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Iron deficiency is a prevalent nutritional deficit associated with organ damage and dysfunction. Recent research increasingly associates iron deficiency with bone metabolism dysfunction, although the precise underlying mechanisms remain unclear. Some studies have proposed that iron-dependent methylation-erasing enzyme activity regulates cell proliferation and differentiation under physiological or pathological conditions. However, it remains uncertain whether iron deficiency inhibits the activation of quiescent mesenchymal stem cells (MSCs) by affecting histone demethylase activity. In our study, we identified KDM4D as a key player in the activation of quiescent MSCs. Under conditions of iron deficiency, the H3K9me3 demethylase activity of KDM4D significantly decreased. This alteration resulted in increased heterochromatin with H3K9me3 near the PIK3R3 promoter, suppressing PIK3R3 expression and subsequently inhibiting the activation of quiescent MSCs via the PI3K-Akt-Foxo1 pathway. Iron-deficient mice displayed significantly impaired bone marrow MSCs activation and decreased bone mass compared to normal mice. Modulating the PI3K-Akt-Foxo1 pathway could reverse iron deficiency-induced bone loss.

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Regulation of gene expression through histone modifications underlies cell homeostasis and differentiation. Kdm4d and Kdm4dl exhibited a high degree of similarity and demethylated H3K9me3. However, the physiological functions of these proteins remain unclear. In this study, we generated Kdm4dl mutant mice and found that Kdm4dl was dispensable for mouse development. However, through the generation of Kdm4d mutant mice, we unexpectedly found that Kdm4d mutant male mice were subfertile because of impaired sperm motility. The absence of Kdm4d was associated with an altered distribution of H3K9me3 in round spermatids, suggesting that the Kdm4d-mediated adjustment of H3K9me3 levels is required to generate motile sperm. Further analysis revealed that the absence of Kdm4d did not affect the functionality of sperm nuclei in generating offspring. As KDM4D is specifically expressed in the human testes, our results suggest that KDM4D expression may be a risk factor for human infertility.

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Purpose: This study aimed to explore the effects of elevated KDM4D expression and potential therapeutic effects of Lycium barbarum polysaccharide (LBP) on pterygium. Methods: The expression levels of KDM4D in the primary pterygium (n = 29) and normal conjunctiva (n = 14) were detected by immunohistochemistry. The effects of KDM4D on pterygium fibroblasts were detected by the CCK-8 assay, liquid chromatography-mass spectrometry assay, flow cytometry, and scratch wound healing assay. The relative expression of KDM4D in pterygium fibroblasts stimulated by interleukin (IL)-1ß, IL-6, IL-8, and LBP was detected by quantitative real-time PCR and Western blot. The effects of LBP on pterygium fibroblasts were detected using flow cytometry and scratch wound healing assays. Results: The expression level of KDM4D in pterygium was higher than that in normal conjunctiva. KDM4D increased the cell viability of pterygium fibroblasts. The differentially expressed genes identified in the LM-MS assay enriched in "actin filament organization" and "apoptosis." KDM4D promoted migration and inhibited apoptosis of pterygium fibroblasts in vitro. Inflammatory cytokines, including IL-1ß, IL-6, and IL-8, enhanced the expression of KDM4D in pterygium fibroblasts. LBP inhibited the expression of KDM4D in pterygium fibroblasts and decreased their cell viability. Moreover, LBP attenuated the KDM4D effects on migration and apoptosis of pterygium fibroblasts. Conclusions: Elevated KDM4D expression is a risk factor for pterygium formation. LBP inhibits the expression of KDM4D in pterygium fibroblasts and may be a potential drug for delaying pterygium development.

