Uncovering the Epigenetic Pathomechanism in 13q14.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 487-487
Author(s):  
Daniel Mertens ◽  
Melanie Ruppel ◽  
Angela Philippen ◽  
Verena Fleig ◽  
Bianca Brakel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Tumor Suppressor ◽  
Candidate Genes ◽  
Critical Region ◽  
Replication Timing ◽  
Complete Loss ◽  
Gene Copy ◽  
Active Copy ◽  
Suppressor Mechanism

Abstract INTRODUCTION: Deletions in chromosomal band 13q14.3 distal to RB1 occur in a variety of human neoplasms like B-cell chronic lymphocytic leukaemia (CLL), indicating a tumor suppressor mechanism in this region. Intriguingly, several characteristics of the region of interest point to an epigenetic pathomechanism: candidate genes lack point mutations, yet these genes are downregulated in tumors, the presence of large non-coding RNA genes in 13q14.3 is reminiscent of imprinted regions where only one gene copy is active. The data we show here led us to propose a novel oncogenic mechanism where already in healthy tissue only one gene copy is active while one gene copy is randomly chosen for silencing. Loss of the single active copy is then sufficient for complete loss of gene function in tumor cells. Currently we are trying to identify the (epi-)genetic element that controls the whole locus. AIM: Identification of the epigenetic regulatory mechanism localized in 13q14.3. METHODS and RESULTS: We performed FISH analyses of hematopoietic and non-hematopoietic cell lines to assess replication timing and chromatin packaging of the critical region. In line with an imprinting mechanism, we find that the two copies of the critical region replicate asynchronously and/or show delayed chromatid segregation, suggesting differential chromatin packaging of the two copies of 13q14.3. Next, we found by sequencing of SNPs that 13q14.3 candidate genes are expressed from one copy only in healthy probands. However, expression originated from either the maternal or paternal copy, excluding an imprinting mechanism. We could also show a functional interconnection of DNA methylation and gene expression, as demethylating agents and histone hyperacetylation induced biallelic expression. However, replication timing was not affected. Currently we are employing array- and capillary electrophoresis-based analysis of DNA-methylation (aPRIMES and bioCOBRA) and chromatin-immunoprecipitation on arrayed CpG-libraries (chIP on chip) with antibodies specific for histone modifications in order to identify the epigenetic element regulating the critical region. CONCLUSIONS: We propose that differential replication timing represents an early epigenetic mark that distinguishes the two copies of 13q14.3, resulting in differential chromatin packaging and monoallelic expression. This has profound effects for the tumor suppressor mechanism localized in 13q14.3: Deletion of the single active copy of the region at 13q14.3, which is detected in more than 50% of CLL tumors, will suffice for complete loss of tumor suppressor function, as the remaining gene copies are epigentically silenced. In addition, we are currently identifying the locus control region that orchestrates gene expression in the critical region. Thus, we provide a model for the pathomechanism of 13q14.3 in CLL by the interaction of genetic lesions and epigenetic silencing.

Epimutation of the Tumor Suppressor Mechanism in 13q14.3 Involves Monoallelic Expression, Non-Coding RNA Genes and Deregulation of NFkB Signalling

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 783-783
Author(s):  
Daniel Mertens ◽  
Angela Philippen ◽  
Nupur Bhattacharya ◽  
Cordula Tschuch ◽  
Melanie Ruppel ◽  
...  
Keyword(s):  
Dna Methylation ◽  
B Cells ◽  
Tumor Suppressor ◽  
Candidate Genes ◽  
Target Genes ◽  
Gene Copy ◽  
Loss Of Function ◽  
Non Coding Rna ◽  
Suppressor Mechanism ◽  
Rna Genes

