Effect of alpha-difluoromethylornithine on DNA methylation in murine erythroleukaemic cells. Relationship to stimulation of induced differentiation.
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ABSTRACT:
SUBMITTER: Papazafiri P
PROVIDER: S-EPMC1147730 | biostudies-other | 1987 Mar
REPOSITORIES: biostudies-other
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Project description:Polyamine spermidine is essential for the proliferation of eukaryotic cells. Administration of polyamine biosynthesis inhibitor α-difluoromethylornithine (DFMO) induces cytostasis that occurs in two phases; the early phase which can be reversed by spermidine, spermine, and some of their analogs, and the late phase which is characterized by practically complete depletion of cellular spermidine pool. The growth of cells at the late phase can be reversed by spermidine and by very few of its analogs, including (S)-1-methylspermidine. It was reported previously (Witherspoon et al. Cancer Discovery 3(9); 1072-81, 2013) that DFMO treatment leads to depletion of cellular thymidine pools, and that exogenous thymidine supplementation partially prevents DFMO-induced cytostasis without affecting intracellular polyamine pools in HT-29, SW480, and LoVo colorectal cancer cells. Here we show that thymidine did not prevent DFMO-induced cytostasis in DU145, LNCaP, MCF7, CaCo2, BT4C, SV40MES13, HepG2, HEK293, NIH3T3, ARPE19 or HT-29 cell lines, whereas administration of functionally active mimetic of spermidine, (S)-1-methylspermidine, did. Thus, the effect of thymidine seems to be specific only for certain cell lines. We conclude that decreased polyamine levels and possibly also distorted pools of folate-dependent metabolites mediate the anti-proliferative actions of DFMO. However, polyamines are necessary and sufficient to overcome DFMO-induced cytostasis, while thymidine is generally not.
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Project description:Genomic levels of DNA methylation undergo widespread alterations in early embryonic development. However, changes in embryonic methylation have proven difficult to study at the level of single-copy genes due to the small amount of tissue available for assay. This study provides the first detailed analysis of the methylation state of a tissue-specific gene through early development and differentiation. Using bisulfite sequencing, we mapped the methylation profile of the tissue-specific mouse skeletal alpha-actin promoter at all stages of development, from gametes to postimplantation embryos. We show that the alpha-actin promoter, which is fully methylated in the sperm and essentially unmethylated in the oocyte, undergoes a general demethylation from morula to blastocyst stages, although the blastula is not completely demethylated. Remethylation of the alpha-actin promoter occurs after implantation in a stochastic pattern, with some molecules being extensively methylated and others sparsely methylated. Moreover, we demonstrate that tissue-specific expression of the skeletal alpha-actin gene in the adult mouse does not correlate with the methylation state of the promoter, as we find a similar low level of methylation in both expressing and one of the two nonexpressing tissues tested. However, a subset of CpG sites within the skeletal alpha-actin promoter are preferentially methylated in liver, a nonexpressing tissue.
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Project description:Several lines of evidence indicate that DNA methylation plays a role in the transcriptional regulation of the murine alpha 1(I) collagen gene. To study the molecular mechanisms involved, a reporter gene construct containing the alpha 1(I) promoter and part of the first exon linked to the luciferase gene (Col3luc) was methylated in vitro and transfected into murine fibroblasts and embryonal carcinoma cells. Methylation resulted in repression of the alpha 1(I) promoter in both cell types, although it was less pronounced in embryonal carcinoma cells than in fibroblasts. The extent of repression depended on the density of methylation. DNase footprint and mobility shift assays indicated that the trans-acting factors binding to the alpha 1(I) promoter and first exon are ubiquitous factors and that their DNA binding is not inhibited by methylation. Transfection of Col3luc into Drosophila SL2 cells together with expression vectors for the transcription factors Sp1 and NF-1 showed that DNA methylation also inhibits the alpha 1(I) promoter in nonvertebrate cells, although to a much lesser extent than in murine cells. However, Sp1 and NF-1 transactivated the unmethylated and methylated reporter gene in SL2 cells equally well, confirming that these factors can bind and transactivate methylated DNA and indicating that DNA methylation represses the alpha 1(I) promoter by an indirect mechanism. This was further confirmed by cotransfection experiments with unspecific methylated competitor DNA which partially restored the activity of the methylated alpha 1(I) promoter. Our results suggest that DNA methylation can inhibit promoter activity by an indirect mechanism independent of methyl-C-binding proteins and that in vertebrate cells, chromatin structure and methyl-C-binding proteins cooperatively mediate the transcriptional inhibitory effect of DNA methylation.
