Demethylation Of Histone H3 Lysine Research Articles

Central carbon metabolism exhibits unique characteristics during the handling of fungal patterns by monocyte-derived dendritic cells

Monocyte-derived dendritic cells (MDDCs) are key players in the defense against fungal infection because of their outstanding capacity for non-opsonic phagocytosis and phenotypic plasticity. Accordingly, MDDCs rewire metabolism to meet the energetic demands for microbial killing and biomass synthesis required to restore homeostasis. It has been commonplace considering the metabolic reprogramming a mimicry of the Warburg effect observed in tumor cells. However, this may be an oversimplification since the offshoots of glycolysis and the tricarboxylic acid (TCA) cycle are connected in central carbon metabolism. Zymosan, the external wall of Saccharomyces cerevisiae, contains β-glucan and α-mannan chains that engage the C-type lectin receptors dectin-1/2 and Toll-like receptors. This makes it an optimal fungal surrogate for experimental research. Using real-time bioenergetic assays and [U–13C]glucose labeling, central hubs connected to cytokine expression were identified. The pentose phosphate pathway (PPP) exhibited a more relevant capacity to yield ribose-5-phosphate than reducing equivalents of NADPH, as judged from the high levels of isotopologues showing 13C-labeling in the ribose moiety and the limited contribution of the oxidative arm of the PPP to the production of ROS by NADPH oxidases (NOX). The finding of 13C-label in the purine ring and in glutathione unveiled the contribution of serine-derived glycine to purine ring and glutathione synthesis. Serine synthesis also supported the TCA cycle. Zymosan exhausted NAD+ and ATP, consistent with intracellular consumption and/or extracellular export. Poly-ADP-ribosylated proteins detected in the nuclear fractions of MDDCs did not show major changes upon zymosan stimulation, which suggests its dependence on constitutive Fe(II)/2-oxoglutarate-dependent demethylation of 5-methylcytosine by TET translocases and/or demethylation of histone H3 lysine 27 by JMJD demethylases rather than on NOX activities. These results disclose a unique pattern of central carbon metabolism following fungal challenge, characterized by the leverage of glycolysis offshoots and an extensive recycling of NAD+ and poly(ADP-ribose).

Epigenetic regulation of Cebpb activation by pY19-Caveolin-2 at the nuclear periphery in association with the nuclear lamina

Here, we show that Caveolin-2 (Cav-2) is an epigenetic regulator for adipogenesis. Upon adipogenic stimulation, inner nuclear membrane (INM)-targeted pY19-Cav-2 interacted with lamin A/C to disengage the repressed Cebpb promoter from lamin A/C, which facilitated the Cebpb promoter association with lamin B1. Consequently, pY19-Cav-2 recruited lysine demethylase 4b (KDM4b) for demethylation of histone H3 lysine 9 trimethylation (H3K9me3) and histone acetyltransferase GCN5 for acetylation of H3K27, and subsequently RNA polymerase II (Pol II) on Cebpb promoter for epigenetic activation of Cebpb, to initiate adipogenesis. Cav-2 knock-down abrogated the Cebpb activation and blocked the Pparg2 and Cebpa activation. Re-expression of Cav-2 restored Cebpb activation and adipogenesis in Cav-2-deficient preadipocytes. Our data identify a new mechanism by which the epigenetic activation of Cebpb is controlled at the nuclear periphery to promote adipogenesis.

H3K4 di-methylation governs smooth muscle lineage identity and promotes vascular homeostasis by restraining plasticity.

Epigenetic mechanisms contribute to the regulation of cell differentiation and function. Vascular smooth muscle cells (SMCs) are specialized contractile cells that retain phenotypic plasticity even after differentiation. Here, by performing selective demethylation of histone H3 lysine 4 di-methylation (H3K4me2) at SMC-specific genes, we uncovered that H3K4me2 governs SMC lineage identity. Removal of H3K4me2 via selective editing in cultured vascular SMCs and in murine arterial vasculature led to loss of differentiation and reduced contractility due to impaired recruitment of the DNA methylcytosine dioxygenase TET2. H3K4me2 editing altered SMC adaptative capacities during vascular remodeling due to loss of miR-145 expression. Finally, H3K4me2 editing induced a profound alteration of SMC lineage identity by redistributing H3K4me2 toward genes associated with stemness and developmental programs, thus exacerbating plasticity. Our studies identify the H3K4me2-TET2-miR145 axis as a central epigenetic memory mechanism controlling cell identity and function, whose alteration could contribute to various pathophysiological processes.