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Objective:To explore the inhibitory mechanism of herb cake-partitioned moxibustion on tumor growth in colitis-associated colorectal cancer(CAC)based on histone lysine demethylase 4D(KDM4D). Methods:Inbred male Sprague-Dawley rats were randomly divided into a normal group,a CAC group,a herb cake-partitioned moxibustion group,and an inhibitor group.Except the normal group,rats in the other three groups were treated with azoxymethane(AOM)combined with dextran sulfate sodium(DSS)to make CAC rat models.Rats in the normal group and the CAC group did not receive interventions;rats in the herb cake-partitioned moxibustion group received moxibustion at Qihai(CV6)and bilateral Tianshu(ST25),2 cones for one point each time,once a day for 30 d with 1-day rest every week;rats in the inhibitor group received intraperitoneal injection of KDM4D inhibitor,5-chloro-8-hydroxyquinoline(5-c-8HQ),once a day for 30 d.After intervention,the general condition,colon length,tumor number and volume,and histopathological colon changes were observed.The expression of adenomatous polyposis coli(APC),axis inhibitor(Axin),cyclin D1,matrix metalloproteinase(MMP)-7 and MMP-9 mRNAs were detected by real-time quantitative polymerase chain reaction.The proliferating cell nuclear antigen(PCNA),cleaved caspase3,KDM4D,APC,and Axin proteins were detected by immunohistochemistry. Results:Compared with the normal group,the general condition was poor,the colon length was significantly shortened(P<0.01),the number and volume of colonic tumors were increased(P<0.01),the structure of glandular duct was obviously disordered with"back-to-back"and cowall phenomenon,and also high-grade adenocarcinoma formed;the protein expression levels of PCNA and KDM4D were significantly increased(P<0.01),while cleaved caspase3,APC,and Axin were significantly reduced(P<0.01);the mRNA expression levels of cyclin D1,MMP-7,and MMP-9 were significantly increased(P<0.01),while APC and Axin were significantly reduced(P<0.01)in the CAC group.Compared with the CAC group,the general condition was improved,the length of colon was significantly increased(P<0.01),the number and volume of the colonic tumors were reduced(P<0.05),and the colon tissues showed epithelial cell proliferation with enlarged and deep staining nuclei,dysplasia and inflammatory cell infiltration;the protein expression levels of PCNA and KDM4D were significantly reduced(P<0.01),while the cleaved caspase3,APC,and Axin were significantly increased(P<0.01);the mRNA expression levels of cyclin D1,MMP-7,and MMP-9 were reduced(P<0.05),while the APC and Axin were increased(P<0.05)in the colon tissues of rats in the herb cake-partitioned moxibustion group and the inhibitor group. Conclusion:Herb cake-partitioned moxibustion regulated abnormally expressed KDM4D in CAC rats,activated APC and Axin,the upstream molecules of Wnt/β-catenin pathway,inhibited abnormally activated downstream molecules of Wnt/β-catenin pathway.This may be a key mechanism of herb cake-partitioned moxibustion in inhibiting CAC tumor growth.

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How the function of the JMJD2D epigenetic regulator is regulated or whether it plays a role in prostate cancer has remained elusive. We found that JMJD2D was overexpressed in prostate tumors, stimulated prostate cancer cell growth and became methylated by SET7/9 on K427. Mutation of this lysine residue in JMJD2D reduced the ability of DU145 prostate cancer cells to grow, invade and form tumors and elicited extensive transcriptomic changes. This included downregulation of CBLC, a ubiquitin ligase gene with hitherto unknown functions in prostate cancer, and upregulation of PLAGL1, a transcription factor with reported tumor suppressive characteristics in the prostate. Bioinformatic analyses indicated that CBLC expression was elevated in prostate tumors. Further, downregulation of CBLC largely phenocopied the effects of the K427 mutation on DU145 cells. In sum, these data have unveiled a novel mode of regulation of JMJD2D through lysine methylation, illustrated how this can affect oncogenic properties by influencing expression of the CBLC gene, and established a pro-tumorigenic role for CBLC in the prostate. A corollary is that JMJD2D and CBLC inhibitors could have therapeutic benefits in the treatment of prostate and possibly other cancers.

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INTRODUCTION: Tanshinone I (Tan I) is one of the bioactive components of Salvia miltiorrhiza. Whether it inhibits gastric cancer through ferroptosis has not been reported. This study aimed to confirm the effect of Tan I on ferroptosis in gastric cancer cells. METHODS: AGS and HGC27 cells were treated with Tan I. First, oxidative stress-related parameters and the expression of ferroptosis-related proteins were examined. Combined with a ferroptosis inhibitor, Tan I was found to inhibit gastric cancer cells via the ferroptosis pathway. Finally, with bioinformatics analysis, the target protein of Tan I was identified. RESULTS: Tan I significantly inhibited the expression level of GPX4. This molecule also increased ROS, MDA, and Fe2+ contents and decreased GSH enzyme activity. Therefore, we hypothesized that Tan I may inhibit gastric cancer cells by inducing ferroptosis. Western blotting results showed that Tan I inhibited the expression levels of the ferroptosis resistance-related proteins GPX4, SLC7A11, and FTH1, while the pro-ferroptosis-related proteins TFR1 and ACSL4 were significantly upregulated. A ferroptosis inhibitor effectively reversed these regulatory effects of Tan I in gastric cancer. With these data combined with the bioinformatics analysis, KDM4D was identified as a key regulatory target of Tan I. Mechanistically, Tan I induced positive regulation of ferroptosis resistance-related indicators by inhibiting KDM4D to upregulate p53 protein expression. Overexpression of KDM4D significantly reversed the effect of Tan I-induced ferroptosis resistance in gastric cancer cells. CONCLUSIONS: Tan I induced ferroptosis inhibition in gastric cancer by regulating the KDM4D/p53 pathway.

