Spermatogenesis is a fundamental process that requires a tightly controlled epigenetic event in spermatogonial stem cells (SSCs). The mechanisms underlying the transition from SSCs to sperm are largely unknown. Most studies utilize gene knockout mice to explain the mechanisms. However, the production of genetically engineered mice is costly and time-consuming. In this study, we presented a convenient research strategy using an RNA interference (RNAi) and testicular transplantation approach. Histone H3 lysine 9 (H3K9) methylation was dynamically regulated during spermatogenesis. As Jumonji domain-containing protein 1A (JMJD1A) and Jumonji domain-containing protein 2C (JMJD2C) demethylases catalyze histone H3 lysine 9 dimethylation (H3K9me2), we firstly analyzed the expression profile of the two demethylases and then investigated their function. Using the convenient research strategy, we showed that normal spermatogenesis is disrupted due to the downregulated expression of both demethylases. These results suggest that this strategy might be a simple and alternative approach for analyzing spermatogenesis relative to the gene knockout mice strategy.
Spermatogenesis/physiology* ; Animals ; Male ; Mice ; Epigenesis, Genetic ; Jumonji Domain-Containing Histone Demethylases/metabolism* ; Histones/metabolism* ; RNA Interference ; Testis/metabolism* ; Methylation ; Mice, Knockout ; Histone Demethylases

Given that ovarian stimulation is vital for assisted reproductive technology (ART) and results in elevated serum estrogen levels, exploring the impact of elevated estrogen exposure on oocytes and embryos is necessary. We investigated the effects of various ovarian stimulation treatments on oocyte and embryo morphology and gene expression using a mouse model and estrogen-treated mouse embryonic stem cells (mESCs). Female C57BL/6J mice were subjected to two types of conventional ovarian stimulation and ovarian hyperstimulation; mice treated with only normal saline served as controls. Hyperstimulation resulted in high serum estrogen levels, enlarged ovaries, an increased number of aberrant oocytes, and decreased embryo formation. The messenger RNA (mRNA)‍-sequencing of oocytes revealed the dysregulated expression of lysine-specific demethylase 6b (Kdm6b), which may be a key factor indicating hyperstimulation-induced aberrant oocytes and embryos. In vitro, Kdm6b expression was downregulated in mESCs treated with high-dose estrogen; treatment with an estrogen receptor antagonist could reverse this downregulated expression level. Furthermore, treatment with high-dose estrogen resulted in the upregulated expression of histone H3 lysine 27 trimethylation (H3K27me3) and phosphorylated H2A histone family member X (γ-H2AX). Notably, knockdown of Kdm6b and high estrogen levels hindered the formation of embryoid bodies, with a concomitant increase in the expression of H3K27me3 and γ-H2AX. Collectively, our findings revealed that hyperstimulation-induced high-dose estrogen could impair the demethylation of H3K27me3 by reducing Kdm6b expression. Accordingly, Kdm6b could be a promising marker for clinically predicting ART outcomes in patients with ovarian hyperstimulation syndrome.
Female ; Mice ; Demethylation/drug effects* ; Embryonic Stem Cells ; Estrogens/administration & dosage* ; Gene Expression/drug effects* ; Histones/metabolism* ; Jumonji Domain-Containing Histone Demethylases/metabolism* ; Mice, Inbred C57BL ; Oocytes ; Ovary/drug effects* ; Reproductive Techniques, Assisted ; Animals

Tissue interactions play a crucial role in tooth development. Notably, extracellular vesicle-mediated interactions between the mandible and tooth germ are considered essential. Here, we revealed that mandible extracellular vesicles could modulate the proliferation and differentiation of dental mesenchymal cells by regulating the histone demethylase KDM2B. Further investigation showed that mandible derived extracellular vesicles could deliver miR-206 to KDM2B, thereby regulating tooth development. An animal study demonstrated that the miR-206/KDM2B pathway affected tooth morphogenesis and mineralization after eight weeks of subcutaneous transplantation in nude mice. In conclusion, this study suggested that the mandible played a critical role in tooth morphogenesis and mineralization, which could be a potential therapeutic target for abnormal tooth development and an alternative model for tooth regeneration.
Animals ; Extracellular Vesicles/metabolism* ; Jumonji Domain-Containing Histone Demethylases/metabolism* ; Swine ; MicroRNAs/metabolism* ; Mandible ; Mice, Nude ; Odontogenesis/physiology* ; Swine, Miniature ; Mice ; Cell Differentiation ; Cell Proliferation

