Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B.

Testicular germ cell tumors (TGCTs) can be treated with cisplatin-based therapy. However, a clinically significant number of cisplatin-resistant patients die from progressive disease as no effective alternatives exist. Curative cisplatin therapy results in acute and life-long toxicities in the young TGCT patient population providing a rationale to decrease cisplatin exposure. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response to chemotherapy. Hence, targeting epigenetic pathways with "epidrugs" is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we demonstrate for the first time that targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 can treat both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier genes, including BRD4, lysine demethylases, chromodomain helicase DNA binding proteins, and lysine methyltransferases, were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Our findings demonstrate that targeting polycomb demethylases is a new potent pharmacologic strategy for treating cisplatin resistant TGCTs that warrants clinical development.

Ribosomal S6 kinase (RSK) plays a critical role in DNA damage response via the phosphorylation of histone lysine demethylase KDM4B.

BACKGROUND: Epigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage. METHODS: KDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay. RESULTS: RSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation. CONCLUSIONS: Overall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy.

Unraveling the Role of JMJD1B in Genome Stability and the Malignancy of Melanomas.

Genome instability relies on preserving the chromatin structure, with any histone imbalances threating DNA integrity. Histone synthesis occurs in the cytoplasm, followed by a maturation process before their nuclear translocation. This maturation involves protein folding and the establishment of post-translational modifications. Disruptions in this pathway hinder chromatin assembly and contribute to genome instability. JMJD1B, a histone demethylase, not only regulates gene expression but also ensures a proper supply of histones H3 and H4 for the chromatin assembly. Reduced JMJD1B levels lead to the cytoplasmic accumulation of histones, causing defects in the chromatin assembly and resulting in DNA damage. To investigate the role of JMJD1B in regulating genome stability and the malignancy of melanoma tumors, we used a JMJD1B/KDM3B knockout in B16F10 mouse melanoma cells to perform tumorigenic and genome instability assays. Additionally, we analyzed the transcriptomic data of human cutaneous melanoma tumors. Our results show the enhanced tumorigenic properties of JMJD1B knockout melanoma cells both in vitro and in vivo. The γH2AX staining, Micrococcal Nuclease sensitivity, and comet assays demonstrated increased DNA damage and genome instability. The JMJD1B expression in human melanoma tumors correlates with a lower mutational burden and fewer oncogenic driver mutations. Our findings highlight JMJD1B's role in maintaining genome integrity by ensuring a proper histone supply to the nucleus, expanding its function beyond gene expression regulation. JMJD1B emerges as a crucial player in preserving genome stability and the development of melanoma, with a potential role as a safeguard against oncogenic mutations.

Yam Carbon Dots Promote Bone Defect Repair by Modulating Histone Demethylase 4B.

Introduction: Chronic apical periodontitis is a typical inflammatory disease of the oral cavity, the pathology is characterized by an inflammatory reaction with bone defects in the periapical area. Chinese medicine is our traditional medicine, Carbon Dots (CDs) are a new type of nanomaterials. The purpose of this study was to prepare Yam Carbon Dots (YAM-CDs) to investigate the mechanism of action of YAM-CDs on bone differentiation in vivo and in vitro. Methods: We characterized YAM-CDs using transmission electron microscopy (TEM), Fourier Transform Infrared Spectrometer (FTIR), X-Ray Diffraction (XRD) and photoluminescence (PL). CCK-8 assay, Real-time qPCR, and Western Blot were conducted using bone marrow mesenchymal stem cells (BMSCs) to verify that YAM-CDs promote osteoblast differentiation. In addition, we investigated the role of YAM-CDs in promoting bone formation in an inflammatory setting in an in vivo mouse model of cranial defects. Results: The results of TEM and PL showed that the YAM-CDs mostly consisted of the components C1s, O1s, and N1s. Additionally the average sizes of YAM-CDs were 2-6 nm. The quantum yield was 4.44%, with good fluorescence stability and biosafety. Real-time qPCR and Western blot analysis showed that YAM-CDs promoted osteoblast differentiation under an inflammatory environment by regulating expression of histone demethylase 4B (KDM4B). In vivo, results showed that YAM-CDs effectively repaired cranial bone defects in a mouse model and reduced the expression of inflammatory factors under the action of lipopolysaccharides (LPS). Conclusion: YAM-CDs promoted the proliferation and differentiation of osteoblasts by regulating the expression of KDM4B to repair cranial bone defects in mice under an LPS-induced inflammatory milieu, which will provide a new idea for the treatment of clinical periapical inflammation and other bone defect diseases.

