In Vitro Histone Demethylase Assays

2015 ◽  
pp. 109-122
Author(s):  
Kenji Kokura ◽  
Lidong Sun ◽  
Jia Fang
Keyword(s):  
Histone Demethylase

scholarly journals Histone demethylase PHF8 accelerates the progression of colorectal cancer and can be regulated by miR-488 in vitro

2017 ◽  
Vol 16 (4) ◽  
pp. 4437-4444 ◽  
Author(s):  
Yao Lv ◽  
Yan Shi ◽  
Quanli Han ◽  
Guanghai Dai
Keyword(s):  
Colorectal Cancer ◽  
Histone Demethylase

scholarly journals A CRISPR/Cas9 screen identifies the histone demethylase MINA53 as a novel HIV-1 latency-promoting gene (LPG)

2019 ◽  
Vol 47 (14) ◽  
pp. 7333-7347 ◽  
Author(s):  
Huachao Huang ◽  
Weili Kong ◽  
Maxime Jean ◽  
Guillaume Fiches ◽  
Dawei Zhou ◽  
...  
Keyword(s):  
Local Level ◽  
Histone Demethylase ◽  
Host Factors ◽  
Epigenetic Mark ◽  
Combination Antiretroviral Therapy ◽  
Downstream Effectors ◽  
Infected Cells ◽  
Latent Hiv ◽  
Hiv 1

AbstractAlthough combination antiretroviral therapy is potent to block active replication of HIV-1 in AIDS patients, HIV-1 persists as transcriptionally inactive proviruses in infected cells. These HIV-1 latent reservoirs remain a major obstacle for clearance of HIV-1. Investigation of host factors regulating HIV-1 latency is critical for developing novel antiretroviral reagents to eliminate HIV-1 latent reservoirs. From our recently accomplished CRISPR/Cas9 sgRNA screens, we identified that the histone demethylase, MINA53, is potentially a novel HIV-1 latency-promoting gene (LPG). We next validated MINA53’s function in maintenance of HIV-1 latency by depleting MINA53 using the alternative RNAi approach. We further identified that in vitro MINA53 preferentially demethylates the histone substrate, H3K36me3 and that in cells MINA53 depletion by RNAi also increases the local level of H3K36me3 at LTR. The effort to map the downstream effectors unraveled that H3K36me3 has the cross-talk with another epigenetic mark H4K16ac, mediated by KAT8 that recognizes the methylated H3K36 and acetylated H4K16. Removing the MINA53-mediated latency mechanisms could benefit the reversal of post-integrated latent HIV-1 proviruses for purging of reservoir cells. We further demonstrated that a pan jumonji histone demethylase inhibitor, JIB-04, inhibits MINA53-mediated demethylation of H3K36me3, and JIB-04 synergizes with other latency-reversing agents (LRAs) to reactivate latent HIV-1.

Identification of a Novel Epigenetic Regulator That Acts As a Tumor Suppressor in the Development of MLL AML Leukemic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3923-3923
Author(s):  
Hangyu Yi ◽  
Halina Leung ◽  
Aliaksei Holik ◽  
Florida Voli ◽  
Marie-Liesse Asselin-Labat ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tumor Suppressor ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Histone Demethylase ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Promising Therapeutic Approach

