scholarly journals therascreenPITX2 RGQ PCR assay for the assessment of PITX2 DNA-methylation status to investigate the role of the transcription factor PITX2 and the regulation of the Wnt/ß-catenin pathway in pathophysiological processes.

2018 ◽  
Author(s):  
Jonathan Perkins ◽  
Rudolf Napieralski ◽  
Gabriele Schricker ◽  
Elodie Piednoir ◽  
Olivia Manner ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factor ◽  
Methylation Status ◽  
Pcr Assay

scholarly journals Title Page Title: The role of DNA methylation in the accumulation of iridoid glycosides in Rehmannia glutinosa

2021 ◽  
Author(s):  
Tianyu Dong ◽  
Xiaoyan Wei ◽  
Qianting Qi ◽  
Peilei Chen ◽  
Yanqing Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Secondary Metabolites ◽  
Iridoid Glycosides ◽  
Methylation Status ◽  
Plant Secondary Metabolites ◽  
Dna Demethylation ◽  
Terpene Biosynthesis ◽  
The Relationship

Abstract Background: Epigenetic regulation plays a significant role in the accumulation of plant secondary metabolites. The terpenoids are the most abundant in the secondary metabolites of plants, iridoid glycosides belong to monoterpenoids which is one of the main medicinal components of R.glutinosa. At present, study on iridoid glycosides mainly focuses on its pharmacology, accumulation and distribution, while the mechanism of its biosynthesis and the relationship between DNA methylation and plant terpene biosynthesis are seldom reports. Results: The research showed that the expression of DXS, DXR, 10HGO, G10H, GPPS and accumulation of iridoid glycosides increased at first and then decreased with the maturity of R.glutinosa, and under different concentrations of 5-azaC, the expression of DXS, DXR, 10HGO, G10H, GPPS and the accumulation of total iridoid glycosides were promoted, the promotion effect of low concentration (15μM-50μM) was more significant, the content of genomic DNA 5mC decreased significantly, the DNA methylation status of R.glutinosa genomes was also changed. DNA demethylation promoted gene expression and increased the accumulation of iridoid glycosides, but excessive demethylation inhibited gene expression and decreased the accumulation of iridoid glycosides. Conclusion: The analysis of DNA methylation, gene expression, and accumulation of iridoid glycoside provides insights into accumulation of terpenoids in R.glutinosa and lays a foundation for future studies on the effects of epigenetics on the synthesis of secondary metabolites.

Genome-Wide DNA Methylation Profiling of Hematopoietic Stem and Progenitor Cells Reveals Over-Representation of ETS Transcrition Factor Binding Sites

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 211-211
Author(s):  
Amber Hogart ◽  
Jens Lichtenberg ◽  
Subramanian Ajay ◽  
Elliott Margulies ◽  
David M. Bodine
Keyword(s):  
Dna Methylation ◽  
Transcription Factor ◽  
Cpg Islands ◽  
Hematopoietic Cells ◽  
Cell Type ◽  
Hematopoietic Stem ◽  
Genome Wide ◽  
Cell Type Specific ◽  
Methylation Patterns