[Effect of JMJD3-IRF4 Signaling Pathway-Mediated Macrophage Polarization on the Malignant Biological Behavior of Multiple Myeloma Cells].

OBJECTIVE: To investigate the effect of macrophage polarization mediated by Jumonji domain containing-3 (JMJD3)-interferon regulatory factor 4 (IRF4) signaling pathway on the malignant biological behavior of multiple myeloma (MM) cells. METHODS: THP-1 monocytes were induced to differentiate into macrophages by phorbol myristate acetate (PMA). THP-1 macrophages were divided into control group (normal culture), M2 induction group ï¼»added recombinant human interleukin (IL) -4, IL-13 proteinsï¼½, M2+JMJD3 protein group (added recombinant human IL-4, IL-13 and JMJD3 proteins) and M2+JMJD3 inhibitor group (added recombinant human IL-4, IL-13 proteins and JMJD3 inhibitor), the proportion of CD206+ cells was detected by flow cytometry, the levels of IL-10 and transforming growth factor-ß (TGF-ß) in the culture supernatant were detected by ELISA assay, the expression levels of arginase-1 (Arg-1), JMJD3 and IRF4 mRNA were detected by real-time quantitative PCR (qRT-PCR), and the expression levels of Arg-1, JMJD3 and IRF4 proteins were detected by Western blot. Correspondingly, human MM cells U266 were cultured with THP-1 macrophage culture supernatant of each group, Methyl thiazolyl tetrazolium (MTT) method and plate colony formation assay were used to detect cell proliferation, cell apoptosis was detected by flow cytometry, Western blot was used to detect the expression levels of apoptosis-promoting protein Bcl-2-associated X protein (Bax) and cleaved caspase-3 in cells, and Transwell assay was used to detect cell migration and invasion. RESULTS: Compared with the control group, the proportion of CD206+ cells in THP-1 macrophages, the mRNA and protein expression levels of Arg-1, JMJD3 and IRF4, and the levels of IL-10 and TGF-ß in the cell culture supernatant in M2 induction group were significantly increased (P <0.001), meanwhile, the proliferation activity and the number of clones of U266 cells were significantly increased (P <0.01), the apoptosis rate and the expression levels of apoptosis-promoting protein Bax and cleaved caspase-3 were significantly decreased (P <0.001), the numbers of migrated cells and invasive cells were increased (P <0.001). Compared with M2 induction group, the proportion of CD206+ cells in THP-1 macrophages, the mRNA and protein expression levels of Arg-1, JMJD3 and IRF4, and the levels of IL-10 and TGF-ß in the cell culture supernatant in M2+JMJD3 protein group were further increased (P <0.01), meanwhile, the proliferation activity and the number of clones of U266 cells were further increased (P <0.05), the apoptosis rate and the expression levels of apoptosis-promoting protein Bax and cleaved caspase-3 were further decreased (P <0.01), the numbers of migrated cells and invasive cells were further increased (P <0.001); However, the change trends of the above indexes in M2+JMJD3 inhibitor group were opposite to those in M2+JMJD3 protein group. CONCLUSION: M2 polarization of macrophages mediated by JMJD3-IRF4 signaling pathway can promote the proliferation, migration and invasion of MM cells, and inhibit cell apoptosis.