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Project description:Epigenetic modifications introduce an additional layer of regulation that drastically expands the instructional capability of the human genome. The regulatory consequences of DNA methylation is context dependent; it can induce, enhance, and suppress gene expression, or have no effect on gene regulation. Therefore, it is essential to account for the genomic location of its occurrence and the protein factors it associates with to improve our understanding of its function and effects. Here, we use ENCODE ChIP-seq and DNase I hypersensitivity data, along with large-scale breast cancer genomic data from The Cancer Genome Atlas (TCGA) to computationally dissect the intricacies of DNA methylation in regulation of cancer transcriptomes. In particular, we identified a relationship between estrogen receptor α (ERα) activity and DNA methylation patterning in breast cancer. We found compelling evidence that methylation status of DNA sequences at ERα binding sites is tightly coupled with ERα activity. Furthermore, we predicted several transcription factors including FOXA1, GATA1, and SUZ12 to be associated with breast cancer by examining the methylation status of their binding sites in breast cancer. Lastly, we determine that methylated CpGs highly correlated with gene expression are enriched in regions 1kb or more downstream of TSSs, suggesting more significant regulatory roles for CpGs distal to gene TSSs. Our study provides novel insights into the role of ERα in breast cancers.
| S-EPMC4121363 | biostudies-literature
Project description:OBJECTIVES:Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized. MATERIALS AND METHODS:Murine-induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated. RESULTS:Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac-specific markers, GATA4, Nkx2.5 and Tnnt2. Although ?-AR signalling is the foremost signalling pathway in cardiomyocytes, EPI-enhanced cardiac differentiation depended more on ?-AR signalling than ?-AR signalling. In addition, selective activation of ?1 -AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of ?2 - or ?-AR signalling induced no or less differentiation, respectively. EPI- and ?1 -AR-dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK-ERK1/2 pathway and regulation of miPS cell-cycle progression. CONCLUSIONS:These results demonstrate that activation of ARs, particularly of ?1 -ARs, promoted miPSC differentiation into cardiac lineages via MEK-ERK1/2 signalling.
| S-EPMC6529101 | biostudies-literature
Project description:Nucleosomes compact and regulate access to DNA in the nucleus, and are composed of approximately 147 bases of DNA wrapped around a histone octamer. Here we report a genome-wide nucleosome positioning analysis of Arabidopsis thaliana using massively parallel sequencing of mononucleosomes. By combining this data with profiles of DNA methylation at single base resolution, we identified 10-base periodicities in the DNA methylation status of nucleosome-bound DNA and found that nucleosomal DNA was more highly methylated than flanking DNA. These results indicate that nucleosome positioning influences DNA methylation patterning throughout the genome and that DNA methyltransferases preferentially target nucleosome-bound DNA. We also observed similar trends in human nucleosomal DNA, indicating that the relationships between nucleosomes and DNA methyltransferases are conserved. Finally, as has been observed in animals, nucleosomes were highly enriched on exons, and preferentially positioned at intron-exon and exon-intron boundaries. RNA polymerase II (Pol II) was also enriched on exons relative to introns, consistent with the hypothesis that nucleosome positioning regulates Pol II processivity. DNA methylation is also enriched on exons, consistent with the targeting of DNA methylation to nucleosomes, and suggesting a role for DNA methylation in exon definition.
| S-EPMC2964354 | biostudies-literature
Project description:BACKGROUND: Synucleinopathy disorders are characterized by aggregates of α-synuclein (α-syn), which engage microglia to elicit a neuroinflammatory response. However, the influence α-syn has on DNA methylation and gene expression patterns for other genes has not been thoroughly explored. Our objective is to determine gene expression and methylation changes in microglia induced by aggregate α-syn.METHODS: Transgenic mThy1-Asyn mice overexpressing human α-syn are a model of synucleinopathy. Microglia from 3 and 13-month-old mice were used to isolate nucleic acids for methylated DNA and RNA-sequencing. α-Syn-induced changes in gene expression and genomic methylation were determined and examined for functional enrichment followed by network analysis to further elucidate possible connections within the data.RESULTS: Microglial DNA isolated from our 3-month cohort had 5,315 differentially methylated gene (DMG) changes, while RNA levels demonstrated a change in 119 differentially expressed genes (DEGs) between mThy1-Asyn mice and wild-type littermate controls. The 3-month DEGs and DMGs were highly associated with adhesion and migration signaling, suggesting a phenotypic transition from resting to active microglia. We observed 3,742 DMGs and 3,766 DEGs in 13-month mThy1-Asyn mice.These genes were often related to adhesion, migration, cell cycle, cellular metabolism, and immune response. Network analysis also showed increased cell mobility and inflammatory functions at 3 months, shifting to cell cycle, immune response, and metabolism changes at 13 months.CONCLUSION: We observed significant α-syn-induced methylation and gene expression changes in microglia. Our data suggest that α-syn overexpression initiates microglial activation leading to neuroinflammation and cellular metabolic stresses, which is associated with disease progression.