Abstract Abstract 211 The hematopoietic system is ideal for the study of epigenetic changes in primary cells because hematopoietic cells representing distinct stages of hematopoiesis can be enriched and isolated by differences in surface marker expression. DNA methylation is an essential epigenetic mark that is required for normal development. Conditional knockout of the DNA methyltransferase enzymes in the mouse hematopoietic compartment have revealed that methylation is critical for long-term renewal and lineage differentiation of hematopoietic stem cells (Broske et al 2009, Trowbridge el al 2009). To better understand the role of DNA methylation in self-renewal and differentiation of hematopoietic cells, we characterized genome-wide DNA methylation in primary cells representing three distinct stages of hematopoiesis. We isolated mouse hematopoietic stem cells (HSC; Lin- Sca-1+ c-kit+), common myeloid progenitor cells (CMP; Lin- Sca-1- c-kit+), and erythroblasts (ERY; CD71+ Ter119+). Methyl Binding Domain Protein 2 (MBD2) is an endogenous reader of DNA methylation that recognizes DNA with a high concentration of methylated CpG residues. Recombinant MBD2 enrichment of DNA followed by massively-parallel sequencing was used to map and compare genome-wide DNA methylation patterns in HSC, CMP and ERY. Two biological replicates were sequenced for each cell type with total read counts ranging from 32,309,435–46,763,977. Model-based analysis of ChIP Seq (MACS) with a significance cutoff of p<10−5 was used to determine statistically significant peaks of methylation in each replicate. Globally, the number of methylation peaks was highest in HSC (85,797peaks), lower in CMP (50,638 peaks), and lowest in ERY (27,839 peaks). Comparison of the peaks in HSC, CMP and ERY revealed that only 2% of the peaks in CMP or ERY are absent in HSC indicating that the vast majority of methylation in HSC is lost during differentiation. Comparison of methylation with genomic features revealed that CpG islands associated with promoters are hypomethylated, while many non-promoter CpG islands are methylated. Furthermore, methylation of non-promoter associated CpG islands occurs infrequently in cell-type specific peaks but is more abundant in common methylation peaks. When the DNA methylation patterns were compared to mRNA expression, we found that as expected, proximal promoter sequences of expressed genes were hypomethylated in all three cell types, while methylation in the gene body positively correlated with gene expression in HSC and CMP. Utilizing de novo motif discovery we found a subset of transcription factor consensus binding motifs that were overrepresented in methylated sequences. Motifs for several ETS transcription factors, including GABPalpha and ELF1 were found to be overrepresented in cell-type specific as well as common methylated regions. Other transcription factor consensus sites, such as the NFAT factors involved in T-cell activation, were specifically overrepresented in the methylated promoter regions of CMP and ERY. Comparison of our methylation data with the occupancy of hematopoietic transcription factors in the HPC7 cell line, which is similar to CMP (Wilson et al 2010), revealed a significant anti-correlation between DNA methylation and the binding of Fli1, Lmo2, Lyl1, Runx1, and Scl. Our genome-wide survey provides new insights into the role of DNA methylation in hematopoiesis. Firstly, the methylation of CpG islands is associated with the most primitive hematopoietic cells and is unlikely to drive hematopoietic differentiation. We feel that the elevated genome-wide DNA methylation in HSC compared to CMP and ERY, combined with the positive association between gene body methylation and gene expression demonstrates that DNA methylation is a mark of cellular plasticity in HSC. Finally, the finding that transcription factor binding sites are over represented in the methylated sequences of the genome leads us to conclude that DNA methylation modulates key hematopoietic transcription factor programs that regulate hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

DNA Methylation Profile of Runx1 Regulatory Regions Is Correlated with Transition From Primitive to Definitive Hematopoietic Potential In Vitro and In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 389-389
Author(s):  
Beau Webber ◽  
Michelina Iacovino ◽  
Michael Kyba ◽  
Bruce R. Blazar ◽  
Jakub Tolar
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Transcription Factor ◽  
Fetal Liver ◽  
Methylation Status ◽  
Differential Methylation ◽  
Promoter Usage ◽  
Highly Correlated

Abstract Abstract 389 Introduction: The Runt-related transcription factor Runx1 (AML1) is a central regulator of mammalian hematopoiesis and is required for the generation of hematopoietic stem cells (HSC) from hemogenic endothelium in the embryo. It has been shown that Runx1 is alternatively expressed from two promoters in a temporal fashion, and that their differential activities are influenced by a conserved intronic enhancer (+23) element. Intriguingly, promoter usage follows a pattern whereby the proximal (P2) initiates early in primitive hematopoiesis, while the distal (P1) becomes active later at the time of HSC emergence and is the predominant isoform expressed in fetal liver and adult HSC. While some transcription factor binding sites and cis-regulatory elements have been identified, an explanation for the alternative promoter usage remains elusive. We hypothesized that this regulation may be at the level of chromatin accessibility, and therefore investigated the DNA methylation status of Runx1 cis-elements. Methods/Results: We analyzed bisulfite-treated genomic DNA from E14.5 fibroblast (MEF), E8.5 yolk sac CD41+ (YS), E14.5 fetal liver Lin-Sca-1+CD48-CD150+ (FL), and adult marrow Lin-cKit+Sca-1+ (KLS); representing non-hematopoietic, primitive hematopoietic, and two stages of definitive HSC respectively. In addition, we also examined methylation in hematopoietic populations derived in vitro from murine embryonic stem cells (mESC). Initial exploratory analysis focused on classically defined CpG islands upstream of each promoter, however no significant differential methylation was observed within these regions. Subsequent analysis focused on CpGs near the transcription start site (TSS) and within the +23 enhancer. The P2 promoter was uninformative as it was unmethylated in all populations analyzed, whereas methylation within the +23 enhancer differentiated between hematopoietic and non-hematopoietic cell populations. At the P1 promoter, methylation status was remarkably correlated with primitive vs. definitive status. P1 was highly methylated in MEFs (77%), mESC embryoid body (EB) derived cKit+CD41+ (66%), and E8.5 YS CD41+ (58%); but significantly less methylated in vivo in FL HSC (8.1%) and adult KLS cells (18%). We are currently using this correlation of demethylation and definitive HSC potential to identify conditions that may drive definitive HSC generation from mESC-derived blood progenitors. Since overexpression of HoxB4 coupled with OP9 co-culture is the only confirmed method capable of producing definitive HSC from mESC, and HoxB4 has been shown to bind within the P1 promoter region of Runx1, we cultured HoxB4 or control EB-derived hematopoietic progenitors on OP9 stroma. We observed progressive demethylation in the HoxB4 arm: after 6 days of co-culture 47% vs. 71% in controls, and after 11 days 27% in the HoxB4 arm while the control population failed to proliferate past day 6. Isoform specific RT-PCR confirmed that HoxB4 overexpression resulted in Runx1 expression from the P1 promoter whereas the control vector did not. Within P1, we identify a single CpG that is most highly correlated with definitive HSC potential in vivo, and most significantly demethylated upon HoxB4 overexpression in vitro. Conclusions: These data indicate that differential methylation occurs at Runx1 regulatory regions during hematopoietic development in vitro and in vivo. The +23 enhancer is demethylated in cells with hematopoietic potential, whereas demethylation of the Runx1 P1 promoter is highly correlated with definitive HSPC populations and is promoted in vitro by HoxB4. These data are the first to identify a role for DNA methylation in the regulation of alternative promoter usage at the Runx1 locus, and may serve as a novel biomarker of HSC potential during embryonic development. Disclosures: No relevant conflicts of interest to declare.