A functional LSD1 coregulator screen reveals a novel transcriptional regulatory cascade connecting R-loop homeostasis with epigenetic regulation.

The lysine specific demethylase 1 (LSD1) plays a pivotal role in cellular differentiation by regulating the expression of key developmental genes in concert with different coregulatory proteins. This process is impaired in different cancer types and incompletely understood. To comprehensively identify functional coregulators of LSD1, we established a novel tractable fluorescent reporter system to monitor LSD1 activity in living cells. Combining this reporter system with a state-of-the-art multiplexed RNAi screen, we identify the DEAD-box helicase 19A (DDX19A) as a novel coregulator and demonstrate that suppression of Ddx19a results in an increase of R-loops and reduced LSD1-mediated gene silencing. We further show that DDX19A binds to tri-methylated lysine 27 of histone 3 (H3K27me3) and it regulates gene expression through the removal of transcription promoting R-loops. Our results uncover a novel transcriptional regulatory cascade where the downregulation of genes is dependent on the LSD1 mediated demethylation of histone H3 lysine 4 (H3K4). This allows the polycomb repressive complex 2 (PRC2) to methylate H3K27, which serves as a binding site for DDX19A. Finally, the binding of DDX19A leads to the efficient removal of R-loops at active promoters, which further de-represses LSD1 and PRC2, establishing a positive feedback loop leading to a robust repression of the target gene.

Histone methylation and demethylation are implicated in the transient and sustained activation of the interleukin-1β gene in murine macrophages.

Macrophages play a crucial role in the development of atherosclerosis. To explore the mechanism by which macrophages attain a proinflammatory phenotype for a sustained period, we stimulated macrophages with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) and measured the interleukin-1β (IL-1β) expression. The IL-1β expression increased transiently, and its expression lasted for, at least, 1week after the cessation of LPS and IFN-γ stimulation. At the promoter region of the IL-1β gene, the demethylation of histone H3 lysine 27 (H3K27) was significantly induced for 1week after transient stimulation with LPS and IFN-γ. The expression of H3K27 demethylases ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) and jumonji domain-containing 3 (JMJD3) increased significantly for 1week after transient stimulation with LPS and IFN-γ. When the UTX expression was inhibited by using small interfering RNA (siRNA) for UTX, the IL-1β expression was significantly suppressed in both transient and sustained phases, whereas siRNA for JMJD3 significantly inhibited only the sustained phase of the IL-1β expression. These results suggested that H3K27 demethylation was implicated in the transient and sustained increase in the IL-1β expression after LPS and IFN-γ stimulation.

METTL14-mediated N6-methyladenosine modification of SOX4 mRNA inhibits tumor metastasis in colorectal cancer

BackgroundColorectal cancer (CRC) is one of the leading causes of tumor-related death worldwide, and its main cause of death is distant metastasis. Methyltransferase-like 14(METTL14), a major RNA N6-adenosine methyltransferase, is involved in tumor progression via regulating RNA function. The goal of the study is to uncover the biological function and molecular mechanism of METTL14 in CRC.MethodsQuantitative real-time PCR (qRT-PCR), western blot and immunohistochemical (IHC) assays were employed to detect METTL14 and SOX4 in CRC cell lines and tissues. The biological functions of METTL14 were demonstrated using in vitro and in vivo experiments. Chromatin immunoprecipitation (ChIP), Transcrptomic RNA sequencing (RNA-Seq), m6A-RNA immunoprecipitation sequencing (MeRIP-Seq), RNA immunoprecipitation and luciferase reporter assays were used to explore the mechanism of METTL14 action.ResultsMETTL14 expression was significantly downregulated in CRC and decreased METTL14 was associated with poor overall survival (OS). Both the univariate and multivariate Cox regression analysis indicated that METTL14 was an independent prognostic factor in CRC. Moreover, lysine-specific histone demethylase 5C(KDM5C)-mediated demethylation of histone H3 lysine 4 tri-methylation(H3K4me3) in the promoter of METTL14 inhibited METTL14 transcription. Functionally, we verified that METTL14 inhibited CRC cells migration, invasion and metastasis through in vitro and in vivo assays, respectively. Furthermore, we identified SRY-related high-mobility-group box 4(SOX4) as a target of METTL14-mediated m6A modification. Knockdown of METTL14 markedly abolished SOX4 mRNA m6A modification and elevated SOX4 mRNA expression. We also revealed that METTL14-mediated SOX4 mRNA degradation relied on the YTHDF2-dependent pathway. Lastly, we demonstrated that METTL14 might inhibit CRC malignant process partly through SOX4-mediated EMT process and PI3K/Akt signals.ConclusionsDecreased METTL14 facilitates tumor metastasis in CRC, suggesting that METTL14 might be a potential prognostic biomarker and effective therapeutic target for CRC.Graphical abstract