Impact of Hypoxia and the Levels of Transcription Factor HIF-1α and JMJD1A on Epithelial-Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma Cell Lines.

BACKGROUND/AIM: This study aimed to assess the impact of hypoxia on epithelial-mesenchymal transition (EMT) in head and neck squamous cell carcinoma (HNSCC), focusing on the involvement of transcription factors hypoxia inducible factor 1 (HIF-1α) and Jumonji Domain-Containing Protein 1A (JMJD1A). MATERIALS AND METHODS: FaDu and Cal33 cell lines were subjected to hypoxic and normoxic conditions. Cell proliferation was quantified electronically, while PCR and western blot analyses were used to measure mRNA and protein levels of HIF-1α, JMJD1A, and EMT markers. EMT was further characterized through immunofluorescence, migration, and invasion assays. RESULTS: Hypoxic conditions significantly reduced cell proliferation after 48 hours in both cell lines. HIF-1α mRNA levels increased initially during short-term hypoxia but declined thereafter, while JMJD1A mRNA levels showed a sustained increase with prolonged hypoxia. Western blot analysis revealed contrasting trends in protein levels. EMT marker expression varied markedly over time at both the mRNA and protein levels, suggesting EMT induction in hypoxia within 24 hours. Immunofluorescence, migration, and invasion assays supported these findings. CONCLUSION: The study provides evidence of hypoxia-induced EMT in HNSCC, although conflicting results suggest a complex interplay among molecular regulators involved in this process.

Enriched H3K27Me3 on BMP4 suppresses the osteoblastic differentiation potential of BMSCs in diabetes mellitus.

Diabetes mellitus has been widely acknowledged to have a negative effect on the osteoblastic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). However, the underlying epigenetic mechanisms associated with this process remain to be elucidated. The goal of the present study was to investigate the effect of diabetes mellitus on the osteoblastic differentiation of BMSCs and assess the role of histone methylation in the observed phenomena. The osteoblastic differentiation ability of BMSCs was shown to be decreased in diabetes mellitus, as indicated by alkaline phosphatase activity and the mRNA levels of osteoblast-related genes. Furthermore, diabetes mellitus caused an increased expression of the histone methylase EZH2 and the levels of H3K27Me3 and decreased the expression of the histone demethylase KDM6B, as demonstrated by qRT-PCR and western blotting. Furthermore, immunofluorescence staining suggested that both EZH2 and H3K27Me3 were primarily localized in the nucleus. In addition, chromatin immunoprecipitation assays indicated an increased presence of H3K27Me3 on the promoter region of the BMP4 gene. In summary, in the present study, we demonstrated that the osteoblastic differentiation of BMSCs is dramatically reduced in diabetes mellitus. In addition, upregulation of EZH2 expression and downregulation of KDM6B expression may not be enough to eliminate transcriptional repression mediated by H3K27Me3 on the promoter region of the BMP4 gene during the osteoblastic differentiation of BMSCs in diabetes mellitus.

Inhibition of KDM4A restricts SQLE transcription and induces oxidative stress imbalance to suppress bladder cancer.

In clinical practice, the limited efficacy of standard comprehensive therapy for advanced bladder cancer and the lack of targeted treatment options are well recognized. Targeting abnormal epigenetic modifications in tumors has shown considerable potential in cancer therapy. Through drug screening in tumor organoids, we identified that ML324, a histone lysine demethylase 4A (KDM4A) inhibitor, exhibits potent antitumor effects in both in vitro and in vivo cancer models. Mechanistically, Kdm4a demethylates H3K9me3, leading to chromatin opening and increased accessibility of Gabpa to the squalene epoxidase (Sqle) gene promoter, resulting in transcriptional activation. Inhibition of Kdm4a downregulates Sqle transcription, blocking cholesterol synthesis and causing squalene (SQA) accumulation. This process induces reactive oxygen species (ROS) clearance and suppresses JNK/c-Jun phosphorylation, ultimately inducing apoptosis. Furthermore, ML324 treatment significantly inhibited tumor growth in bladder cancer patient-derived xenograft (PDX) models. Our findings reveal the presence of a Kdm4a-Sqle-ROS-JNK/c-Jun signaling axis that regulates oxidative stress balance, offering a novel strategy for targeted therapy in bladder cancer.

