• Journal of Ginseng Research
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• v.44 no.1
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• pp.58-66
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• 2020

Background: The biological and pharmacological effects of BST204, a fermented ginseng extract, have been reported in various disease conditions. However, its molecular action in metabolic disease remains poorly understood. In this study, we identified the antiadipogenic activity of BST204 resulting from its inhibition of the S6 kinase 1 (S6K1) signaling pathway. Methods: The inhibitory effects of BST204 on S6K1 signaling were investigated by immunoblot, nuclear fractionation, immunoprecipitation analyses. The antiadipogenic effect of BST204 was evaluated by measuring mRNA levels of adipogenic genes and by chromatin immunoprecipitation and quantitative real-time polymerase chain reaction analysis. Results: Treatment with BST204 inhibited activation and nuclear translocation of S6K1, further decreasing the interaction between S6K1 and histone H2B in 10T1/2 mesenchymal stem cells. Subsequently, phosphorylation of H2B at serine 36 (H2BS36p) by S6K1 was reduced by BST204, inducing an increase in the mRNA expression of Wnt6, Wnt10a, and Wnt10b, which disturbed adipogenic differentiation and promoted myogenic and early osteogenic gene expression. Consistently, BST204 treatment during adipogenic commitment suppressed the expression of adipogenic marker genes and lipid drop formation. Conclusion: Our results indicate that BST204 blocks adipogenesis of mesenchymal stem cells through the inhibition of S6K1-mediated histone phosphorylation. This study suggests the potential therapeutic strategy using BST204 to combat obesity and musculoskeletal diseases.

• Journal of Life Science
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• v.31 no.11
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• pp.995-1003
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• 2021

Chromatin remodeling regulates gene expression through epigenetic mechanisms. Aberrations in histone modification have been associated with depression-like behaviors in animal models. Additionally, growing evidence also indicates that epigenetic modification is associated with depression. p11 (S100A10) has been implicated in the pathophysiology of depression both in human and rodent models. In the present study, we investigated alterations in histone acetylation and methylation at the promoter of the p11 gene in the hippocampus of mice subjected to chronic unpredictable stress (CUS). C57BL/6 mice were exposed to CUS daily for 3 weeks. Depression-like behaviors were measured with the forced swimming test (FST). The levels of hippocampal p11 expression were analyzed by quantitative real-time polymerase chain reaction (PCR) and Western blotting. The levels of acetylated and methylated histone H3 at the promoter of p11 were measured by chromatin immunoprecipitation followed by real-time PCR. CUS-exposed mice displayed depression-like behaviors with prolonged immobility in FST. CUS led to significant decreases in the expression of p11 at both protein and mRNA levels. Meanwhile, there was a decrease in histone H3 acetylation (Ac-H3) and H3-K4 trimethylation (H3K4met3) and an increase in H3-K27 trimethylation (H3K27met3) at the p11 promoter. These results indicate that chronic stress causes the epigenetic suppression of p11 expression in the hippocampus.

• Animal Bioscience
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• v.37 no.6
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• pp.1021-1030
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• 2024

Objective: R-loops are DNA:RNA triplex hybrids, and their metabolism is tightly regulated by transcriptional regulation, DNA damage response, and chromatin structure dynamics. R-loop homeostasis is dynamically regulated and closely associated with gene transcription in mouse zygotes. However, the factors responsible for regulating these dynamic changes in the R-loops of fertilized mouse eggs have not yet been investigated. This study examined the functions of candidate factors that interact with R-loops during zygotic gene activation. Methods: In this study, we used publicly available next-generation sequencing datasets, including low-input ribosome profiling analysis and polymerase II chromatin immunoprecipitation-sequencing (ChIP-seq), to identify potential regulators of R-loop dynamics in zygotes. These datasets were downloaded, reanalyzed, and compared with mass spectrometry data to identify candidate factors involved in regulating R-loop dynamics. To validate the functions of these candidate factors, we treated mouse zygotes with chemical inhibitors using in vitro fertilization. Immunofluorescence with an anti-R-loop antibody was then performed to quantify changes in R-loop metabolism. Results: We identified DEAD-box-5 (DDX5) and histone deacetylase-2 (HDAC2) as candidates that potentially regulate R-loop metabolism in oocytes, zygotes and two-cell embryos based on change of their gene translation. Our analysis revealed that the DDX5 inhibition of activity led to decreased R-loop accumulation in pronuclei, indicating its involvement in regulating R-loop dynamics. However, the inhibition of histone deacetylase-2 activity did not significantly affect R-loop levels in pronuclei. Conclusion: These findings suggest that dynamic changes in R-loops during mouse zygote development are likely regulated by RNA helicases, particularly DDX5, in conjunction with transcriptional processes. Our study provides compelling evidence for the involvement of these factors in regulating R-loop dynamics during early embryonic development.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.10
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• pp.1383-1391
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• 2016