Glioblastoma is the most common primary cranial malignancy, and chemotherapy remains an important tool for its treatment. Sanggenon C(San C), a class of natural flavonoids extracted from Morus plants, is a potential antitumor herbal monomer. In this study, the effect of San C on the growth and proliferation of glioblastoma cells was examined by methyl thiazolyl tetrazolium(MTT) assay and 5-bromodeoxyuridinc(BrdU) labeling assay. The effect of San C on the tumor cell cycle was examined by flow cytometry, and the effect of San C on clone formation and self-renewal ability of tumor cells was examined by soft agar assay. Western blot and bioinformatics analysis were used to investigate the mechanism of the antitumor activity of San C. In the presence of San C, the MTT assay showed that San C significantly inhibited the growth and proliferation of tumor cells in a dose and time-dependent manner. BrdU labeling assay showed that San C significantly attenuated the DNA replication activity in the nucleus of tumor cells. Flow cytometry confirmed that San C blocked the cell cycle of tumor cells in G_0/G_1 phase. The soft agar clone formation assay revealed that San C significantly attenuated the clone formation and self-renewal ability of tumor cells. The gene set enrichment analysis(GSEA) implied that San C inhibited the tumor cell division cycle by affecting the myelocytomatosis viral oncogene(MYC) signaling pathway. Western blot assay revealed that San C inhibited the expression of cyclin through the regulation of the MYC signaling pathway by lysine demethylase 4B(KDM4B), which ultimately inhibited the growth and proliferation of glioblastoma cells and self-renewal. In conclusion, San C exhibits the potential antitumor activity by targeting the KDM4B-MYC axis to inhibit glioblastoma cell growth, proliferation, and self-renewal.
Humans ; Glioblastoma/genetics* ; Bromodeoxyuridine/therapeutic use* ; Signal Transduction ; Proto-Oncogene Proteins c-myc/metabolism* ; Agar ; Cell Proliferation ; Cell Line, Tumor ; Apoptosis ; Jumonji Domain-Containing Histone Demethylases/metabolism*

Osteoarthritis (OA) is a prevalent joint disease with no effective treatment strategies. Aberrant mechanical stimuli was demonstrated to be an essential factor for OA pathogenesis. Although multiple studies have detected potential regulatory mechanisms underlying OA and have concentrated on developing novel treatment strategies, the epigenetic control of OA remains unclear. Histone demethylase JMJD3 has been reported to mediate multiple physiological and pathological processes, including cell differentiation, proliferation, autophagy, and apoptosis. However, the regulation of JMJD3 in aberrant force-related OA and its mediatory effect on disease progression are still unknown. In this work, we confirmed the upregulation of JMJD3 in aberrant force-induced cartilage injury in vitro and in vivo. Functionally, inhibition of JMJD3 by its inhibitor, GSK-J4, or downregulation of JMJD3 by adenovirus infection of sh-JMJD3 could alleviate the aberrant force-induced chondrocyte injury. Mechanistic investigation illustrated that aberrant force induces JMJD3 expression and then demethylates H3K27me3 at the NR4A1 promoter to promote its expression. Further experiments indicated that NR4A1 can regulate chondrocyte apoptosis, cartilage degeneration, extracellular matrix degradation, and inflammatory responses. In vivo, anterior cruciate ligament transection (ACLT) was performed to construct an OA model, and the therapeutic effect of GSK-J4 was validated. More importantly, we adopted a peptide-siRNA nanoplatform to deliver si-JMJD3 into articular cartilage, and the severity of joint degeneration was remarkably mitigated. Taken together, our findings demonstrated that JMJD3 is flow-responsive and epigenetically regulates OA progression. Our work provides evidences for JMJD3 inhibition as an innovative epigenetic therapy approach for joint diseases by utilizing p5RHH-siRNA nanocomplexes.
Cartilage, Articular/pathology* ; Chondrocytes/metabolism* ; Down-Regulation ; Epigenesis, Genetic ; Humans ; Jumonji Domain-Containing Histone Demethylases/metabolism* ; Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism* ; Osteoarthritis/pathology* ; RNA, Small Interfering/pharmacology*