H3K9me3 demethylation by JMJD2B is regulated by pirfenidone resulting in improved NASH.

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.

An RNA damage response network mediates the lethality of 5-FU in colorectal cancer.

5-fluorouracil (5-FU), a major anti-cancer therapeutic, is believed to function primarily by inhibiting thymidylate synthase, depleting deoxythymidine triphosphate (dTTP), and causing DNA damage. Here, we show that clinical combinations of 5-FU with oxaliplatin or irinotecan show no synergy in human colorectal cancer (CRC) trials and sub-additive killing in CRC cell lines. Using selective 5-FU metabolites, phospho- and ubiquitin proteomics, and primary human CRC organoids, we demonstrate that 5-FU-mediated CRC cell killing primarily involves an RNA damage response during ribosome biogenesis, causing lysosomal degradation of damaged rRNAs and proteasomal degradation of ubiquitinated ribosomal proteins. Tumor types clinically responsive to 5-FU treatment show upregulated rRNA biogenesis while 5-FU clinically non-responsive tumor types do not, instead showing greater sensitivity to 5-FU's DNA damage effects. Finally, we show that treatments upregulating ribosome biogenesis, including KDM2A inhibition, promote RNA-dependent cell killing by 5-FU, demonstrating the potential for combinatorial targeting of this ribosomal RNA damage response for improved cancer therapy.

Design, synthesis and biological evaluation of 4,6-diarylquinoxaline-based KDM4D inhibitors.

Histone lysine demethylase 4D (KDM4D) is a critical player in the regulation of tumorigenesis, emerging as a potential target for developing anti-tumor agents. In this study, a series of KDM4D inhibitors containing the 4,6-diarylquinoxaline scaffold were prepared based on the previously discovered hit compound QD-1. Among these inhibitors, 33a was the most potent compound, with an IC50 value of 0.62 µM. In an in vitro assay, 33a showed a superior ability to inhibit the viability of liver cancer Huh-7 cells with IC50 = 5.23 µM. 33a exhibits significant effects in inhibiting cell cycle progression and proliferation of liver cancer cells, as well as suppressing cell migration. This work provided a promising scaffold for developing KDM4D inhibitors, as well as a lead compound for the development of anti-tumor drugs targeting KDM4D.

Overexpression of Kdm6b induces testicular differentiation in a temperature-dependent sex determination system.

In reptiles, such as the red-eared slider turtle ( Trachemys scripta elegans), gonadal sex determination is highly dependent on the environmental temperature during embryonic stages. This complex process, which leads to differentiation into either testes or ovaries, is governed by the finely tuned expression of upstream genes, notably the testis-promoting gene Dmrt1 and the ovary-promoting gene Foxl2. Recent studies have identified epigenetic regulation as a crucial factor in testis development, with the H3K27me3 demethylase KDM6B being essential for Dmrt1 expression in T. s. elegans. However, whether KDM6B alone can induce testicular differentiation remains unclear. In this study, we found that overexpression of Kdm6b in T. s. elegans embryos induced the male development pathway, accompanied by a rapid increase in the gonadal expression of Dmrt1 at 31°C, a temperature typically resulting in female development. Notably, this sex reversal could be entirely rescued by Dmrt1 knockdown. These findings demonstrate that Kdm6b is sufficient for commitment to the male pathway, underscoring its role as a critical epigenetic regulator in the sex determination of the red-eared slider turtle.

AUP1 transcriptionally activated by KDM5B reprograms lipid metabolism to promote the malignant progression of cervical cancer.