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5'-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the -134 to -19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

• Molecules and Cells
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• v.23 no.1
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• pp.23-29
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• 2007

Cyclic AMP-responsive element binding protein (CREB) is known to be associated with angiogenesis. In the present study we investigated the possible role of CREB in the expression of vascular endothelial growth factor (VEGF) by mouse macrophages. Over-expression of CREB increased VEGF secretion by cells of the RAW264.7 mouse macrophage cell line. It also increased the promoter activity of a mouse reporter driven by the VEGF promoter, while a dominant negative CREB (DN-CREB) abrogated the activity, suggesting that CREB mediates VEGF transcription. Forskolin, an adenylyl cyclase activator, stimulated VEGF transcription, and the PKA inhibitor H89 abolished this effect. IFN-${\gamma}$, a potent cytokine, stimulated VEGF expression only in part through the PKA-CREB pathway. These results indicate that PKA phosphorylates CREB and so induces VEGF gene expression. An analysis of mutant promoters revealed that one of the putative CREB responsive elements (CREs), at -399 ~ -388 in the promoter, is critical for CREB-mediated VEGF promoter activity, and the significance of this CRE was confirmed by chromatin immunoprecipitation assays.

• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.189-196
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• 2017

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with formation of Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. Replication and transcription activator (RTA) genes are expressed upon reactivation of KSHV, which displays a biphasic life cycle consisting of latent and lytic replication phases. RTA protein expression results in KSHV genome amplification and successive viral lytic gene expression. Transcriptional activity of viral lytic genes is regulated through epigenetic modifications. In Raji cells latently infected with Epstein-Barr virus, various modifications, such as acetylation and methylation, have been identified at specific lysine residues in histone H4 during viral reactivation, supporting the theory that expression of specific lytic genes is controlled by histone modification processes. Data obtained from chromatin immunoprecipitation and quantitative real-time PCR analyses revealed alterations in the H4K8ac and H4K20me3 levels at lytic gene promoters during reactivation. Our results indicate that H4K20me3 is associated with the maintenance of latency, while H4K8ac contributes to KSHV reactivation in infected TREx BCBL-1 RTA cells.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.6
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• pp.2565-2570
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• 2014

H19 is an imprinted oncofetal gene, and loss of imprinting at the H19 locus results in over-expression of H19 in cancers. Aflatoxin B1(AFB1) is regarded as one of the most dangerous carcinogens. Exposure to AFB1 would most easily increase susceptibility to diseases such as hepatocellular carcinoma(HCC) but any possible relationship between AFB1 and H19 is not clear. In present study, we found that AFB1 could up-regulate the expression of H19 and promote cell growth and invasion by hepatocellular carcinoma HepG2 cells. Knocking down H19 RNA co ld reverse the effects of AFB1 on cell growth and invasion. In addition, AFB1 induced the expression of E2F1 and its knock-down could down-regulate H19 expression and suppress cell growth and invasion in hepatocellular carcinoma HepG2 cells. Furthermore, E2F1 over-expression could up-regulate H19 expression and promote cell growth and invasion, with binding to the H19 promoter being demonstrated by chromatin immunoprecipitation assays (ChIP). In summary, our results suggested that aflatoxin B1could promote cell growth and invasion in hepatocellular carcinoma HepG2 cells through actions on H19 and E2F1.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.1
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• pp.359-365
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• 2013