The histone demethylase JMJD2A promotes glioma cell growth via targeting Akt-mTOR signaling

BackgroundA number of JmjC domain-containing histone demethylases have been identified and biochemically characterized in mammalian models and humans. JMJD2A is a transcriptional co-factor and enzyme that catalyzes the demethylation of histone H3 lysine 9 and 36 (H3K9 and H3K36). Here in this study, we reported the role of JMJD2A in human glioma.MethodsQuantitative real-time PCR and western blot were performed to analyzed JMJD2A expression in glioma. Log-rank was performed to plot the survival curve. JMJD2A was knocked or overexpressed with lentivirus. Cell proliferation and colony formation were performed to assess the effects of JMJD2A on glioma cell growth. Xenograft experiment was performed the evaluate the growth rate of glioma cells in vivo. The signaling pathway was analyzed with western blot and mTOR was inhibited with rapamycin.ResultsQuantitative real-time PCR and western blot experiments revealed higher expression of JMJD2A and lower levels of H3K9me3/H3K36me3 in glioma tissues than that in normal brain tissues. We showed that knockdown of JMJD2A expression attenuated the growth and colony formation in three lines of glioma cells (U251, T98G, and U87MG), whereas JMJD2A overexpression resulted in opposing effects. Furthermore, we performed in vivo xenograft experiments and our data demonstrated that JMJD2A knockdown reduced the growth of glioma T98G cells in vivo. Further mechanism study implicated that JMJD2A activated the Akt-mTOR pathway and promoted protein synthesis in glioma cells via promoting phosphoinositide-dependent kinase-1 (PDK1) expression. The activation of the Akt-mTOR pathway was also validated in human glioma tissues. Finally, we showed that inhibition of mTOR with rapamycin blocked the effects of JMJD2A on protein synthesis, cell proliferation and colony formation of glioma cells.ConclusionsThese findings demonstrated that JMJD2A regulated glioma growth and implicated that JMJD2A might be a promising target for intervention.

ZHX2 restricts hepatocellular carcinoma by suppressing stem cell-like traits through KDM2A-mediated H3K36 demethylation.

BackgroundLiver cancer stem cells (CSCs) are critical determinants of HCC relapse and therapeutic resistance, but the mechanisms underlying the maintenance of CSCs are poorly understood. We aimed to explore the role of tumor repressor Zinc-fingers and homeoboxes 2 (ZHX2) in liver CSCs.MethodsCD133+ or EPCAM+ stem-like liver cancer cells were sorted from tumor tissues of HCC patients and HCC cell lines by flow cytometry. In addition, sorafenib-resistant cells, tumor-sphere forming cells and side population (SP) cells were respectively cultured and isolated as hepatic CSCs. The tumor-initiating and chemoresistance properties of ZHX2-overexpressing and ZHX2-knockdown cells were analyzed in vivo and in vitro. Microarray, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and ChIP-on-chip analyses were performed to explore ZHX2 target genes. The expression of ZHX2 and its target gene were determined by quantitative RT-PCR, western blot, immunofluorescence and immunohistochemical staining in hepatoma cells and tumor and adjacent tissues from HCC patients.ResultsZHX2 expression was significantly reduced in liver CSCs from different origins. ZHX2 deficiency led to enhanced liver tumor progression and expansion of CSC populations in vitro and in vivo. Re-expression of ZHX2 restricted capabilities of hepatic CSCs in supporting tumor initiation, self-renewal and sorafenib-resistance. Mechanically, ZHX2 suppressed liver CSCs via inhibiting KDM2A-mediated demethylation of histone H3 lysine 36 (H3K36) at the promoter regions of stemness-associated transcription factors, such as NANOG, SOX4 and OCT4. Moreover, patients with lower expression of ZHX2 and higher expression of KDM2A in tumor tissues showed significantly poorer survival.ConclusionZHX2 counteracts stem cell traits through transcriptionally repressing KDM2A in HCC. Our data will aid in a better understanding of molecular mechanisms underlying HCC relapse and drug resistance.