Cervical cancer is one of the reproductive malignancies threatening women's lives worldwide. In the present study, it was aimed to explore the role and mechanism of ancient ubiquitous protein 1 (AUP1) in cervical cancer. Through bioinformatics analysis, AUP1 expression in cervical cancer tissues and the correlation between AUP1 and the prognosis of patients were analyzed. AUP1 expression in several cervical cancer cell lines was detected. Following the co­transfection of short hairpin RNA specific to AUP1 with or without lysine demethylase 5B (KDM5B) overexpression plasmids in SiHa cells, the proliferation and apoptosis of SiHa cells were detected. Additionally, wound healing and Transwell assays were used to detect SiHa cell migration and invasion. Cellular lipid droplets level was detected using the Oil red O staining. Meantime, the levels of triglyceride, cholesterol, oxygen consumption rates and expression of lipid metabolism­related proteins were detected to assess the lipid metabolism in SiHa cells. Then, the luciferase reporter assay and ChIP assay were used to verify the binding between KDM5B and AUP1. Finally, the effects of AUP1 and KDM5B on the growth and lipid metabolism in SiHa tumor­bearing mice were measured. AUP1 was significantly upregulated in cervical cancer tissues and cells. AUP1 interference inhibited the malignant biological behaviors and lipid metabolism reprogramming of SiHa cells, which was blocked by KDM5B overexpression. Moreover, KDM5B could transcriptionally activate AUP1 and upregulate AUP1 expression. Furthermore, AUP1 knockdown transcriptionally regulated by KDM5B limited the tumor growth and suppressed the lipid metabolism reprogramming in vivo. Collectively, AUP1 could be transcriptionally activated by KDM5B to reprogram lipid metabolism, thereby promoting the progression of cervical cancer. These findings reveal possible therapeutic strategies in targeting metabolic pathways.

Multiple Comprehensive Analyses Identify Lysine Demethylase KDM as a Potential Therapeutic Target for Pancreatic Cancer.

Pancreatic cancer (PC) is a challenging and heterogeneous disease with a high mortality rate. Despite advancements in treatment, the prognosis for PC patients remains poor, with a high chance of disease recurrence. Biomarkers are crucial for diagnosing cancer, predicting patient prognosis and selecting treatments. However, the current lack of effective biomarkers for PC could contribute to the insufficiency of existing treatments. These findings underscore the urgent need to develop novel strategies to fight this disease. This study utilized multiple comprehensive bioinformatic analyses to identify potential therapeutic target genes in PC, focusing on histone lysine demethylases (KDMs). We found that high expression levels of KDM family genes, particularly KDM1A, KDM5A and KDM5B, were associated with improved overall survival in the cohort. Furthermore, the infiltration of various immune cells, including B cells, neutrophils, CD8+ T cells, dendritic cells, and macrophages, was positively correlated with KDM1A, KDM5A, and KDM5B expression. Moreover, MetaCore pathway analysis revealed interesting connections between KDM1A and the cell cycle and proliferation, between KDM5A and DNA damage and double-strand break repair through homologous recombination, and between KDM5B and WNT/ß-catenin signaling. These findings suggest that KDM1A, KDM5A and KDM5B may serve as promising biomarkers and therapeutic targets for PC, a disease of high importance due to its aggressive nature and urgent need for novel biomarkers to improve diagnosis and treatment.

CircMAPK1 induces cell pyroptosis in sepsis-induced lung injury by mediating KDM2B mRNA decay to epigenetically regulate WNK1.