Abstract Deletions in chromosomal band 13q14.3 occur in a variety of human neoplasms like chronic lymphocytic leukaemia (CLL), indicating a tumor suppressor mechanism (TSM) in this region. Intriguingly, several characteristics of the region of interest point to an epigenetic pathomechanism: candidate protein-coding genes and non-coding RNA genes including miR15a and miR16-1 lack point mutations in the majority of patients, yet these genes are significantly downregulated in almost all CLL patients the presence of large non-coding RNA genes in 13q14.3 is reminiscent of imprinted regions where only one gene copy is active. We have recently shown that already in healthy tissue only one gene copy of 13q14.3 is active while one gene copy is randomly chosen for silencing. Thus, loss of the single active copy is sufficient for complete loss of gene function in tumor cells. In order to elucidate the epigenetic regulatory mechanism, we analysed DNA- and Histone-methylation of all CpG islands in the region in non-malignant B-cells and CLL cells. Using aPRIMES and ChIP-qPCR as screening tools, BioCOBRA as a quantitative high-throughput method and bisulfite sequencing for validation, we could identify two candidate regulatory elements with abnormal chromatin in CLL patients (n=80, median 57% DNA-methylation, range 0–100%) as compared to healthy probands (n=20, median 88% DNA-methylation, range 74–100%, p<0.003). Interestingly, this epimutation can be found in all cytogenetic subgroups of CLL patients and is independent of IgV(H) mutation status, making it a prime candidate for an underlying epigenetic defect in CLL. Pilot studies suggest that this epimutation regulates gene expression of the critical region via large non-coding RNA genes. In order to find out how loss of function of the 13q14 genes could result in the pathophenotype of CLL cells, we overexpressed and knocked-down RFP2, C13ORF1, KPNA3 and the largen non-coding RNA gene Dleu2 in two different cell lines and used custom oligonucleotide microarrays and timecourse experiments (n=68 array hybridizations) to identify genes that were subsequently deregulated and thus potential target genes. Less than 1% of genes represented on the arrays were significantly deregulated (median 211/25100 genes, range 44–370), showing the high specificity of the procedure. Using ingenuity pathway analyses, we found that modulation of the expression of 13q14.3 candidate genes deregulates most significantly NFkB target genes and components of the NFkB pathway itself. For a detailed validation analysis we focused on RFP2 and could show that it robustly and quickly induces NFkB activity in fibroblasts (HeLa), kidney cells (HEK-293) and CLL cell lines (Granta-591). However, analyses by oligonucleotide ELISA, Western Blot and EMSA-Band-Shift assays suggest that activation of NFkB occurs not via modulation of components of the canonical or non-canonical NFkB signalling pathways. Therefore, we propose a model for the TSM in 13q14.3 where in healthy B-cells, only one gene copy is active while the second is epigenetically silenced expression of candidate genes is deregulated in CLL cells by epimutation that is present in all cytogenetic subgroups and that this loss of function of 13q14 candidate genes results in deregulation of the NFkB signalling pathway which will change the activation level of CLL cells and their sensitivity to induction of apoptosis.

scholarly journals CG gene body DNA methylation changes and evolution of duplicated genes in cassava

2015 ◽  
Vol 112 (44) ◽  
pp. 13729-13734 ◽  
Author(s):  
Haifeng Wang ◽  
Getu Beyene ◽  
Jixian Zhai ◽  
Suhua Feng ◽  
Noah Fahlgren ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Expression Patterns ◽  
Regulation Of Gene Expression ◽  
Gene Copy ◽  
Gene Body ◽  
Duplicated Genes ◽  
Gene Body Methylation ◽  
Substantial Impact ◽  
Tropical Regions

DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for cassava (Manihot esculenta, cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plant Arabidopsis thaliana. As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components of Arabidopsis DNA methylation pathways was identified. Methylation of LTR transposons (GYPSY and COPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from whole-genome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits.

Detection and Analysis of RNAs Expression Profile for Methylated Candidate Tumor Suppressor Genes in Nasopharyngeal Carcinoma

2019 ◽  
Vol 19 (6) ◽  
pp. 772-782
Author(s):  
Shuang Zhao ◽  
Ye Zhang ◽  
Xujun Liang ◽  
Maoyu Li ◽  
Fang Peng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Nasopharyngeal Carcinoma ◽  
Tumor Suppressor ◽  
Promoter Methylation ◽  
Tumor Suppressor Genes ◽  
Enrichment Analysis ◽  
Pathway Enrichment Analysis ◽  
Suppressor Genes ◽  
Multiple Tumor

