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MSP analysis of DAZAP2 promoter methylation status in multiple myeloma cell lines KM3, MM.1S, OPM-2, and ARH-77. MSP was performed with the specific primers to amplify the target sequence and a fragment with length of 214 bp was obtained. M: 100 bp DNA ladder plus marker; U: unmethylated sequence; M: methylated sequence; M + : positive methylation control; U + : negative methylation control. doi:10.1371/journal.pone.0040475.g002
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Our previous studies had shown that DAZAP2 was profoundly downregulated in bone marrow mononuclear cells from multiple myeloma patients. In this report, we analyzed epigenetic changes in multiple myeloma cell lines to understand the molecular mechanisms underlying the downregulation of DAZAP2. Four multiple myeloma cell lines, KM3, MM.1S, OPM-2 and...
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Citations
... Recent methylome analysis of monozygotic twins discordant for childhood psychotic symptoms identified a differentially methylated site located closest to DAZAP2 among the top ten most associated findings with the phenotype, however the probe was located -19713 bp from DAZAP2 TSS, while our probe was located closer to the gene, within the -1500 bp region from its TSS 102 . Differential methylation of DAZAP2 promoter region was shown to regulate its expression in multiple myeloma cell lines 103 , and its decreased expression levels were found to contribute to pathogenesis of this cancer 104 . Therefore, possible link between DAZAP2 and depression symptomatology requires further investigation. ...
Depression is a severe and debilitating mental disorder diagnosed by evaluation of affective, cognitive and physical depression symptoms. Severity of these symptoms strongly impacts individual’s quality of life and is influenced by a combination of genetic and environmental factors. One of the molecular mechanisms allowing for an interplay between these factors is DNA methylation, an epigenetic modification playing a pivotal role in regulation of brain functioning across lifespan. The aim of this study was to investigate if there are DNA methylation signatures associated with depression symptomatology in order to identify molecular mechanisms contributing to pathophysiology of depression. We performed an epigenome-wide association study (EWAS) of continuous depression symptomatology score measured in a cohort of 724 monozygotic Danish twins (346 males, 378 females). Through EWAS analyses adjusted for sex, age, flow-cytometry based blood cell composition, and twin relatedness structure in the data we identified depression symptomatology score to be associated with blood DNA methylation levels in promoter regions of neuropsin (KLK8, p-value = 4.7 × 10−7) and DAZ associated protein 2 (DAZAP2, p-value = 3.13 × 10−8) genes. Other top associated probes were located in gene bodies of MAD1L1 (p-value = 5.16 × 10−6), SLC29A2 (p-value = 6.15 × 10−6) and AKT1 (p-value = 4.47 × 10−6), all genes associated before with development of depression. Additionally, the following three measures (a) DNAmAge (calculated with Horvath and Hannum epigenetic clock estimators) adjusted for chronological age, (b) difference between DNAmAge and chronological age, and (c) DNAmAge acceleration were not associated with depression symptomatology score in our cohort. In conclusion, our data suggests that depression symptomatology score is associated with DNA methylation levels of genes implicated in response to stress, depressive-like behaviors, and recurrent depression in patients, but not with global DNA methylation changes across the genome.
... DAZAP2 is necessary for the FGF-dependent posterior patterning. In contrast to the FGF activity, dazap2's introduction of hoxb9 is not congested by the loss of canonical Wnt signaling and the increment in the level of dazap2 changes neural modeling and induces posterior neural markers [29]. The molecular-based functions of daz and dazl proteins were observed by studying RNAs and proteins which come in contact with DAZ and DAZL. ...
Non-obstructive azoospermia is a severe infertility factor. Currently, the etiology of this condition remains elusive with several possible molecular pathway disruptions identified in the post-meiotic spermatozoa. In the presented study, in order to identify all possible candidate genes associated with azoospermia and to map their relationship, we present the first protein-protein interaction network related to azoospermia and analyze the complex effects of the related genes systematically. Using Online Mendelian Inheritance in Man, the Human Protein Reference Database and Cytoscape, we created a novel network consisting of 209 protein nodes and 737 interactions. Mathematical analysis identified three proteins, ar, dazap2, and esr1, as hub nodes and a bottleneck protein within the network. We also identified new candidate genes, CREBBP and BCAR1, which may play a role in azoospermia. The gene ontology analysis suggests a genetic link between azoospermia and liver disease. The KEGG analysis also showed 45 statistically important pathways with 31 proteins associated with colorectal, pancreatic, chronic myeloid leukemia and prostate cancer. Two new genes and associated diseases are promising for further experimental validation.