BACKGROUND: Macrophage pyroptosis is a pivotal inflammatory mechanism in sepsis-induced lung injury, however, the underlying mechanisms remain inadequately elucidated. METHODS: Lipopolysaccharides (LPS)/adenosine triphosphate (ATP)-stimulated macrophages and cecal ligation and puncture (CLP)-induced mouse model for sepsis were established. The levels of key molecules were examined by qRT-PCR, Western blotting, immunohistochemistry (IHC) and ELISA assay. The subcellular localization of circMAPK1 was detected by RNA fluorescence in situ hybridization (FISH). Cell viability, LDH release and caspase-1 activity were monitored by CCK-8, LDH assays, and flow cytometry. The bindings between KDM2B/H3K36me2 and WNK1 promoter was detected by chromatin immunoprecipitation (ChIP) assay and luciferase assay, and associations among circMAPK1, UPF1 and KDM2B mRNA were assessed by RNA pull-down or RNA immunoprecipitation (RIP) assays. The pathological injury of lung tissues was evaluated by lung wet/dry weight ratio and hematoxylin and eosin (H&E) staining. RESULTS: CircMAPK1 was elevated in patients with septic lung injury. Knockdown of circMAPK1 protected against LPS/ATP-impaired cell viability and macrophage pyroptosis via WNK1/NLRP3 axis. Mechanistically, loss of circMAPK1 enhanced the association between KDM2B and WNK1 promoter to promote the demethylation of WNK1 and increase its expression. CircMAPK1 facilitated KDM2B mRNA decay by recruiting UPF1. Functional experiments showed that silencing of KDM2B or WNK1 counteracted circMAPK1 knockdown-suppressed macrophage pyroptosis. In addition, silencing of circMAPK1 alleviated CLP-induced lung injury in mice via KDM2B/WNK1/NLRP3 axis. CONCLUSION: CircMAPK1 exacerbates sepsis-induced lung injury by destabilizing KDM2B mRNA to suppress WNK1 expression, thus facilitating NLRP3-driven macrophage pyroptosis.

ß-Adrenergic Signal and Epigenomic Regulatory Process for Adaptive Thermogenesis.

Activation of ß-adrenergic (ß-AR) signaling induces fight-or-flight stress responses which include enhancement of cardiopulmonary function, metabolic regulation, and muscle contraction. Classical dogma for ß-AR signaling has dictated that the receptor-mediated response results in an acute and transient signal. However, more recent studies support more wide-ranging roles for ß-AR signaling with long-term effects including cell differentiation that requires precisely timed and coordinated integration of many signaling pathways that culminate in precise epigenomic chromatin modifications. In this chapter, we discuss cold stress/ß-AR signaling-induced epigenomic changes in adipose tissues that influence adaptive thermogenesis. We highlight recent studies showing dual roles for the histone demethylase JMJD1A as a mediator of both acute and chronic thermogenic responses to cold stress, in two distinct thermogenic tissues, and through two distinct molecular mechanisms. ß-AR signaling not only functions through transient signals during acute stress responses but also relays a more sustained signal to long-term adaptation to environmental changes.

Calcium modulates the tethering of BCOR-PRC1.1 enzymatic core to KDM2B via liquid-liquid phase separation.

Recruitment of non-canonical BCOR-PRC1.1 to non-methylated CpG islands via KDM2B plays a fundamental role in transcription control during developmental processes and cancer progression. However, the mechanism is still largely unknown on how this recruitment is regulated. Here, we unveiled the importance of the Poly-D/E regions within the linker of BCOR for its binding to KDM2B. Interestingly, we also demonstrated that these negatively charged Poly-D/E regions on BCOR play autoinhibitory roles in liquid-liquid phase separation (LLPS) of BCORANK-linker-PUFD/PCGF1RAWUL. Through neutralizing negative charges of these Poly-D/E regions, Ca2+ not only weakens the interaction between BCOR/PCGF1 and KDM2B, but also promotes co-condensation of the enzymatic core of BCOR-PRC1.1 with KDM2B into liquid-like droplet. Accordingly, we propose that Ca2+ could modulate the compartmentation and recruitment of the enzymatic core of BCOR-PRC1.1 on KDM2B target loci. Thus, our finding advances the mechanistic understanding on how the tethering of BCOR-PRC1.1 enzymatic core to KDM2B is regulated.