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OBJECTIVE: Gastrointestinal stromal tumors (GISTs) can rapidly proliferate through angiogenesis. Previous studies indicated the potential influence of microRNA on the progression of tumor immature angiogenesis. This study aimed to explore the specific mechanism by which microRNA-409-5p (miR-409-5p) contributes to GIST. METHODS: To identify genes potentially involved in the development and progression of GIST, the differences of miR-409-5p between tumors and adjacent tissues were first analyzed. Following this analysis, target genes were predicted. To further investigate the function of miRNA in GIST cells, two GIST cell lines (GIST-T1 and GIST882) were transfected with lentiviruses that stably expressed miR-409-5p and scrambled miRNA (negative control). Later, the cells were subjected to Western blotting and ELSA to determine any differences in angiogenesis-related genes. RESULTS: In GISTs, there was a decrease in the expression levels of miR-409-5p compared to the adjacent tissues. It was observed that the upregulation of miR-409-5p in GIST cell lines effectively inhibited the proteins hypoxia-inducible transcription factor 1ß (HIF1ß) and vascular endothelial growth factor A (VEGF-A). Further investigations revealed that miR-409-5p acted as an inhibitor of angiogenesis by binding to the 3'-UTR of Lysine-specific demethylase 4D (KDM4D) mRNA. Moreover, the combination of miR-409-5p with imatinib enhanced its inhibitory effect on angiogenesis. CONCLUSION: This study demonstrated that the miRNA-409-5p/KDM4D/HIF1ß/VEGF-A signaling pathway could serve as a novel target for the development of therapeutic strategies for the treatment of imatinib-resistance in GIST patients.

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Oxidative damage to skin fibroblast cells is a causative factor in many skin diseases. Previous studies have reported that lysine-specific demethylase 4D (Kdm4d) is involved in DNA replication, but its role on antioxidant capacity remains unclear. In the present study, we used goat fibroblast cells (GFCs) as the research model and identified 504 up-regulated and 1013 down-regulated genes following the knockdown of Kdm4d, respectively. The down-regulated genes of this enzyme were found to be enriched in the cell cycle, DNA replication, mitotic processes, and the oxidative phosphorylation pathway, as previously revealed from gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and gene set enrichment analysis (GSEA), suggesting vital roles of the Kdm4d enzyme in the cell cycle and in antioxidant regulation. To this end, we found the cell proliferation rate was significantly decreased after the knockdown of Kdm4d. Moreover, both the mRNA and protein expression levels of superoxide dismutase 2 (SOD2), one of the major antioxidant enzymes, was decreased, while the reactive oxygen species (ROS) level was significantly increased in Kdm4d knocked-down cells. In addition, the expression of γH2A histone family member X (γH2AX) increased significantly, indicating the presence of DNA double-strand breaks after the knockdown of the Kdm4d enzyme. In conclusion, the knockdown of Kdm4d inhibited DNA replication and the cell cycle, repressed the expression of SOD2, and increased the generation of ROS, which led to the production of DNA damage in GFCs. Our data will be helpful for understanding the mechanism underlying antioxidant capacity regulation in fibroblast cells.

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Introduction: Transcriptional activation depends on the interplay of chromatin modifiers to establish a permissive epigenetic landscape. While histone 3 lysine 9 (H3K9) methylation has long been associated with gene repression, there is limited evidence to support a role for H3K9 demethylases in gene activation. Methods: We leveraged knockdown and overexpression of JMJD2d / Kdm4d in mouse embryonic fibroblasts, coupled with extensive epigenomic analysesm to decipher the role of histone 3 lysine 9 demethylases in the innate immune response. Results: Here we describe the H3K9 demethylase Kdm4d/JMJD2d as a positive regulator of type I interferon responses. In mouse embryonic fibroblasts (MEFs), depletion of JMJD2d attenuates the transcriptional response, conferring increased viral susceptibility, while overexpression of the demethylase results in more robust IFN activation. We find that the underlying mechanism of JMJD2d in type I interferon responses consists of an effect both on the transcription of enhancer RNAs (eRNAs) and on dynamic H3K9me2 at associated promoters. In support of these findings, we establish that JMJD2d is associated with enhancer regions throughout the genome prior to stimulation but is redistributed to inducible promoters in conjunction with transcriptional activation. Discussion: Taken together, our data reveal JMJD2d as a chromatin modifier that connects enhancer transcription with promoter demethylation to modulate transcriptional responses.