scholarly journals DNA Methylation and Gene Expression Profiling of Ewing Sarcoma Primary Tumors Reveal Genes That Are Potential Targets of Epigenetic Inactivation

Sarcoma ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Nikul Patel ◽  
Jennifer Black ◽  
Xi Chen ◽  
A. Mario Marcondes ◽  
William M. Grady ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Cell Lines ◽  
Ewing Sarcoma ◽  
Methylation Status ◽  
Primary Cultures ◽  
Human Mesenchymal Stem Cell ◽  
Primary Tumors ◽  
Hypermethylated Genes

The role of aberrant DNA methylation in Ewing sarcoma is not completely understood. The methylation status of 503 genes in 52 formalin-fixed paraffin-embedded EWS tumors and 3 EWS cell lines was compared to human mesenchymal stem cell primary cultures (hMSCs) using bead chip methylation analysis. Relative expression of methylated genes was assessed in 5-Aza-2-deoxycytidine-(5-AZA)-treated EWS cell lines and in a cohort of primary EWS samples and hMSCs by gene expression and quantitative RT-PCR. 129 genes demonstrated statistically significant hypermethylation in EWS tumors compared to hMSCs. Thirty-six genes were profoundly methylated in EWS and unmethylated in hMSCs. 5-AZA treatment of EWS cell lines resulted in upregulation of expression of hundreds of genes including 162 that were increased by at least 2-fold. The expression of 19 of 36 candidate hypermethylated genes was increased following 5-AZA. Analysis of gene expression from an independent cohort of tumors confirmed decreased expression of six of nineteen hypermethylated genes (AXL, COL1A1, CYP1B1, LYN, SERPINE1,) andVCAN. Comparing gene expression and DNA methylation analyses proved to be an effective way to identify genes epigenetically regulated in EWS. Further investigation is ongoing to elucidate the role of these epigenetic alterations in EWS pathogenesis.

scholarly journals Genetic and Epigenetic Alterations Induced by Pesticide Exposure: Integrated Analysis of Gene Expression, microRNA Expression and DNA Methylation Datasets

2021 ◽  
Vol 18 (16) ◽  
pp. 8697
Author(s):  
Federica Giambò ◽  
Gian Leone ◽  
Giuseppe Gattuso ◽  
Roberta Rizzo ◽  
Alessia Cosentino ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Pesticide Exposure ◽  
Methylation Status ◽  
Microrna Expression ◽  
Integrated Analysis ◽  
Epigenetic Alterations ◽  
Expression Levels ◽  
Prediction Tools