Histone H3K9 Methylation Features under Hypoxic Conditions after the HIF1A Knockdown in Mesenchymal Stromal Cells In Vitro.

The effects of HIF1A knockdown by RNA interference on the histone H3K9 methylation in human umbilical cord mesenchymal stromal cells in vitro under conditions of 24-h exposure to hypoxia (1% O2) were studied. Evaluation of transcriptional activity of genes involved in the regulation of H3K9 methylation (KDM3A, KDM4A, and EHMT2) and the cytofluorimetric analysis of the expression of the corresponding antigens and H3K9 methylation level demonstrated a pronounced stimulating effect of hypoxic exposure. Moreover, the expression of KDM4A and EHMT2 was regulated by HIF1A-mediated mechanism, unlike KDM3A; the level of the corresponding proteins depended on HIF1A. In addition, the HIF-1-dependent regulation of KDM3A, KDM4A, and EHMT2/G9a, and directly the H3K9 methylation level in mesenchymal stromal cells also took place under normoxia conditions.

Understanding the Role of the SMN Complex Component GEMIN5 and Its Functional Relationship with Demethylase KDM6B in the Flunarizine-Mediated Neuroprotection of Motor Neuron Disease Spinal Muscular Atrophy.

Dysregulated RNA metabolism caused by SMN deficiency leads to motor neuron disease spinal muscular atrophy (SMA). Current therapies improve patient outcomes but achieve no definite cure, prompting renewed efforts to better understand disease mechanisms. The calcium channel blocker flunarizine improves motor function in Smn-deficient mice and can help uncover neuroprotective pathways. Murine motor neuron-like NSC34 cells were used to study the molecular cell-autonomous mechanism. Following RNA and protein extraction, RT-qPCR and immunodetection experiments were performed. The relationship between flunarizine mRNA targets and RNA-binding protein GEMIN5 was explored by RNA-immunoprecipitation. Flunarizine increases demethylase Kdm6b transcripts across cell cultures and mouse models. It causes, in NSC34 cells, a temporal expression of GEMIN5 and KDM6B. GEMIN5 binds to flunarizine-modulated mRNAs, including Kdm6b transcripts. Gemin5 depletion reduces Kdm6b mRNA and protein levels and hampers responses to flunarizine, including neurite extension in NSC34 cells. Moreover, flunarizine increases the axonal extension of motor neurons derived from SMA patient-induced pluripotent stem cells. Finally, immunofluorescence studies of spinal cord motor neurons in Smn-deficient mice reveal that flunarizine modulates the expression of KDM6B and its target, the motor neuron-specific transcription factor HB9, driving motor neuron maturation. Our study reveals GEMIN5 regulates Kdm6b expression with implications for motor neuron diseases and therapy.

α-Ketoglutarate promotes cardiomyocyte proliferation and heart regeneration after myocardial infarction.

The neonatal mammalian heart can regenerate following injury through cardiomyocyte proliferation but loses this potential by postnatal day 7. Stimulating adult cardiomyocytes to reenter the cell cycle remains unclear. Here we show that cardiomyocyte proliferation depends on its metabolic state. Given the connection between the tricarboxylic acid cycle and cell proliferation, we analyzed these metabolites in mouse hearts from postnatal day 0.5 to day 7 and found that α-ketoglutarate ranked highest among the decreased metabolites. Injection of α-ketoglutarate extended the window of cardiomyocyte proliferation during heart development and promoted heart regeneration after myocardial infarction by inducing adult cardiomyocyte proliferation. This was confirmed in Ogdh-siRNA-treated mice with increased α-ketoglutarate levels. Mechanistically, α-ketoglutarate decreases H3K27me3 deposition at the promoters of cell cycle genes in cardiomyocytes. Thus, α-ketoglutarate promotes cardiomyocyte proliferation through JMJD3-dependent demethylation, offering a potential approach for treating myocardial infarction.