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The abnormal modification of histone is an important factor restricting development of porcine cloned embryos. Overexpression of histone H3K9me3 demethylase KDM4 family can effectively improve the developmental efficiency of cloned embryos. In order to explore the effects of overexpression of H3K9me3 demethylase on the development of porcine cloned embryos, KDM4A mRNA and KDM4D mRNA were injected respectively into porcine cloned embryos at the 1-cell stage and 2-cell stage to detect the blastocyst rate; 2-cell stage cloned embryos injected with KDM4A mRNA and embryo injection water (the control group) at the 1-cell stage were collected to detect the expression level of H3K9me3, and 4-cell stage cloned embryos were collected for single cell transcriptome sequencing, then the sequencing data was analyzed with KEGG and GO. The results showed that the blastocyst rate of porcine cloned embryos injected with KDM4A mRNA at 1-cell stage was significantly higher than that of the control group (25.32 ± 0.74% vs 14.78 ± 0.87%), while cloned embryos injected with KDM4D mRNA had a similar blastocyst rate with cloned embryos in control group (16.27 ± 0.77% vs 14.78 ± 0.87%). Porcine cloned embryos injected with KDM4A mRNA and KDM4D mRNA at 2-cell stage had a similar blastocyst rate with cloned embryos in control group (32.18 ± 1.67%, 30.04 ± 0.91% vs 31.22 ± 1.40%). The expression level of H3K9me3 in cloned embryos injected with KDM4A mRNA at 1-cell stage was lower than that in control group. There were 133 differentially expressed genes detected by transcriptome sequencing, including 52 up-regulated genes and 81 down-regulated genes. Pathways enriched by GO analyses were mainly related to protein localization. Pathways enriched by KEGG analyses were related to cellular senescence and acute myeloid leukemia. These results suggest that overexpression of histone H3K9me3 demethylase KDM4A can significantly improve the developmental efficiency of porcine cloned embryos.

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OBJECTIVES: Stem cells of the apical papilla (SCAPs) provide promising candidates for dental pulp regeneration. Despite great advances in the transcriptional controls of the SCAPs fate, little is known about the regulation of SCAP differentiation. MATERIALS AND METHODS: Short hairpin RNAs and full-length RNA were used to deplete or overexpress lysine demethylase 4D (KDM4D) gene expression. Western blotting, real-time RT-PCR, alizarin red staining, and scratch migration assays were used to study the role of KDM4D and the ribosomal protein encoded by RPS5 in SCAPs. RNA microarray, chromatin Immunoprecipitation (ChIP), and co-immunoprecipitation (Co-IP) assays were performed to explore the underlying molecular mechanisms. RESULTS: KDM4D enhanced the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. The microarray results revealed that 88 mRNAs were differentially expressed in KDM4D-overexpressed SCAPs. ChIP results showed knock-down of KDM4D increased the level of H3K9me2 and H3K9me3 in CNR1 promoter region. There were 37 possible binding partners of KDM4D. KDM4D was found to combine with RPS5, which also promoted the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. CONCLUSIONS: KDM4D promoted the osteo/dentinogenic differentiation and migration potential of SCAPs in combination with RPS5, which provides a therapeutic clue for improving SCAPs-based dental tissue regeneration.

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Posttranslational modifications (PTMs) of histones are well-established contributors in a variety of biological functions, especially tumorigenesis. Histone demethylase JMJD2D (also known as KDM4D), a member of the JMJD2 subfamily, promotes gene transcription by antagonizing H3K9 methylation. JMJD2D is an epigenetic factor coordinating androgen receptor activation, DNA damage repair, DNA replication, and cell cycle regulation. Recently, the oncogenic role of JMJD2D in colorectal cancer (CRC) and hepatocellular cancer (HCC) has been recognized. JMJD2D serves as a coactivator of ß-catenin, Gli1/2, HIF1α, STAT3, IRF1, TCF4, and NICD or an antagonist of p53 to promote the progression of CRC and HCC. In this review, we summarize the molecular mechanisms of JMJD2D in promoting the progression of CRC and HCC as well as the constructive role of its targeting inhibitors in suppressing tumorigenesis and synergistically enhancing the efficacy of anti-PD-1/PD-L1 immunotherapy.