... It would be interesting to determine how DAZAP2 is transcriptionally regulated. Indeed, promoter methylation resulting in DAZAP2 downregulation has been described in multiple myeloma (37,39); perhaps an aberrant downregulation of DAZAP2 is present in atopic asthmatics. Furthermore, identifying potential tyrosine kinases or the status of the phosphorylated IL-25R may serve as important therapeutic targets or potential biomarkers, respectively. ...
IL-25 promotes type 2 immunity by inducing the expression of Th2-associated cytokines. Although it is known that the IL-25R (IL-17RB) recruits the adaptor protein ACT1, the IL-25R signaling mechanism remains poorly understood. While screening for IL-25R components, we found that IL-25 responses were impaired in Traf4 (-/-) cells. Administering IL-25 to Traf4 (-/-) mice resulted in blunted airway eosinophilia and Th2 cytokine production. Notably, IL-25R recruitment of TRAF4 was required for the ACT1/IL-25R interaction. Mechanistically, TRAF4 recruited the E3-ligase SMURF2, to degrade the IL-25R-inhibitory molecule DAZAP2. Silencing Dazap2 increased ACT1/IL-25R interaction and IL-25 responsiveness. Moreover, a tyrosine within the IL-25R elicited DAZAP2 interference. This study indicates that TRAF4-SMURF2-mediated DAZAP2 degradation is a crucial initiating event for the IL-25 response.
Copyright © 2015 by The American Association of Immunologists, Inc.
... Methylation of DKK1 promoters occurs in several MM cell lines and MM cells from advanced MM patients. Promoter demethylation of DKK1 restores its expression, which results in inhibition of β-catenin/TCF-mediated gene transcription in MM cell lines (Luo et al. 2012;Kocemba et al. 2012). DKK1 is highly expressed in primary MM, and its expression is downregulated or completely suppressed in the advanced stage (Luo et al. 2012;Kocemba et al. 2012). ...
... Promoter demethylation of DKK1 restores its expression, which results in inhibition of β-catenin/TCF-mediated gene transcription in MM cell lines (Luo et al. 2012;Kocemba et al. 2012). DKK1 is highly expressed in primary MM, and its expression is downregulated or completely suppressed in the advanced stage (Luo et al. 2012;Kocemba et al. 2012). Aberrant methylation of DKK1 is a reason for DKK1 silencing in the advanced stage, which unleashes the expression of β-catenin/ TCF and activates the Wnt signaling pathway (Kocemba et al. 2012). ...
Multiple myeloma (MM) is a B cell malignancy characterized by accumulation of malignant plasma cells in the bone marrow. Pathogenesis of MM involves a complex pattern of structural and numerical chromosomal aberrations. In addition, epigenetic changes such as DNA methylation and histone modifications may play a role in this disease by affecting the expression of different genes. This article reviews recent findings on the role of epigenetic alterations in MM pathogenesis, which affect the expression of cell cycle regulatory molecules, apoptosis, DNA repair system, CD markers, cell signaling pathways as well as tumor suppressor miRNAs. Given these results, it can be stated that these epigenetic changes play an important role in the initiation and progression of MM. Therefore, understanding the impact of epigenetics in MM pathogenesis in each stage of disease progression can help develop therapeutic targets to increase survival and reduce drug resistance.
... Region2 and Region3 were verified by automated sequencing. The respective plasmid was linearized by Xho I and subjected to in vitro the methylation assay as described in our previous work [21]. The methylation products were then digested by Kpn I and inserted into the pGL4.17 ...
Organic cation/carnitine transporter 2 (OCTN2) is responsible for the cellular uptake of the antineoplastic agent, oxaliplatin. Epigenetic modification is a possible mechanism of altered drug-transporter expression in cancers, leading to altered efficacy of chemotherapeutic drugs. However, the mechanisms governing OCTN2 regulation are not completely understood. In this study, the low levels of OCTN2 in HepG2 and LS174T cells were elevated by the demethylating reagent, decitabine (DCA). To further reveal the epigenetic mechanism of down-regulation of OCTN2, we found that Region-1 within the OCTN2 promoter (spanning -354 to +85) was a determinant of OCTN2 expression in a luciferase reporter assay. Moreover, methylation-specific PCR (MSP) and bisulfite genomic sequencing showed that the degree of individual methylated CpG sites within this region was inversely correlated with the levels of OCTN2 in different cancer cells. Application of DCA to HepG2 and LS174T cells reversed the hypermethylation status of the OCTN2 promoter and increased OCTN2 expression, enhancing cellular uptake of oxaliplatin. Thus, we identified that promoter methylation is responsible for epigenetic down-regulation of OCTN2 in HepG2 and LS174T cells. Given the essential role of OCTN2 in cancer cell uptake of chemotherapeutics, and thus treatment efficacy, pretreatment with a demethylating reagent is a possible strategy for optimizing pharmacotherapies against cancers.