ZHX2 Restricts Hepatocellular Carcinoma by Suppressing Stem Cell-Like Traits Through KDM2A-Mediated H3K36 Demethylation

Background: Liver cancer stem cells (CSCs) are critical determinants of cancer relapse and therapeutic resistance, but the mechanisms underlying the maintenance of CSCs are poorly understood. We aimed to explore the role of tumor repressor Zinc-fingers and homeoboxes (ZHX2) in liver CSCs. Methods: CD133+ or EPCAM+ stem-like liver cancer cells were sorted from tumor tissues of HCC patients and cell lines by flow cytometry. Also, sorafenib-resistant cells, tumor-sphere forming cells and side population (SP) cells were respectively cultured and isolated as hepatic CSCs. The tumor-initiating and chemoresistance properties of ZHX2-overexpressing and ZHX2-knockdown cells were analyzed in vivo and in vitro. Microarray, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and ChIP-on-chip analyses were performed to explore ZHX2 target genes. The expression of ZHX2 and its target gene were determined by quantitative RT-PCR, western blot, immunofluorescence and immunohistochemical staining in hepatoma cells and tumor and adjacent tissues from HCC patients. Results: The expression of ZHX2 was reduced in liver CSCs from different origins. ZHX2 deficiency led to enhanced liver tumor progression and expansion of CSC populations in vitro and in vivo. Re-expression of ZHX2 restricted capabilities of CSCs in supporting tumor initiation, self-renew and sorafenib-resistance. Mechanically, ZHX2 suppressed liver CSCs via inhibiting KDM2A-mediated demethylation of histone H3 lysine 36 (H3K36) at the promoter regions of stemness-associated transcription factors, such as NANOG, SOX4 and OCT4. Moreover, patients with lower expression of ZHX2 and higher expression of KDM2A in tumor tissues showed significant poor survival. Conclusion: ZHX2 counteracts stem cell traits through transcriptionally repressing KDM2A in HCC. Our data will aid in a better understanding of molecular mechanisms underlying HCC relapse and drug resistance. Funding Statement: This work was supported by grants from the National Natural Science Fund for Outstanding Youth Fund (81425012), the National Key Research and Development Program (No.2016YFE0127000), the National Science Foundation of China (No. 81830017, 81370527, 81702647), Taishan Scholarship (No.tspd20181201), Shandong Provincial Key Innovation project (No.2018FYJH0503) and Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Informed consent was obtained from all patients before the study was initiated with approval of the Shandong University Medical Ethics Committee in accordance with the Declaration of Helsinki. All animal procedures were performed in according to protocols approved by the Shandong University Animal Care Committee and conducted with an animal ethical approval.

