subunit, Embryonic Ectoderm Development (EED). In addition, in vitro PAMPA assays suggested passive BBB permeability of SG-8. In cell-based assays, SG-8 and the structurally related EZH2i PF-06726304 displayed lower cytotoxicity than Tazemetostat in both primary (A375) and metastatic (Colo-679) human melanoma cells. Western blot analysis showed that SG-8 and PF-06726304 markedly reduced EED protein levels and, to a lesser extent, EZH2 levels, without affecting total H3K27me3, consistent with preserved canonical PRC2 activity. Instead, treatment with both compounds—most prominently SG-8—was associated with reduced phosphorylation levels of EZH2 (Ser21) and its upstream regulator Akt (Ser473), suggesting that modulation of the Akt–EZH2 signaling axis may at least partially contribute to their anti-melanoma activity.

Methylation of histone H3 at lysine 36 (H3K36me), a widely-distributed chromatin mark, largely results from association of the lysine methyltransferase (KMT) SET-2 with RNA polymerase II (RNAPII), but most eukaryotes also have additional H3K36me KMTs that act independently of RNAPII. These include the orthologs of ASH1, which are conserved in animals, plants, and fungi but whose function and control are poorly understood. We found that Neurospora crassa has just two H3K36 KMTs, ASH1 and SET-2, and were able to explore the function and distribution of each enzyme independently. While H3K36me deposited by SET-2 marks active genes, inactive genes are modified by ASH1 and its activity is critical for their repression. ASH1-marked chromatin can be further modified by methylation of H3K27, and ASH1 catalytic activity modulates the accumulation of H3K27me2/3 both positively and negatively. These findings provide new insight into ASH1 function, H3K27me2/3 establishment, and repression in facultative heterochromatin. Not all genes in a cell’s DNA are active all the time. There are several ways to control this activity. One is by altering how the DNA is packaged into cells. DNA strands are wrapped around proteins called histones to form nucleosomes. Nucleosomes can then be packed together tightly, to restrict access to the DNA at genes that are not active, or loosely to allow access to the DNA of active genes. Chemical marks, such as methyl groups, can be attached to particular sites on histones to influence how they pack together. One important site for such marks is known as position 36 on histone H3, or H3K36 for short. Correctly adding methyl groups to this site is critical for normal development, and when this process goes wrong it can lead to diseases like cancer. An enzyme called SET-2 oversees the methylation of H3K36 in fungi, plants and animals. However, many species have several other enzymes that can also add methyl groups to H3K36, and their roles are less clear. A type of fungus called Neurospora crassa contains just two enzymes that can add methyl groups to H3K36: SET-2, and another enzyme called ASH1. By performing experiments that inactivated SET-2 and ASH1 in this fungus, Bicocca et al. found that each enzyme works on a different set of genes. Genes in regions marked by SET-2 were accessible for the cell to use, while genes marked by ASH1 were inaccessible. ASH1 also affects whether a methyl group is added to another site on histone H3. This mark is important for controlling the activity of genes that are critical for development. ASH1 is found in many other organisms, including humans. The results presented by Bicocca et al. could therefore be built upon to understand the more complicated systems for regulating H3K36 methylation in other species. From there, we can investigate how to intervene when things go wrong during developmental disorders and cancer.