Abstract Acute myeloid leukemia (AML) is still a deadly form of leukemia due to frequent relapse caused by the persistence of drug-resistant leukemic stem cells (LSCs). We have previously demonstrated a crucial role for β-catenin signaling in regulating LSCs and identified GPR84 as an important β-catenin regulator in the maintenance of mixed-lineage leukemia (MLL) LSCs (Wang et al., Science 2010; Dietrich et al., Blood 2014). Hence, targeting LSCs by pharmacological inhibition of GPR84/β-catenin signaling represents a promising therapeutic approach. In collaboration with a pharmaceutical company that has developed a novel GPR84 antagonist (GP), we investigated the effect of GP in MLL pre-leukemic stem cell (pre-LSC) function. GP (20 μM) significantly inhibited the colony forming ability of MLL pre-LSCs (P < 0.0001) but had little effect on normal hematopoietic stem cells. Quantitative RT-PCR and western blot analysis confirmed GP-induced downregulation of GPR84 target genes, including Hoxa5, Hoxa7 and Meis1a, indicating GP-induced inhibition of GPR84 signaling. To further examine the mechanism of GPR84 inhibition on MLL pre-LSCs, we evaluated several epigenetic regulators (i.e. JMJD1c and EZH2) known to promote leukemogenesis (Zhu et al., ‎J Clin Invest 2016; Tanaka et al., Blood 2012). Western blot analysis showed that inhibition of GPR84 signaling did not alter the expression of JMJD1c or EZH2. However, we observed a significant increase in the expression of a novel and not-yet-characterized histone demethylase (HD) in AML. To investigate the role of HD in AML leukemogenesis, we overexpressed HD in MLL pre-LSCs and subsequent serial replating assay showed a marked reduction in colony forming ability (P < 0.005), indicating impaired self-renewal in vitro. Consistent with our in vitro observations, in vivo transplantation in syngeneic mice revealed a significant delay in leukemia onset and increase in mouse survival (P < 0.001). We next performed western blot analysis to examine the demethylase activity of HD, and our data revealed that HD overexpression caused a substantial reduction in global histone 3 lysine 36 dimethylation (H3K36me2), an epigenetic mark normally associated with transcriptional activation and elongation. In order to identify genes regulated by HD through demethylation of H3K36me2, we performed H3K36me2 ChIP-seq on HD overexpressing MLL pre-LSCs. Our analysis identified several genes including anti-apoptotic protein Mcl-1 and angiogenic receptor Nrp1, which are known to be involved in AML leukemogenesis, with decreased H3K36me2 mark on both the transcriptional start site and gene body. Subsequent western blot analysis confirmed the decreased expression of both Mcl-1 and Nrp1 in HD overexpressing pre-LSCs. Given the prominent roles of anti-apoptosis and angiogenesis in the development of hematologic malignancies such as leukemia, we are currently evaluating these mechanisms caused by HD overexpression in an important subtype of AML. Taken together, our study identifies a novel histone demethylase that acts downstream of GPR84 signaling to function as a potent tumor suppressor in the development of MLL LSCs. Disclosures No relevant conflicts of interest to declare.

scholarly journals OTUB2 Facilitates Tumorigenesis of Gastric Cancer Through Promoting KDM1A-Mediated Stem Cell-Like Properties

2021 ◽  
Vol 11 ◽  
Author(s):  
Guangming Liu ◽  
Wei Guo ◽  
Junjie Qin ◽  
Zhiliang Lin
Keyword(s):  
Gastric Cancer ◽  
Stem Cell ◽  
Survival Rates ◽  
Histone Demethylase ◽  
Deubiquitinating Enzyme ◽  
Stem Cell Markers ◽  
Loss Of Function ◽  
Sphere Formation

Otubain 2 (OTUB2), a deubiquitinating enzyme, overexpression is considered to predict poor outcome in various cancers. However, the function and potential regulatory mechanisms of OTUB2 in gastric cancer (GC) progression remains unclear. To determine how OTUB2 participate in GC progression, the gain and loss of-function experiments were conducted in vivo and in vitro. We found that OTUB2 was upregulated in GC samples (n=140) and cells. Moreover, the overall, first progression and post progression survival rates of GC patients with high OTUB2 expression showed a poorer prognosis than that in those patients with low OTUB2 expression. Down-regulation of OTUB2 suppressed sphere formation and reduced expression of stem cell markers in GC cells. Furthermore, OTUB2-silenced GC cells also showed a decreased proliferation, invasion, migration, and in vivo tumorigenic ability. However, OTUB2 overexpression showed the opposite effects. Notably, we demonstrated that OTUB2 increased lysine-specific histone demethylase 1A (KDM1A) expression through deubiquitination. KDM1A, a demethylase known to promote demethylation of downstream genes, was identified to promote the maintenance of cancer stem cell characteristics. Moreover, the alterations caused by OTUB2 overexpression were partly inversed by KDM1A knockdown and in turn KDM1A overexpression reversed the changes induced by OTUB2 shRNA. Taken together, we demonstrate that OTUB2 may serve as a vital driver in GC tumorigenesis by enhancing KDM1A-mediated stem cell-like properties.

scholarly journals The Histone Demethylase LSD1/ΚDM1A Mediates Chemoresistance in Breast Cancer via Regulation of a Stem Cell Program

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1585 ◽  
Author(s):  
John Verigos ◽  
Panagiotis Karakaidos ◽  
Dimitris Kordias ◽  
Alexandra Papoudou-Bai ◽  
Zoi Evangelou ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Drug Resistance ◽  
Tumor Cells ◽  
Histone Demethylase ◽  
Small Subset ◽  
Tumor Initiating Cells ◽  
Female Population ◽  
Number Of Patients