Abstract GMM-022: IDH1 IS A PRO-SENESCENT THERAPY IN HIGH-GRADE SEROUS OVARIAN CANCER

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. High-grade serous carcinoma (HGSC) is the most frequently diagnosed and fatal histosubtype of EOC. We found that HGSC cell lines exhibit increased TCA cycle metabolites compared to fallopian tube cells, the proposed site of HGSC origin. Therefore, we propose that inhibition of TCA cycle metabolism may be a novel therapeutic strategy for HGSC. To determine which TCA cycle metabolism enzymes are dysregulated in ovarian cancer, we performed an unbiased qPCR screen of all 27 enzymes in the pathway. Our results indicate that isocitrate dehydrogenase I (IDH1) expression is significantly altered in EOC cell lines compared to fallopian tube cells. IDH1 catalyzes the conversion of isocitrate to alpha ketoglutarate (αKG) in a reversible reaction. It is well-known that IDH1 mutations play a role in cancer; however, recent publications suggest wildtype IDH1 overexpression promotes primary glioblastoma progression in the absence of mutation. Wildtype IDH1 and its role in metabolism and epigenetics has never been investigated in HGSC. To determine whether IDH1 plays a functional role in HGSC, we used both genetic and pharmacological approaches to suppress IDH1 in multiple HGSC cell lines. Knockdown or inhibition of IDH1 using the small molecule inhibitor GSK864 significantly decreased HGSC cell proliferation. Mechanistically, this was due to induction of senescence, a stable cell cycle arrest. We next aimed to determine the molecular mechanism underlying senescence induction due to inhibition of IDH1. Increased histone methylation of proliferation promoting genes (i.e., CCNA2 and PCNA) is a characteristic of senescence. αKG acts as a cofactor for the Jumonji C (JmjC) histone demethylase family, suggesting that suppression of αKG may affect the methylation status of histones. αKG levels were significantly decreased in IDH1 knockdown cells. Therefore, we hypothesized that decreasing αKG levels in HGSC may modulate the epigenome to induce senescence. Chromatin immunoprecipitation (ChIP) experiments showed an increase in repressive H3K9me2 histone methylation at proliferation-promoting gene loci (CCNA2 and PCNA) when IDH1 was knocked down. This correlated with a decrease in mRNA of both genes. These data suggest that knockdown of IDH1 induces senescence of HGSC cells by increased histone methylation of proliferation promoting genes. Finally, we aimed to determine the JmjC demethylase family member that is inhibited by IDH1 knockdown. The JmjC KDM4 family modulates histone demethylation of histone H3 lysine 9. KDM4A is upregulated in EOC and correlates with worse overall survival. Interestingly, knockdown studies of KDM4A phenocopied those of IDH1 knockdown. Future studies are aimed to dissect the role of KDM4A in senescence induction. Altogether, these data suggest that targeting the metabolic enzyme IDH1 in HGSC induces senescence through epigenetic reprogramming and may be a novel metabolic therapy for HGSC patients. Citation Format: Erika S Dahl, Nathaniel W Snyder, Katherine M Aird. IDH1 IS A PRO-SENESCENT THERAPY IN HIGH-GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-022.

SUN-210 THE HISTONE DEMETHYLASE JUMONJI DOMAIN-CONTAINING PROTEIN 3 ACTS AS AN EPIGENETIC SUPPRESSOR OF RENAL FIBROSIS

The histone demethylase Jumonji domain-containing protein 3 (JMJD3) is the enzyme that induces demethylation of histone H3 lysine 27 (H3K27) and its activation is involved in the tumor genesis. Although activation of H3K27 methyltransferase has been shown to be required for renal interstitial fibroblast activation and renal fibrogenesis, the role of JMJD3 in these processes remains elusive.

KDM3A-mediated demethylation of histone H3 lysine 9 facilitates the chromatin binding of Neurog2 during neurogenesis.

Neurog2 is a crucial regulator of neuronal fate specification and differentiation in vivo and in vitro However, it remains unclear how Neurog2 transactivates neuronal genes that are silenced by repressive chromatin. Here, we provide evidence that the histone H3 lysine 9 demethylase KDM3A facilitates the Xenopus Neurog2 (formerly known as Xngnr1) chromatin accessibility during neuronal transcription. Loss-of-function analyses reveal that KDM3A is not required for the transition of naive ectoderm to neural progenitor cells but is essential for primary neuron formation. ChIP series followed by qPCR analyses reveal that Neurog2 promotes the removal of the repressive H3K9me2 marks and addition of active histone marks, including H3K27ac and H3K4me3, at the NeuroD1 and Tubb2b promoters; this activity depends on the presence of KDM3A because Neurog2, via its C-terminal domain, interacts with KDM3A. Interestingly, KDM3A is dispensable for the neuronal transcription initiated by Ascl1, a proneural factor related to neurogenin in the bHLH family. In summary, our findings uncover a crucial role for histone H3K9 demethylation during Neurog2-mediated neuronal transcription and help in the understanding of the different activities of Neurog2 and Ascl1 in initiating neuronal development.