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Macroautophagy/autophagy is a conserved eukaryotic process to mediate the degradation of cell organelles and protein aggregates, which participates in a variety of cellular responses, including immune signal transduction. KDM4D functions as an important histone demethylase to regulate gene transcription by inhibiting histone H3K9 trimethylation. Whether autophagy epigenetically regulates the immune response via modulating the stability and activity of KDM4D remains largely unclear. Recently, we identified TRIM14 (tripartite motif-containing 14) as an epigenetic regulator, which recruits USP14 and BRCC3 to form a regulatory complex, and promotes an inflammation response through inhibiting OPTN-mediated autophagic degradation of KDM4D.

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Autophagy is a fundamental cellular process of protein degradation and recycling that regulates immune signaling pathways via multiple mechanisms. However, it remains unclear how autophagy epigenetically regulates the immune response. Here, we identified TRIM14 as an epigenetic regulator that reduces histone H3K9 trimethylation by inhibiting the autophagic degradation of the histone demethylase KDM4D. TRIM14 recruited the deubiquitinases USP14 and BRCC3 to cleave the K63-linked ubiquitin chains of KDM4D, which prevented KDM4D from undergoing optineurin (OPTN)-mediated selective autophagy. Tripartite motif-containing 14 (TRIM14) deficiency in dendritic cells significantly impaired the expression of the KDM4D-directed proinflammatory cytokines interleukin 12 (Il12) and Il23 and protected mice from autoimmune inflammation. Taken together, these findings highlight the cross-talk between epigenetic regulation and autophagy and suggest TRIM14 is a potential target of therapeutic intervention for inflammation-related diseases.

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BACKGROUND: Increasing researches have been reported that epigenetic alterations play critical roles in ESCC development. However, the role of the histone demethylase KDM4D in ESCC tumorigenesis is poorly investigated. This study aims to discover the underlying mechanisms between KDM4D and ESCC progression. METHODS: CCK-8 assays, clone formation assay and soft-agar assays were performed to assess cell proliferation. Transwell assay was utilized to assess cell migration efficiency, while sphere formation assay was used to evaluate the cell self-renewal ability. Bioinformatic analysis was conducted to identify prognostic factors and predict the potential E3 ubiquitin ligases. In vitro ubiquitination assay was conducted to confirm the regulations between SYVN1 and HMGB1. The mRNA levels or protein levels of genes were detected by real-time PCR and western blot analysis. In vivo tumor xenograft models were used to determine whether the HMGB1 inhibition affected the malignant features of ESCC cells. RESULT: Epigenome screening and low-throughput validations highlighted that KDM4D is a tumor suppressor in ESCC. KDM4D expressed lowly in tumors that predicts poor prognosis. KDM4D deficiency significantly enhanced tumor growth, migration and stemness. Mechanistically, KDM4D transcriptionally activates SYVN1 expressions via H3K9me3 demethylation at the promoter region, thereby triggering the ubiquitin-dependent degradation of HMGB1. Low KDM4D depended on accumulated HMGB1 to drive ESCC progression and aggressiveness. Targeting HMGB1 (Glycyrrhizin) could remarkably suppress ESCC tumor growth in vitro and in vivo, especially in KDM4D-deficient cells. CONCLUSIONS: We systematically identified KDM4D/SYVN1/HMGB1 axis in ESCC progression, proving novel biomarkers and potential therapeutic targets.

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Histone lysine demethylase 4D (KDM4D) plays an important role in the regulation of tumorigenesis, progression and drug resistance and has been considered a potential target for cancer treatment. However, there is still a lack of potent and selective KDM4D inhibitors. In this investigation, we report a new class of KDM4D inhibitors containing the 2-(aryl(pyrrolidine-1-yl)methyl)phenol scaffold, identified through AlphaLisa-based screening, structural optimization, and structure-activity relationship analyses. Among these inhibitors, 24s was the most potent, with an IC50 value of 0.023 ± 0.004 µM. This compound exhibited more than 1500-fold selectivity towards KDM4D versus KDM4A as well as other JMJD subfamily members, indicating good selectivity for KDM4D. Kinetic analysis indicated that 24s did not occupy the 2-oxoglutarate binding pocket. In an in vitro assay, 24s significantly suppressed the proliferation and migration of colorectal cancer (CRC) cells. Overall, this study has identified a good tool compound to explore the biological function of KDM4D and a good lead compound for drug discovery targeting KDM4D.