The Hippo signaling pathway plays a critical role in both normal animal physiology and pathogenesis. Since pharmacological interventions targeting this pathway have diverse clinical implications, a better understanding of its regulation in various conditions and organisms is crucial. Here, we identified deleted in azoospermia-associated protein 2 (DAZAP2) in Chinese mitten crab (Eriocheir sinensis), designated EsDAZAP2, as a Hippo-regulatory protein highly similar to proteins in various species of insects, fish, and mammals. We found that a bacterial infection significantly induces EsDAZAP2 expression and an EsDAZAP2 knockdown both suppresses antimicrobial peptide (AMP) expression in vitro, and results in increased viable bacterial counts and mortality in vivo, suggesting that EsDAZAP2 plays a critical role in innate immunity. Using yeast two-hybrid screening and co-immunoprecipitation assays, we found that EsDAZAP2 regulates the Toll pathway rather than the immune deficiency (Imd) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways. Our findings also demonstrate that EsDAZAP2 binds to the Hippo protein, Salvador (Sav). Moreover, by examining the regulation of Dorsal, a trasnscription factor that regulates AMP expression in E. sinensis, we provide experimental evidence indicating that EsDAZAP2 promotes Hippo pathway activation in innate immunity, with EsDAZAP2 and Hippo binding to different Sav domains. To the best of our knowledge, this is the first report of a DAZAP2-regulated Hippo signaling pathway operating in animal innate immunity.
Multiple myeloma (MM) is hematological malignancy characterized by clonal proliferation of malignant plasma cells in the bone marrow environment. Previously, we identified DAZAP2 as a candidate cancer suppressor gene, the downregulation of which is regulated by its own promoter methylation status. In the current study, we analyzed the DAZAP2 promoter in MM cell lines KM3, MM.1S, OPM-2, and ARH77 by bisulfite genomic sequencing assay. We identified the binding site for transcription factor cyclic adenosine monophosphate response element binding (CREB) in the DAZAP2 promoter CpG2, and we found that hypermethylation of the CREB binding motif in the DAZAP2 promoter is responsible for the reduced DAZAP2 expression in MM cells. Later we checked the p38/MAPK signaling cascade, which is reported to regulate expression and function of CREB. Our results showed that the p38/MAPK signaling pathway drives the expression of DAZAP2 by phosphorylation of CREB, and hypermethylation of CREB binding motif in DAZAP2 promoter can inhibit binding of CREB to the latter, thus downregulating DAZAP2 expression. Moreover, treating the MM cells with 5-aza-2′ deoxycytidine to demethylate DAZAP2 promoter restored the binding of CREB to its binding motif, and thus upregulated DAZAP2 expression. Our results not only identified DAZAP2 as a new downstream target of p38/MAPK/CREB signaling cascade, but we also clarified that the downregulation of DAZAP2 in MM cells is caused by hypermethylation of CREB binding motif in its own promoter region, which implies that demethylation of DAZAP2 promoter can be a novel therapeutic strategy for MM treatment.
Introduction:
Clonal plasma cells in multiple myeloma (MM) are typified by their nearly universal aneuploidy and the presence of recurrent chromosomal aberrations reflecting their chromosomal instability. Multiple myeloma is also recognized to be heterogeneous with distinct molecular subgroups. Deep genome sequencing studies have recently revealed an even wider heterogeneity and genomic instability with the identification of a complex mutational landscape and a branching pattern of clonal evolution.
Areas covered:
Despite the lack of full understanding of the exact mechanisms driving the genomic instability in MM, recent observations have correlated these abnormalities with impairments in the DNA damage repair machinery as well as epigenetic changes. These mechanisms and the resulting therapeutic implications will be the subject of this review.
Expert opinion:
By providing growth advantage of the fittest clone and promoting the acquisition of drug resistance, genomic instability is unarguably beneficial to MM cells, however, it may also well be its Achilles heal by creating exploitable vulnerabilities. As such, targeting presumptive DNA repair defects and other oncogenic addictions represent a promising area of clinical investigation. In particular, by inducing gene or pathway dependencies not present in normal cells, genomic instability can generate targets of contextual synthetic lethality in MM cells.