Vitamin C induces specific demethylation of H3K9me2 in mouse embryonic stem cells via Kdm3a/b

BackgroundHistone methylation patterns regulate gene expression and are highly dynamic during development. The erasure of histone methylation is carried out by histone demethylase enzymes. We had previously shown that vitamin C enhances the activity of Tet enzymes in embryonic stem (ES) cells, leading to DNA demethylation and activation of germline genes.ResultsWe report here that vitamin C induces a remarkably specific demethylation of histone H3 lysine 9 dimethylation (H3K9me2) in naïve ES cells. Vitamin C treatment reduces global levels of H3K9me2, but not other histone methylation marks analyzed, as measured by western blot, immunofluorescence and mass spectrometry. Vitamin C leads to widespread loss of H3K9me2 at large chromosomal domains as well as gene promoters and repeat elements. Vitamin C-induced loss of H3K9me2 occurs rapidly within 24 h and is reversible. Importantly, we found that the histone demethylases Kdm3a and Kdm3b are required for vitamin C-induced demethylation of H3K9me2. Moreover, we show that vitamin C-induced Kdm3a/b-mediated H3K9me2 demethylation and Tet-mediated DNA demethylation are independent processes at specific loci. Lastly, we document Kdm3a/b are partially required for the upregulation of germline genes by vitamin C.ConclusionsThese results reveal a specific role for vitamin C in histone demethylation in ES cells and document that DNA methylation and H3K9me2 cooperate to silence germline genes in pluripotent cells.

Jumonji domain containing 2C promotes cell migration and invasion through modulating CUL4A expression in lung cancer.

Jumonji domain containing 2C (JMJD2C), also named as KDM4C, was found to a transcriptional cofactor and enzyme that catalyzes demethylation of histone H3 lysine 9 and 36. Here in this study, we found that the expression of JMJD2C increased in a majority of the human lung cancer tissues examined compared with adjacent tissues. Furthermore, the expression of JMJD2C was found to be higher in metastatic lung cancer tissues than which in non-metastatic lung cancer tissues. Knockdown of JMJD2C inhibited the ability of migration and invasion of lung cancer cells. Moreover, JMJD2C knockdown was proven to inhibit the tumor hepatic metastasis of lung cancer cells in vivo and epithelial-mesenchymal transition (EMT) in vitro. On the contrary, over-expression of JMJD2C was found to promote the ability of migration, invasion and EMT. As to mechanism, knockdown of JMJD2C was found to inhibit the expression of CUL4A while to promote the expression of p53 and p27. Furthermore, we found that JMJD2C regulated the activities of lung cancer cells by directly controlling the expression of CUL4A in JMJD2C over-expression cell line, and interference of CUL4A was found to reverse the ability of migration, invasion and EMT which JMJD2C over-expression bought to. Together, these results of this study not only enriched the JMJD2C biological function of lung cancer, but also illuminated exploring the prevention and treatment of the invasion and metastasis of lung cancer.

Role of UTX in retinoic acid receptor-mediated gene regulation in leukemia.

Human UTX, a member of the Jumonji C family of proteins, associates with mixed-lineage leukemia 3/4 complexes. Stimulation with retinoic acid leads to the recruitment of UTX-containing complexes to HOX genes, which results in demethylation of histone H3 lysine 27 and concomitant methylation of histone H3 lysine 4. Here, we show that UTX interacts with the retinoic acid receptor α (RARα) and that this interaction is essential for proper differentiation of leukemic U937 cells in response to retinoic acid. UTX occupies the promoters of several RAR target genes and regulates their transcriptional output by modulating ASH2L complex recruitment. Overexpression of UTX in promyelocytic NB4 cells results in enhanced cellular differentiation upon retinoic acid treatment. Our results show that UTX is important for RAR-mediated transcription and provide insight into the critical role of cross talk between histone H3 lysine 4 methylation and histone H3 lysine 27 demethylation during cellular differentiation.

THU0464 Lsd1-Mediated Demethylation of Histone H3 Lysine 9 Contributes to Interleukin 1-Induced Microsomal Prostaglandin E Synthase-1 Expression

BackgroundMicrosomal prostaglandin E synthase-1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE2, a critical mediator in the pathophysiology of osteoarthritis (OA). Histone methylation plays an important role in...