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Acute myeloid leukemia (AML) is regarded as a fatal cancer in the world. The overall survival in adult patients with AML is still poor. As lysine demethylases, the KDM4 family is found highly expressed in many kinds of tumors. In this study, we demonstrate that KDM4D is overexpressed in AML and knockdown of KDM4D not only inhibits the proliferation of AML cells, but also induces cell cycle arrest and apoptosis. Furthermore, our research shows that KDM4D can regulate the expression of MCL-1 by demethylating H3K9me3 at the promoter region in AML cells. Besides, we find that high expression of KDM4D is correlated with poor overall survival in AML patients. Taken together, our study demonstrated that KDM4D can promote MCL-1 expression in AML and may serve as a novel target for the treatment of AML.

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Histone lysine demethylase 4D (KDM4D), also known as JMJD2D, plays an important role in cell proliferation and survival and has been associated with several tumor types. KDM4D has emerged as a potential target for the treatment of human cancer. Here, we reported crystal complex structures for two KDM4D inhibitors, OWS [2-(1H-pyrazol-3-yl)isonicotinic acid] and 10r (5-hydroxy-2-methylpyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-3-carbonitrile), which were both determined to 2.0 Å. OWS is a newly discovered KDM4D inhibitor (IC50 = 4.28 µM) and the critical pharmacophores of this compound are confirmed by the complex structure. Compound 10r is a KDM4D inhibitor reported by us previously. To clarify the binding mode in more detail, the crystal structure was determined and the comparison analysis revealed unique interactions that had never been observed before. Overall, our data provide new structural insights for rational design and offer an opportunity for optimization of KDM4D inhibitors.

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Somatic cell nuclear transfer (SCNT) holds vast potential in agriculture. However, its applications are still limited by its low efficiency. Histone 3 lysine 9 trimethylation (H3K9me3) was identified as an epigenetic barrier for this. Histone demethylase KDM4D could regulate the level of H3K9me3. However, its effects on buffalo SCNT embryos are still unclear. Thus, we performed this study to explore the effects and underlying mechanism of KDM4D on buffalo SCNT embryos. The results revealed that compared with the IVF embryos, the expression level of KDM4D in SCNT embryos was significantly lower at 8- and 16-cell stage, while the level of H3K9me3 in SCNT embryos was significantly higher at 2-cell, 8-cell, and blastocyst stage. Microinjection of KDM4D mRNA could promote the developmental ability of buffalo SCNT embryos. Furthermore, the expression level of ZGA-related genes such as ZSCAN5B, SNAI1, eIF-3a, and TRC at the 8-cell stage was significantly increased. Meanwhile, the pluripotency-related genes like POU5F1, SOX2, and NANOG were also significantly promoted at the blastocyst stage. The results were reversed after KDM4D was inhibited. Altogether, these results revealed that KDM4D could correct the H3K9me3 level, increase the expression level of ZGA and pluripotency-related genes, and finally, promote the developmental competence of buffalo SCNT embryos.

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Lysine demethylase 4D (KDM4D) encodes a histone demethylase, which can influence the androgen signaling as well as play an essential role in spermatogenesis. Recently a study has shown that the mRNA of KDM4D is the key to the successful clone of the Macaque monkeys named as "Zhongzhong" and "Huahua," which greatly increased pregnancy rates of female monkey, suggesting that the KDM4D gene may be strongly associated with reproduction. Therefore, the objective of this study was to explore possible single nucleotide polymorphisms (SNPs) within the coding region of KDM4D gene and analyze the associations with testis morphology traits of male pigs. Herein, two SNPs in exon1 of pig KDM4D gene were identified（NC_010451.4:g.1078A > G and NC_010451.4:g.1358G > C）. Among them, the g.1078A > G mutation was located at the JmjN domain which located from 15 to 55 amino acids of KDM4D. Association analyses showed that the g.1078A > G mutation was strongly associated with testis long circumference (TLC), testis short girth (TSG) and testis weight (TW) of Large White (LW) as well as Landrace (LD) (p < 0.05). The AA/AG genotype had a positive effect on testis morphology traits. Briefly, the novel missense mutation g.1078A > G could be a molecular marker for improving testis morphology traits in pig breeding.

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