Breast cancer is the leading cause of cancer death in the female population, despite advances in diagnosis and treatment. The highly heterogeneous nature of the disease represents a major obstacle to successful therapy and results in a significant number of patients developing drug resistance and, eventually, suffering from tumor relapse. Cancer stem cells (CSCs) are a small subset of tumor cells characterized by self-renewal, increased tumor-initiation capacity, and resistance to conventional therapies. As such, they have been implicated in the etiology of tumor recurrence and have emerged as promising targets for the development of novel therapies. Here, we show that the histone demethylase lysine-specific demethylase 1 (LSD1) plays an important role in the chemoresistance of breast cancer cells. Our data, from a series of in vitro and in vivo assays, advocate for LSD1 being critical in maintaining a pool of tumor-initiating cells that may contribute to the development of drug resistance. Combinatory administration of LSD1 inhibitors and anti-cancer drugs is more efficacious than monotherapy alone in eliminating all tumor cells in a 3D spheroid system. In conclusion, we provide compelling evidence that LSD1 is a key regulator of breast cancer stemness and a potential target for the design of future combination therapies.

scholarly journals Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dong Hoon Lee ◽  
Go Woon Kim ◽  
Jung Yoo ◽  
Sang Wu Lee ◽  
Yu Hyun Jeon ◽  
...  
Keyword(s):  
Tumor Suppressor Genes ◽  
Therapeutic Strategy ◽  
Target Gene ◽  
Molecular Mechanisms ◽  
Histone Demethylase ◽  
Wild Type ◽  
P53 Target Gene ◽  
The Stability

AbstractGlioblastoma is the most lethal brain tumor and its pathogenesis remains incompletely understood. KDM4C is a histone H3K9 demethylase that contributes to epigenetic regulation of both oncogene and tumor suppressor genes and is often overexpressed in human tumors, including glioblastoma. However, KDM4C’s roles in glioblastoma and the underlying molecular mechanisms remain unclear. Here, we show that KDM4C knockdown significantly represses proliferation and tumorigenesis of glioblastoma cells in vitro and in vivo that are rescued by overexpressing wild-type KDM4C but not a catalytic dead mutant. KDM4C protein expression is upregulated in glioblastoma, and its expression correlates with c-Myc expression. KDM4C also binds to the c-Myc promoter and induces c-Myc expression. Importantly, KDM4C suppresses the pro-apoptotic functions of p53 by demethylating p53K372me1, which is pivotal for the stability of chromatin-bound p53. Conversely, depletion or inhibition of KDM4C promotes p53 target gene expression and induces apoptosis in glioblastoma. KDM4C may serve as an oncogene through the dual functions of inactivation of p53 and activation of c-Myc in glioblastoma. Our study demonstrates KDM4C inhibition as a promising therapeutic strategy for targeting glioblastoma.

scholarly journals KDM2B promotes cell viability by enhancing DNA damage response in canine hemangiosarcoma

2020 ◽  
Author(s):  
Kevin Christian M. Gulay ◽  
Keisuke Aoshima ◽  
Yuki Shibata ◽  
Hironobu Yasui ◽  
Qin Yan ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Damage Response ◽  
Histone Demethylase ◽  
Tumor Bearing ◽  
Lysine Specific Demethylase ◽  
Damage Response ◽  
Epigenetic Regulators ◽  
Induced Apoptosis

AbstractEpigenetic regulators have been implicated in tumorigenesis of many types of cancer; however, their roles in endothelial cell cancers such as canine hemangiosarcoma (HSA) have not been studied. In this study, we found that lysine-specific demethylase 2B (Kdm2b) was highly expressed in HSA cell lines compared to normal canine endothelial cells. Silencing of Kdm2b in HSA cells resulted to increased cell death in vitro compared to the scramble control by inducing apoptosis through the inactivation of the DNA repair pathways and accumulation of DNA damage. Similarly, doxycycline-induced Kdm2b silencing in tumor xenografts resulted to decreased tumor sizes compared to the scramble control. Furthermore, Kdm2b was also highly expressed in clinical cases of HSA, and its expression levels was higher than in hemangioma, a benign counterpart of HSA. Based on these results, we hypothesized that pharmacological Kdm2b inhibition can also induce HSA cell death and can be used as an alternative treatment for HSA. We treated HSA cells with GSK-J4, a histone demethylase inhibitor, and found that GSK-J4 treatment also induced apoptosis and cell death. On top of that, GSK-J4 treatment in HSA tumor-bearing mice decreased tumor sizes without any obvious side-effects. In this study, we demonstrated that Kdm2b acts as an oncogene in HSA by enhancing DNA damage response and can be used as a biomarker to differentiate HSA from hemangioma. Moreover, we indicated that histone demethylase inhibitor GSK-J4 can be used as a therapeutic alternative to doxorubicin for HSA treatment.