Background The intriguing connection between selenium and cancer resembles a captivating puzzle that keeps researchers engaged and curious. While selenium has shown promise in reducing cancer risks through supplementation, its interaction with epigenetics in cervical cancer remains a fascinating yet largely unexplored realm. Unraveling the intricacies of selenium's role and its interaction with epigenetic factors could unlock valuable insights in the battle against this complex disease. Result Selenium has shown remarkable inhibitory effects on cervical cancer cells in various ways. In in vitro studies, it effectively inhibits the proliferation, migration, and invasion of cervical cancer cells, while promoting apoptosis. Selenium also demonstrates significant inhibitory effects on human cervical cancer-derived organoids. Furthermore, in an in vivo study, the administration of selenium dioxide solution effectively suppresses the growth of cervical cancer tumors in mice. One of the mechanisms behind selenium's inhibitory effects is its ability to inhibit histone demethylases, specifically JMJD3 and UTX. This inhibition is observed both in vitro and in vivo. Notably, when JMJD3 and UTX are inhibited with GSK-J4, similar biological effects are observed in both in vitro and in vivo models, effectively inhibiting organoid models derived from cervical cancer patients. Inhibiting JMJD3 and UTX also induces G2/M phase arrest, promotes cellular apoptosis, and reverses epithelial-mesenchymal transition (EMT). ChIP-qPCR analysis confirms that JMJD3 and UTX inhibition increases the recruitment of a specific histone modification, H3K27me3, to the transcription start sites (TSS) of target genes in cervical cancer cells (HeLa and SiHa cells). Furthermore, the expressions of JMJD3 and UTX are found to be significantly higher in cervical cancer tissues compared to adjacent normal cervical tissues, suggesting their potential as therapeutic targets. Conclusions Our study highlights the significant inhibitory effects of selenium on the growth, migration, and invasion of cervical cancer cells, promoting apoptosis and displaying promising potential as a therapeutic agent. We identified the histone demethylases JMJD3 and UTX as specific targets of selenium, and their inhibition replicates the observed effects on cancer cell behavior. These findings suggest that JMJD3 and UTX could be valuable targets for selenium-based treatments of cervical cancer.

Establishing and maintaining appropriate gene repression is critical for the health and development of multicellular organisms. Histone H3 lysine 27 (H3K27) methylation is a chromatin modification associated with repressed facultative heterochromatin, but the mechanism of this repression remains unclear. We used a forward genetic approach to identify genes involved in transcriptional silencing of H3K27-methylated chromatin in the filamentous fungus Neurospora crassa . We found that the N. crassa homologs of ISWI (NCU03875) and ACF1 (NCU00164) are required for repression of a subset of H3K27-methylated genes and that they form an ACF chromatin-remodeling complex. This ACF complex interacts with chromatin throughout the genome, yet association with facultative heterochromatin is specifically promoted by the H3K27 methyltransferase, SET-7. H3K27-methylated genes that are upregulated when iswi or acf1 are deleted show a downstream shift of the +1 nucleosome, suggesting that proper nucleosome positioning is critical for repression of facultative heterochromatin. Our findings support a direct role of the ACF complex in Polycomb repression. All the cells in an organism contain the exact same DNA, yet each type of cell performs a different role. They achieve this by turning specific genes on or off. To do this, cells wind their genetic code into structures called nucleosomes, which work a bit like spools of thread. Chemical modifications on these nucleosomes can determine whether a cell will use the genes spooled around it or not. In many organisms, cells can turn genes off using a modification called H3K27 methylation. This mark attracts a protein complex called PRC1 that packs the genes away, making them inaccessible to the proteins that would activate them. But the filamentous fungus Neurospora crassa does not produce PRC1. This suggests that this organism must keep genes with the H3K27 mark switched off in a different way. One possibility is that H3K27 methylation somehow leads to changes in the position of nucleosomes on the genome, since having nucleosomes near the beginning of gene sequences can stop the cell from reading the code. One protein complex responsible for positioning nucleosomes is known as the ATP-utilizing chromatin assembly and remodeling factor (ACF) complex, but it remained unknown whether it interacted with H3K27 methylation marks. To investigate further, Wiles et al. generated strains of Neurospora crassa that did not synthesize ACF and discovered that many of their genes, including ones marked with H3K27, were turned on. This was probably because the nucleosomes had shifted out of position, allowing the proteins responsible for activating the genes to gain access to the start of the genes’ sequences. Turning genes on and off at the right time is crucial for development, cell survival, and is key in tissues and organs working properly. Understanding the role of ACF adds to what we know about this complex process, which is involved in many diseases, including cancer.

Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), possesses histone N-methyltransferase (HMT) activity and plays an essential role in cancer initiation and development. The aim of the present study was to investigate the potential of Wedelolactone (WL) to inhibit the methylation activity of EZH2. The mantle cell lymphoma (MCL) cell line, Mino, was treated with WL, while untreated cells were used as control. HMT activity and EZH2 amount were measured in nuclear extracts from WL-treated and control Mino cells. WL was found to target EZH2-mediated histone H3K27 methylation. Along with the inhibition of H3K27 methylation in vitro (IC50=0.3 μM), WL suppressed HMT activity in Mino cells with an IC50 value of 3.2 μM. We detected a reduced amount of EZH2 in Mino cells treated with WL, compared to untreated control cells. This is the first study to show that WL induces inhibition of H3K27 methylation via EZH2 modulation and decreases cell proliferation in MCL, in vitro. WL is proposed as a promising agent and a novel epigenetic approach in MCL investigation and treatment.

Inflammation is a multifaceted defense mechanism of the immune system against infection. Chronic inflammation is intricately linked to all stages of tumorigenesis and is therefore associated with an elevated risk of developing serious cancers. Epigenetic mechanisms have the capacity to trigger inflammation as well as facilitate tumor development and transformation within an inflammatory context. They achieve this by dynamically modulating the expression of both pro—inflammatory and anti—inflammatory cytokines, which in turn sustains chronic inflammation. The aberrant epigenetic landscape reconfigures the transcriptional programs of inflammatory and oncogenic genes. This reconfiguration is pivotal in dictating the biological functions of both tumor cells and immune cells. Aberrant histone H3 lysine 27 site (H3K27) methylation has been shown to be involved in biological behaviors such as inflammation development, tumor progression, and immune response. The establishment and maintenance of this repressive epigenetic mark is dependent on the involvement of the responsible histone modifying enzymes enhancer of zeste homologue 2 (EZH2), jumonji domain containing 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat gene X (UTX) as well as multiple cofactors. In addition, specific pharmacological agents have been shown to modulate H3K27 methylation levels, thereby modulating inflammation and carcinogenesis. This review comprehensively summarises the current characteristics and clinical significance of epigenetic regulation of H3K27 methylation in the context of inflammatory response and tumor progression.

Ankylosing spondylitis (AS) is a common chronic progressive inflammatory autoimmune disease. T helper 17 (Th17) cells are the major effector cells mediating AS inflammation. Histone 3 Lys 27 trimethylation (H3K27me3) is an inhibitory histone modification that silences gene transcription and plays an important role in Th17 differentiation. The objective of this study was to investigate the expression of H3K27me3 in patients with AS and to explore its epigenetic regulation mechanism of Th17 differentiation during AS inflammation. We collected serum samples from 45 patients with AS at various stages and 10 healthy controls to measure their Interleukin-17 (IL-17) levels using ELISA. A quantitative polymerase chain reaction was used to quantify the mRNA levels of RORc and the signaling molecules of the JAK2/STAT3 pathway, JMJD3, and EZH2. Additionally, Western blot analysis was performed to quantify the protein levels of H3K27me3, RORγt, JAK2, STAT3, JMJD3, and EZH2 in cell protein extracts. The results showed that H3K27me3 expression in peripheral blood mononuclear cells (PBMCs) was significantly lower in patients with active AS compared to both the normal control groups and those with stable AS. Moreover, a significant negative correlation was observed between H3K27me3 expression and the characteristic transcription factor of Th17 differentiation, RORγt. We also discovered that patients with active AS exhibited significantly higher levels of JMJD3, an inhibitor of H3K27 demethylase, compared to the normal control group and patients with stable AS, while the expression of H3K27 methyltransferase (EZH2) was significantly lower. These findings suggest that H3K27me3 may be a dynamic and important epigenetic modification in AS inflammation, and JMJD3/EZH2 regulates the methylation level of H3K27me3, which may be one of the key regulatory factors in the pathogenesis of AS. These findings contribute to our understanding of the role of epigenetics in AS and may have implications for the development of novel therapeutic strategies for AS.