2021-07-02 | GSE158981 | GEO
Project description:Differentiation potency of human dental pulp cells (hDPCs) is essential for dentin regeneration. DNA methylation is one of the major epigenetic mechanisms and is suggested to involve in differentiation of hDPCs, the machinery of which includes DNA methyltransferase enzymes (DNMTs) and methyl-CpG-binding domain proteins (MBDs). Our previous study has found that melatonin (MT) promoted hDPC differentiation, but its mechanism remains elusive. We aimed to investigate the role of DNA methylation in the promotion of MT to differentiation of hDPCs in vitro. hDPCs were cultured in basal growth medium (CO) or odontogenic medium (OM) exposed to MT at different concentrations (0, 10-12, 10-10, 10-8, 10-6, 10-4 M). The cell growth was analyzed using Cell Counting Kit-8 assay, and mineralized tissue formation was measured using Alizarin red staining. The expression of the 10 genes (DNMT1, DNMT3A, DNMT3B, MBD1-6, MeCP2) was determined using real-time qPCR and western blotting. The abundance of MeCP2 in the nuclei was evaluated using immunofluorescence analysis. Global methylation level was tested using ELISA. We found that mineralized tissue formation significantly increased in OM with MT at 10-4 M, while the levels of MeCP2 and global DNA methylation level declined. The expression of MBD1, MBD3, and MBD4 significantly increased in OM alone, and the expession of DNMT1 and MBD2 was decreased. These results indicate that MT promotes odontogenic differentiation of hDPCs in vitro by regulating the levels of DNMT1, MeCP2, and global DNA methylation, suggesting that MT-induced DNA methylation machinery may play an important role in tooth regeneration.
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Project description:Nutritional intake impacts the human epigenome by directing epigenetic pathways in normal cell development via as yet unknown molecular mechanisms. Consequently, imbalance in the nutritional intake is able to dysregulate the epigenetic profile and drive cells towards malignant transformation. Here we present a novel epigenetic effect of the essential nutrient, NAD. We demonstrate that impairment of DNMT1 enzymatic activity by NAD-promoted ADP-ribosylation leads to demethylation and transcriptional activation of the CEBPA gene, suggesting the existence of an unknown NAD-controlled region within the locus. In addition to the molecular events, NAD- treated cells exhibit significant morphological and phenotypical changes that correspond to myeloid differentiation. Collectively, these results delineate a novel role for NAD in cell differentiation, and indicate novel nutri-epigenetic strategies to regulate and control gene expression in human cells.
| S-EPMC8616462 | biostudies-literature
Project description:PurposePluronic block copolymers are potent sensitizers of multidrug resistant cancers. SP1049C, a Pluronic-based micellar formulation of doxorubicin (Dox) has completed Phase II clinical trial and demonstrated safety and efficacy in patients with advanced adenocarcinoma of the esophagus and gastroesophageal junction. This study elucidates the ability of SP1049C to deplete cancer stem cells (CSC) and decrease tumorigenicity of cancer cells in vivo.Experimental designP388 murine leukemia ascitic tumor was grown in BDF1 mice. The animals were treated with: (a) saline, (b) Pluronics alone, (c) Dox or (d) SP1049C. The ascitic cancer cells were isolated at different passages and examined for 1) in vitro colony formation potential, 2) in vivo tumorigenicity and aggressiveness, 3) development of drug resistance and Wnt signaling activation 4) global DNA methylation profiles, and 5) expression of CSC markers.ResultsSP1049C treatment reduced tumor aggressiveness, in vivo tumor formation frequency and in vitro clonogenic potential of the ascitic cells compared to drug, saline and polymer controls. SP1049C also prevented overexpression of BCRP and activation of Wnt-?-catenin signaling observed with Dox alone. Moreover, SP1049C significantly altered the DNA methylation profiles of the cells. Finally, SP1049C decreased CD133(+) P388 cells populations, which displayed CSC-like properties and were more tumorigenic compared to CD133(-) cells.ConclusionsSP1049C therapy effectively suppresses the tumorigenicity and aggressiveness of P388 cells in a mouse model. This may be due to enhanced activity of SP1049C against CSC and/or altered epigenetic regulation restricting appearance of malignant cancer cell phenotype.
| S-EPMC3747131 | biostudies-literature