Environmental or occupational exposure to pesticides is considered one of the main risk factors for the development of various diseases. Behind the development of pesticide-associated pathologies, there are both genetic and epigenetic alterations, where these latter are mainly represented by the alteration in the expression levels of microRNAs and by the change in the methylation status of the DNA. At present, no studies have comprehensively evaluated the genetic and epigenetic alterations induced by pesticides; therefore, the aim of the present study was to identify modifications in gene miRNA expression and DNA methylation useful for the prediction of pesticide exposure. For this purpose, an integrated analysis of gene expression, microRNA expression, and DNA methylation datasets obtained from the GEO DataSets database was performed to identify putative genes, microRNAs, and DNA methylation hotspots associated with pesticide exposure and responsible for the development of different diseases. In addition, DIANA-miRPath, STRING, and GO Panther prediction tools were used to establish the functional role of the putative biomarkers identified. The results obtained demonstrated that pesticides can modulate the expression levels of different genes and induce different epigenetic alterations in the expression levels of miRNAs and in the modulation of DNA methylation status.

scholarly journals Gene-associated methylation status of ST14 as a predictor of survival and hormone receptor positivity in breast Cancer

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yang-Hong Dai ◽  
Ying-Fu Wang ◽  
Po-Chien Shen ◽  
Cheng-Hsiang Lo ◽  
Jen-Fu Yang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Methylation ◽  
Prognostic Value ◽  
Potential Role ◽  
Cox Model ◽  
Methylation Status ◽  
Specific Gene ◽  
Molecular Features ◽  
Gene Sets

Abstract Background Genomic profiles of specific gene sets have been established to guide personalized treatment and prognosis for patients with breast cancer (BC). However, epigenomic information has not yet been applied in a clinical setting. ST14 encodes matriptase, a proteinase that is widely expressed in BC with reported prognostic value. Methods In this present study, we evaluated the effect of ST14 DNA methylation (DNAm) on overall survival (OS) of patients with BC as a representative example to promote the use of the epigenome in clinical decisions. We analyzed publicly available genomic and epigenomic data from 1361 BC patients. Methylation was characterized by the β-value from CpG probes based on sequencing with the Illumina Human 450 K platform. Results A high mean DNAm (β > 0.6779) across 34 CpG probes for ST14, as the gene-associated methylation (GAM) pattern, was associated with a longer OS after adjusting age, stage, histology and molecular features in Cox model (p value < 0.001). A high GAM status was also associated with a higher XBP1 expression level and higher proportion of hormone-positive BC (p value < 0.001). Pathway analysis revealed that altered GAM was related to matrisome-associated pathway. Conclusions Here we show the potential role of ST14 DNAm in BC prognosis and warrant further study.

scholarly journals The Methylation Status of the Epigenome: Its Emerging Role in the Regulation of Tumor Angiogenesis and Tumor Growth, and Potential for Drug Targeting

Cancers ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 268 ◽  
Author(s):  
Luciano Pirola ◽  
Oskar Ciesielski ◽  
Aneta Balcerczyk
Keyword(s):  
Dna Methylation ◽  
Tumor Growth ◽  
Tumor Angiogenesis ◽  
Methylation Status ◽  
Angiogenesis Inhibitors ◽  
Post Translational Modifications ◽  
Methylation Patterns ◽  
Cancerous Cells ◽  
Transcriptional State

Approximately 50 years ago, Judah Folkman raised the concept of inhibiting tumor angiogenesis for treating solid tumors. The development of anti-angiogenic drugs would decrease or even arrest tumor growth by restricting the delivery of oxygen and nutrient supplies, while at the same time display minimal toxic side effects to healthy tissues. Bevacizumab (Avastin)—a humanized monoclonal anti VEGF-A antibody—is now used as anti-angiogenic drug in several forms of cancers, yet with variable results. Recent years brought significant progresses in our understanding of the role of chromatin remodeling and epigenetic mechanisms in the regulation of angiogenesis and tumorigenesis. Many inhibitors of DNA methylation as well as of histone methylation, have been successfully tested in preclinical studies and some are currently undergoing evaluation in phase I, II or III clinical trials, either as cytostatic molecules—reducing the proliferation of cancerous cells—or as tumor angiogenesis inhibitors. In this review, we will focus on the methylation status of the vascular epigenome, based on the genomic DNA methylation patterns with DNA methylation being mainly transcriptionally repressive, and lysine/arginine histone post-translational modifications which either promote or repress the chromatin transcriptional state. Finally, we discuss the potential use of “epidrugs” in efficient control of tumor growth and tumor angiogenesis.

scholarly journals DNA Methylation in Osteoarthritis: Current Status and Therapeutic Implications

2018 ◽  
Vol 12 (1) ◽  
pp. 37-49 ◽  
Author(s):  
Antonio Miranda-Duarte
Keyword(s):  
Dna Methylation ◽  
Genetic Factors ◽  
Methylation Status ◽  
Gene Repression ◽  
Current Status ◽  
Multifactorial Disease ◽  
Review Of The Literature ◽  
Therapeutic Implications ◽  
Genome Wide