The high mobility group box 1 (HMGB1) protein is a multifunctional cytokine-like molecule that plays an important role in the pathogenesis of tumors. In this study, real-time polymerase chain reactions and Western blot assays indicated that HMGB1 transcriptional activity and protein level are increased in $Tax^+$-T cells (TaxP). To clarify the mechanisms, a series of HMGB1 deletion reporter plasmids (pHLuc1 to pHLuc6) were transfected into $Tax^-$-T cells (TaxN, Jurkat) and $Tax^+$-T cells (TaxP). We found that promoter activity in $Tax^+$-T cells to be higher than that in $Tax^-$-T cells, indicating a significant increase in pHLuc6. Bay11-7082 (NF-${\kappa}B$ inhibitor) treatment did not block the enhancing effect. Chromatin immunoprecipitation assays revealed that Tax was retained on a HMGB1 promoter fragment encompassing -1163 to -975. Bioinformatics analysis showed six characteristic cis-elements for CdxA, AP-1, AML-1a, USF, v-Myb, and C/EBP in the fragment in question. Mutation of cis-elements for C/EBP reduced significant HMGB1 promoter activity induced by Tax. These findings indicate that Tax enhances the expression of HMGB1 gene at the transcriptional level, possibly by interacting with C/EBP.

• International Journal of Oral Biology
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• v.38 no.1
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• pp.37-42
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• 2013

Receptor activator of NF-${\kappa}B$ ligand (RANKL) is an essential cytokine for osteoclast differentiation, activation and survival. T lymphocytes such as $T_{17}$ cells, a subset of T helper cells that produce IL-17, play an important role in rheumatoid arthritic bone resorption by producing inflammatory cytokines and RANKL. It has not yet been clearly elucidated how T cell activation induces RANKL expression. T cell receptor activation induces the activation of nuclear factor of activated T cell (NFAT) and expression of its target genes. In this study, we examined the role of NFAT in T cell activation-induced RANKL expression. EL-4, a murine T lymphocytic cell line, was used. When T cell activation was induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin, RANKL expression increased in a time-dependent manner. In the presence of cyclosporin, an inhibitor of NFAT activation, this PMA/ionomycin-induced RANKL expression was blocked. Overexpression of either NFATc1 or NFATc3 induced RANKL expression. Chromatin immunoprecipitation results demonstrated that PMA/ionomycin treatment induced the binding of NFATc1 and NFATc3 to the mouse RANKL gene promoter. These results suggest that NFATc1 and NFATc3 mediates T cell receptor activation-induced RANKL expression in T lymphocytes.

• Journal of Gastric Cancer
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• v.11 no.1
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• pp.16-22
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• 2011

Purpose: Eupatilin is an antioxidative flavone and a phytopharmaceutical derived from Artemisia asiatica. It has been reported to possess anti-tumor activity in some types of cancer including gastric cancer. Eupatilin may modulate the angiogenesis pathway which is part of anti-inflammatory effect demonstrated in gastric mucosal injury models. Here we investigated the anti-tumor effects of eupatilin on gastric cancer cells and elucidated the potential underlying mechanism whereby eupatilin suppresses angiogenesis and tumor growth. Materials and Methods: The impact of eupatilin on the expression of angiogenesis pathway proteins was assessed using western blots in MKN45 cells. Using a chromatin immunoprecipitation assay, we tested whether eupatilin affects the recruitment of signal transducer and activator of transcription 3 (STAT3), aryl hydrocarbon receptor nuclear translocator (ARNT) and hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$) to the human VEGF promoter. To investigate the effect of eupatilin on vasculogenesis, tube formation assays were conducted using human umbilical vein endothelial cells (HUVECs). The effect of eupatilin on tumor suppression in mouse xenografts was assessed. Results: Eupatilin significantly reduced VEGF, ARNT and STAT3 expression prominently under hypoxic conditions. The recruitment of STAT3, ARNT and HIF-$1{\alpha}$ to the VEGF promoter was inhibited by eupatilin treatment. HUVECs produced much foreshortened and severely broken tubes with eupatilin treatment. In addition, eupatilin effectively reduced tumor growth in a mouse xenograft model. Conclusions: Our results indicate that eupatilin inhibits angiogenesis in gastric cancer cells by blocking STAT3 and VEGF expression, suggesting its therapeutic potential in the treatment of gastric cancer.