Histone methylation is one of the most widely studied post-transcriptional modifications. It is thought to be an important epigenetic event that is closely associated with cell fate determination and differentiation. To explore the spatiotemporal expression of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) epigenetic marks and methylation or demethylation transferases in tooth organ development, we measured the expression of SET7, EZH2, KDM5B and JMJD3 via immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis in the first molar of BALB/c mice embryos at E13.5, E15.5, E17.5, P0 and P3, respectively. We also measured the expression of H3K4me3 and H3K27me3 with immunofluorescence staining. During murine tooth germ development, methylation or demethylation transferases were expressed in a spatial-temporal manner. The bivalent modification characterized by H3K4me3 and H3K27me3 can be found during the tooth germ development, as shown by immunofluorescence. The expression of SET7, EZH2 as methylation transferases and KDM5B and JMJD3 as demethylation transferases indicated accordingly with the expression of H3K4me3 and H3K27me3 respectively to some extent. The bivalent histone may play a critical role in tooth organ development via the regulation of cell differentiation.
Animals ; Cell Differentiation ; physiology ; DNA-Binding Proteins ; analysis ; Dental Papilla ; embryology ; Embryo, Mammalian ; Enamel Organ ; embryology ; Enhancer of Zeste Homolog 2 Protein ; Epigenesis, Genetic ; physiology ; Gene Expression Regulation, Developmental ; Histone-Lysine N-Methyltransferase ; analysis ; Histones ; metabolism ; Jumonji Domain-Containing Histone Demethylases ; analysis ; Lysine ; metabolism ; Methylation ; Mice ; Mice, Inbred BALB C ; Odontogenesis ; physiology ; Polycomb Repressive Complex 2 ; analysis ; Protein Processing, Post-Translational ; physiology ; Tooth Germ ; embryology

KDM5B is a histone H3K4me2/3 demethylase. The PHD1 domain of KDM5B is critical for demethylation, but the mechanism underlying the action of this domain is unclear. In this paper, we observed that PHD1KDM5B interacts with unmethylated H3K4me0. Our NMR structure of PHD1KDM5B in complex with H3K4me0 revealed that the binding mode is slightly different from that of other reported PHD fingers. The disruption of this interaction by double mutations on the residues in the interface (L325A/D328A) decreases the H3K4me2/3 demethylation activity of KDM5B in cells by approximately 50% and increases the transcriptional repression of tumor suppressor genes by approximately twofold. These findings imply that PHD1KDM5B may help maintain KDM5B at target genes to mediate the demethylation activities of KDM5B.
Binding Sites ; genetics ; Crystallography, X-Ray ; Gene Expression Regulation ; HEK293 Cells ; Histones ; chemistry ; metabolism ; Humans ; Jumonji Domain-Containing Histone Demethylases ; chemistry ; genetics ; metabolism ; Lysine ; chemistry ; metabolism ; Magnetic Resonance Spectroscopy ; Methylation ; Microscopy, Fluorescence ; Models, Molecular ; Mutation ; Nuclear Proteins ; chemistry ; genetics ; metabolism ; Peptides ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; Repressor Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo evaluate the safety of intracytoplasmic sperm injection (ICSI) in the mouse model.

METHODSWe simulated clinical ICSI technology and comprehensively evaluated it by parthenogenetic activation, immunofluorescence, embryo transplantation, examination of early implantation, and measurement of the crown-rump length (CRL).