In Vitro Histone Demethylase Assays

2009 ◽  
pp. 249-261 ◽  
Author(s):  
Kenji Kokura ◽  
Jia Fang
Keyword(s):  
Histone Demethylase

Discovery of Critical Functions of Histone Demethylase, PHF8, in Mediating ATRA Response in APL

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 226-226 ◽  
Author(s):  
Maria Francisca Arteaga ◽  
Jan-Henrik Mikesch ◽  
Chi Wai Eric So
Keyword(s):  
Cellular Transformation ◽  
Histone Demethylase ◽  
Leukemic Cells ◽  
Epigenetic Therapy ◽  
Phosphorylation Sites ◽  
Histone Demethylases ◽  
Atra Treatment ◽  
Resistant Variant ◽  
Resistant Cells

Abstract Abstract 226 Introduction: Transcription deregulation plays a key role in acute leukemogenesis, which is mostly initiated by chimeric transcription factors such as PML–RARa that accounts for almost 100% of acute promyelocytic leukemia (APL). While APL is uniquely sensitive to all trans retinoic acid (ATRA) treatment and has been the paradigm of differentiation/epigenetic therapies, the underlying mechanisms remain largely unknown and are major interests in the field with a potential of extending the success to other hematological malignancies. Previously, others and we have shown that aberrant recruitment of histone deacetylases and polycomb repressive complexes by oligomeric PML-RARa are key for suppression of its downstream targets and promote cellular transformation. ATRA treatment disrupts their bindings and results in de-repression of target genes. However, the identification of the co-activator complex responsible for gene activation upon ATRA treatment remains a major hurdle that significantly hinders the progress of understanding mechanisms underlying the epigenetic therapy. Results and Discussion: Given the critical functions of JmjC-domains containing histone demethylases in mediating transcriptional regulation, we performed a systematic screening for differential interaction between JmjC-histone demethylases and PML–RARa upon ATRA treatment. We identified a highly specific interaction between PML–RARa and Plant Homeodomain Finger 8 (PHF8). To assess its effect on histone methylation, we detected specific reduction of H3K9me2 and enhancement of H3K4me3 in PHF8 transfected cells, consistent with its function as a transcriptional activator. This was further supported by results from chromatin immuno-precipitation (ChIP) assays in human APL cell line (NB4) showing that PHF8 differentially bound and mediated the same histone modifications in RARb promoter and activated its expression. To investigate the functional significance of this interaction, PHF8 was expressed in human NB4 cells harboring PML-RARa or murine primary bone marrow cells transformed by APL fusion proteins. Induction of PHF8 expression significantly decreased their in vitro transformation capacity in the presence of physiological concentrations of ATRA. Conversely, specific down-modulation of PHF8 expression by shRNAs reduced ATRA sensitivity of these cells, suggesting a critical function of PHF8 in mediating ATRA response. We hypothesized that PHF8 may be able to sensitize ATRA resistant cells to the treatment. Hence, we induced expression of PHF8 in the ATRA resistant variant of NB4 line, NB4-MR2. We were able to demonstrate that PHF8 sensitized NB4-MR2 cells to ATRA treatment in vitro. In contrast, NB4-MR2 cells expressing PHF8-F279S, a catalytically inactive mutant could not be sensitized to ATRA treatment, indicating that the enzymatic activity is critical for mediating the ATRA response. Most importantly, NB4-MR2 cells expressing wild type PHF8 were also sensitive to ATRA treatment in vivo and failed to induce leukemia in NOD/SCID mice, which would otherwise succumb to leukemia in a very brief latency. To gain further insights into the molecular regulation of PHF8 in ATRA response, we characterized the potential functions of CDK1-mediated phosphorylation of PHF8. It is known that in ATRA treated leukemic cells Cyclin A translocates into the nucleus where it interacts with CDK1. Activated CDK1 induces phosphorylation of PHF8 at two serine (S33)/ threonine (T84) phosphorylation sites. Our results showed that PHF8 in its phosphorylated form had a much higher binding affinity to PML-RARa. Consistently, ChIP analyses revealed that the binding of PHF8 to the RARb promoter and the resultant activation were significantly augmented when PHF8 was constitutively phosphorylated. Moreover, inhibition of PHF8 dephosphorylation by Okadaic Acid, sensitized NB4-MR2 cells to ATRA treatment. Conclusions: We discovered a novel function of the histone demethylase PHF8 in mediating therapeutic response in both ATRA sensitive and ATRA resistant cells. This function of PHF8 is critically dependent on two phosphorylation sites as well as its histone modification activity. Thus, therapeutic interventions such as phosphatase inhibitors that enhance the PHF8 activity might become useful tools for development of new epigenetic therapies sensitizing leukemic cells to ATRA treatment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Histone demethylase KDM2B upregulates histone methyltransferase EZH2 expression and contributes to the progression of ovarian cancer in vitro and in vivo

2017 ◽  
Vol Volume 10 ◽  
pp. 3131-3144 ◽  
Author(s):  
Yan Kuang ◽  
Fangfang Lu ◽  
Jianfeng Guo ◽  
Hong Xu ◽  
Qi Wang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Histone Methyltransferase ◽  
Histone Demethylase ◽  
Ezh2 Expression
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