Background: Primary Osteoarthritis (OA) is a multifactorial disease in which genetic factors are strongly associated with its development; however, recently it has been observed that epigenetic modifications are also involved in the pathogenesis of OA. DNA methylation is related to gene silencing, and several studies have investigated its role in the loci of different pathways or molecules associated to OA. Objective: This review is focused on the current status of DNA methylation studies related to OA pathogenesis. Method: A review of the literature was conducted on searching in PUBMED for original papers on DNA methylation in OA. Conclusion: The DNA methylation research of loci related to OA pathogenesis has shown a correlation between methylation and gene repression; however, there are some exceptions to this rule. Recently, the development of genome-wide methylation and genome-wide hydroxymethylation profiles has demonstrated that several genes previously associated with OA can have changes in their methylation status, favoring the development of the disease, and these have even shown the role of other epigenetic markers.

Transcription Factor CTCF Inhibits Effects of 5-Azacitidine in MDS/AML Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3848-3848
Author(s):  
Martina Kapalova ◽  
Pavel Burda ◽  
Karin Vargova ◽  
Filipp Savvulidi ◽  
Tomas Zikmund ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Transcription Factor ◽  
High Risk ◽  
Binding Sites ◽  
Conflicts Of Interest ◽  
Methylation Status ◽  
Significant Proportion ◽  
Dna Demethylation ◽  
Ctcf Binding

Abstract Abstract 3848 Introduction: 5-azacitidine (AZA) represents very promising albeit not fully efficient therapy for int-2 and high risk MDS patients. Molecules that interfere with AZA therapy are not known. In significant proportion of MDS patients, PU.1 gene is methylated at −17-kb-located upstream regulatory element (URE) where several key transcription factors regulate PU.1 expression. PU.1 represents major factor that controls normal myeloid differentiation. Methylated URE in MDS progenitors can be efficiently demethylated by AZA leading to restoration of cell differentiation capacity (Curik et al 2012). PU.1 gene contains several binding sites for transcription factor CTCF. CTCF represents very important modulator of gene expression, whose binding to DNA can be prevented by DNA methylation. We herein asked if CTCF regulates PU.1 and if so, whether its association with PU.1 gene coincides with DNA methylation status of MDS blasts. Methods: Human high risk MDS patient CD34+ progenitors and MDS-derived erytroleukaemia OCI-M2 and murine erythroleukaemia cell (MEL) lines were studied by RT-PCR, immunoblotting, and chromatin immunoprecipitation (ChIP) assays. Manipulation of gene expression was done by transfection of cDNA or siRNA. Results: We herein show that CTCF binding sites at PU.1 gene similarly to URE are severely methylated in CD34+ progenitors from high risk MDS patients and MDS-derived erytroleukaemia cell line, and as expected, AZA induced their rapid demethylation. Methylated CTCF binding sites are not occupied by CTCF. However upon AZA-mediated demethylation, CTCF is recruited to the binding sites at PU.1 gene as determined by ChIP. Our other data provided evidence that CTCF interacts with the ISWI ATPse SNF2H (SMARCA5). Indeed, the recruitment of CTCF at PU.1 gene in MDS/AML cells was coincident with recruitment of its interacting partner SMARCA5. In addition, SMARCA5 facilitates CTCF binding to the DNA as demonstrated at ICR locus (near H19 and Igf2 genes) upon siRNA-mediated downregulation of SMARCA5. To understand role of CTCF-SMARCA5 recruitment to the PU.1 gene and its effects on PU.1 expression we upregulated CTCF expression by transfecting an expression plasmid encoding CTCF cDNA and observed that upon increasing CTCF levels the PU.1 protein level was downregulated. Conversely, downregulation of SMARCA5 by siRNA caused upregulation of PU.1 levels. These data indicated that PU.1 is negatively regulated by CTCF and SMARCA5. Furthermore, inhibitory effects of CTCF and SMARCA5 on PU.1 expression were also demonstrated in presence of AZA in MDS cells following DNA demethylation of PU.1 gene. Conclusion: Our results indicate that CTCF and SMARCA5 are cooperating inhibitory factors to downregulate PU.1 and that AZA-mediated demethylation facilitates the CTCF-SMARCA5 binding to PU.1 gene in MDS patients. CTCF and SMARCA5 are novel factors that interfere with positive prodifferentiation effects of AZA. (Grant support: P305/12/1033, UNCE 204021, PRVOUK-P24/LF1/3, SVV-2012–264507, P301/12/P380, GAUK 251070 45410 and 251135 82210). Disclosures: No relevant conflicts of interest to declare.

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