RESULTSICSI significantly reduced the ability of preimplantation embryo development of the mouse, especially after the 8-cell stage (P < 0.01). The fluorescence of H3K9 dimethylation was abnormal at the male pronuclei of the embryos derived from ICSI. Further examination of the development of the transferred ICSI embryos indicated no significant difference in the rate of early implantation at E5. 5 days as compared with normal fertilization (P = 0.6), but the percentage of "normal embryos" was decreased significantly at E9.5 days (P < 0.01). Obvious growth retardation phenotype was observed even in the normal ICSI embryos at E9.5 days.

CONCLUSIONICSI might result in growth retardation of embryos by affecting H3K9 dimethylation in the male pronuclei.

Animals ; Embryonic Development ; Female ; Histones ; metabolism ; Jumonji Domain-Containing Histone Demethylases ; metabolism ; Male ; Methylation ; Mice ; Mice, Inbred ICR ; Pregnancy ; Sperm Injections, Intracytoplasmic ; adverse effects

OBJECTIVETo study the effect of small interfering RNA(siRNA) targeting JARID1B gene on the proliferation and apoptosis in HL-60 acute promyelocytic leukemia cell line, and to explore its mechanisms.

METHODSThe JARID1B siRNA was transfected into HL-60 cells using Lipofectamine(TM) 2000(Lipo) vector. The proliferation inhibition by siRNA targeting JARID1B was detected by MTT, cells apoptosis by flow cytometry, the mRNA expression of JARID1B by RT-PCR, the protein expression of JARID1B, Bcl-2, procaspase-9, procaspase-3, c-myc and P27 and histone methylated H3K4 by Western blot.

RESULTSsiRNA targeting JARID1B upregulated histone methylated H3K4 level, inhibited the proliferation of HL-60 cells, and induced the cells apoptosis. After transfection of siRNA targeting JARID1B at 0, 30, 60, 120 nmmol/L for 24 hours, the apoptotic rate were (11.0 ± 3.6)%, (35.2 ± 5.1)%, (52.7 ± 3.8)%, and (62.0 ± 5.7)% respectively (F = 70.27, P < 0.01). The protein expression of P27 was upregulated, and Bcl-2, procaspase-9, procaspase-3, c-myc was down regulated.

CONCLUSIONSJARID1B siRNA upregulates histone methylated H3K4. It reduces HL-60 cells proliferation and apoptosis by up regulating the p27 expression and down regulating the Bcl-2, procaspase-9, procaspase-3, c-myc expression. It might be a new therapeutic targeting for human leukemia.

Apoptosis ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Gene Expression Regulation, Leukemic ; Gene Targeting ; HL-60 Cells ; Histones ; metabolism ; Humans ; Jumonji Domain-Containing Histone Demethylases ; genetics ; Leukemia ; genetics ; Methylation ; Nuclear Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Repressor Proteins ; genetics

For investigating the effect of Jumonji domain-containing protein-3 (JMJD3) on the behavior of lung cancer cell line, A549 proliferation was measured with EDU staining and flow cytometer after JMJD3 expression plasmid and pcDNA3. 1 transfection at 48h. The migration ability of A549 was tested at the same time. The expression of p21 mRNA was measured with RT-PCR. The results showed that JMJD3 transfection increased the EDU positive cells ratio (JMJD3: 40.75% +/- 2.07%, control: 20.97% +/- 1.5%, P < 0.001). G1 phase cell ration also increased after JMJD3 transfection (JMJD3:47. 80% +/- 1.85%, control: 54.60% +/- 0.95%, P = 0.005). The mRNA expression of p21 decreased in JMJD3 group (JMJD3: 35. 89% +/- 3.71%, control: 91.78% +/- 3.74%, P < 0.001). The distances of migration were (0.47 +/- 0.27) cm and (0.96 +/- 0.40) cm after 24h and 48h with JMJD3 tranfection, compared to (0.57 +/- 0.22)cm and (1.08 +/- 0.33)cm in control, respectively (P > 0.05). JMJD3 promoted the proliferation of A549 and decreased the G1 cell numbers, decreased the p21 mRNA, but had no effect on A549 migration.
Adenocarcinoma ; pathology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Humans ; Jumonji Domain-Containing Histone Demethylases ; pharmacology ; Lung Neoplasms ; pathology ; RNA, Messenger ; genetics ; metabolism ; Transfection