LSD1-mediated demethylation of histone h3 lysine 9 contributes to interleukin 1-induced microsomal prostaglandin e synthase-1 expression in human osteoarthritic chondrocytes

LSD1-mediated demethylation of histone h3 lysine 9 contributes to interleukin 1-induced microsomal prostaglandin e synthase-1 expression in human osteoarthritic chondrocytes

Genetic association analysis of radiographic hip osteoarthritis with established loci for bone mineral density: data from the osteoarthritis initiative

Genetic association analysis of radiographic hip osteoarthritis with established loci for bone mineral density: data from the osteoarthritis initiative

Immunohistochemistry for Histone H3 Lysine 9 Methyltransferase and Demethylase Proteins in Human Melanomas

Methylation and demethylation of histone H3 lysine 9 (H3K9) play a role in the transcriptional regulation of several cancer-related genes and are closely associated with malignant tumor behavior. A novel study has recently demonstrated that SETDB1, a member of the H3K9 methyltransferases, accelerates tumor formation significantly in a zebrafish melanoma model. However, the expression of H3K9 methyltransferases including SETDB1 and demethylases has not been systematically examined in samples of human melanoma. Here, we used immunohistochemistry to examine the expression of the H3K9 methyltransferases, EHMT2 and SETDB1, and a H3K9 demethylase, LSD1, in 67 patients with melanoma. Overexpression of EHMT2, SETDB1, and LSD1 was observed in 14 (21%), 38 (57%), and 53 (79%) of the 67 patients, respectively. A significant relationship was observed between overexpression of EHMT2 or SETDB1 and aggressive tumor behavior such as lymph node metastasis and/or distant metastasis (P < 0.05), whereas no significant relationship was evident for LSD1 immunoreactivity. Univariate log-rank tests demonstrated that patients with melanoma overexpressing EHMT2 had a poorer outcome (P < 0.001), whereas overexpression of SETDB1 or LSD1 had no prognostic impact. These results suggest that overexpression of EHMT2 might be a prognostic marker in patients with melanoma.

Lysine-specific demethylase 1-mediated demethylation of histone H3 lysine 9 contributes to interleukin 1β-induced microsomal prostaglandin E synthase 1 expression in human osteoarthritic chondrocytes.

IntroductionMicrosomal prostaglandin E synthase 1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE2, a critical mediator in the pathophysiology of osteoarthritis (OA). Histone methylation plays an important role in epigenetic gene regulation. In this study, we investigated the roles of histone H3 lysine 9 (H3K9) methylation in interleukin 1β (IL-1β)-induced mPGES-1 expression in human chondrocytes.MethodsChondrocytes were stimulated with IL-1β, and the expression of mPGES-1 mRNA was evaluated using real-time RT-PCR. H3K9 methylation and the recruitment of the histone demethylase lysine-specific demethylase 1 (LSD1) to the mPGES-1 promoter were evaluated using chromatin immunoprecipitation assays. The role of LSD1 was further evaluated using the pharmacological inhibitors tranylcypromine and pargyline and small interfering RNA (siRNA)-mediated gene silencing. The LSD1 level in cartilage was determined by RT-PCR and immunohistochemistry.ResultsThe induction of mPGES-1 expression by IL-1β correlated with decreased levels of mono- and dimethylated H3K9 at the mPGES-1 promoter. These changes were concomitant with the recruitment of the histone demethylase LSD1. Treatment with tranylcypromine and pargyline, which are potent inhibitors of LSD1, prevented IL-1β-induced H3K9 demethylation at the mPGES-1 promoter and expression of mPGES-1. Consistently, LSD1 gene silencing with siRNA prevented IL-1β-induced H3K9 demethylation and mPGES-1 expression, suggesting that LSD1 mediates IL-1β-induced mPGES-1 expression via H3K9 demethylation. We show that the level of LSD1 was elevated in OA compared to normal cartilage.ConclusionThese results indicate that H3K9 demethylation by LSD1 contributes to IL-1β-induced mPGES-1 expression and suggest that this pathway could be a potential target for pharmacological intervention in the treatment of OA and possibly other arthritic conditions.