Background:DNA methylation, which acts as an expression regulator for multiple Tumor Suppressor Genes (TSGs), is believed to play an important role in Nasopharyngeal Carcinoma (NPC) development.Methods:We compared the effects of 5-aza-2-deoxycytidine (decitabine, DAC) on gene expression using RNA sequencing in NPC cells.Results:We analyzed Differentially Expressed Genes (DEGs) in NPC cells using DAC demethylation treatment and found that 2182 genes were significantly upregulated (≥ 2-fold change), suggesting that they may play a key role in cell growth, proliferation, development, and death. For data analysis, we used the Gene Ontology database and pathway enrichment analysis of the DEGs to discover differential patterns of DNA methylation associated with changes in gene expression. Furthermore, we evaluated 74 methylated candidate TSGs from the DEGs in NPC cells and summarized these genes in several important signaling pathways frequently disrupted by promoter methylation in NPC tumorigenesis.Conclusion:Our study analyzes the DEGs and identifies a set of genes whose promoter methylation in NPC cells is reversed by DAC. These genes are potential substrates of DNMT inhibitors and may serve as tumor suppressors in NPC cells.

ABC and GCB DLBCLs Display Unique Biologically Distinct and Clinically Relevant Epigenetic Signatures.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 619-619
Author(s):  
Rita Shaknovich ◽  
Huimin Geng ◽  
Nathalie Johnson ◽  
Leandro Cerchietti ◽  
Maria E Figueroa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Tumor Suppressor ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Methylation Level ◽  
Inverse Correlation ◽  
Gene Set Enrichment Analysis ◽  
Epigenetic Signatures

Abstract Abstract 619 Diffuse Large B-cell Lymphoma (DLBCL) is a complex biological entity with heterogeneous genetic, biological and clinical features. Gene expression profiling studies have attempted to resolve some of this heterogeneity. For example, DLBCL patients harboring gene signatures associated with expression of germinal center B-cell genes (GCB) or activated B-cell genes (ABC) vary in their response rate to standard chemo-immunotherapy regimens. Since epigenetic gene regulation can play a fundamental role in determining the phenotype of normal and malignant tissues we asked whether ABC and GCB DLBCLs also display unique epigenetic signatures that might be clinically useful and further explain the biology of these tumors. For this purpose we examined the DNA methylation level of 50,000 cytosine residues distributed among 14,000 gene promoters in a cohort of 159 patients with DLBCL, all of whom were uniformly treated with R-CHOP, using the HELP assay and high-density oligonucleotide microarrays. For a subset of these patients (n=69), we also performed Affymetrix gene expression profiling. First, a Bayesian predictor of ABC/GCB subtypes was trained from a published expression profiling study of 203 DLBCL patients. The predictor was then applied to our cohort of 69 patients. At a probability cutoff of 0.9, 20 patients were classified as ABC, 40 were classified as GCB and 9 could not be classified (i.e. “type III DLBCL”). As expected from published studies, the differences in progression free survival (PFS) and overall survival (OS) of these ABC vs. GCB patients thus determined was highly significant, with p=0.0026 (log rank) and p=0.043 (log rank) respectively, with a much worse prognosis for ABC patients compared to GCB ones. We then asked whether the ABC and GCB subtypes could be predicted from the DNA methylation profiles of the same 69 patients. By performing a t-test we found that 239 genes were differentially methylated between ABC and GCB (p<0.0001) and also displayed >30% difference in methylation level. This DNA methylation signature was incorporated into a new Bayesian predictor, which we showed to predict ABC and GCB DLBCL subtypes from DNA methylation profiles with a 91% accuracy. Using a cross-validation procedure, we estimated that the classification performance on independent cases to be ∼87%. The predicted ABC and GCB cases retained the clinical predictive power of the gene expression profile when applied to the remaining 90 patients that did not have gene expression profiles, confirming its clinical relevance (difference in PFS p=0.0148, log rank). Gene set enrichment analysis showed that the ABC DNA methylation signature was enriched in genes involved in antigen dependent B and T-cell responses and in TNF inflammatory responses (p<1.01E-4 and <6.01E-4 respectively). A computational analysis of promoter DNA sequences of the genes involved in this signature revealed over-representation of binding sites for transcription factors including MYB, STAT5A, MAZ, and JUN and Sp1; many of these factors have a known role in B cell development and function. The role of Sp1 in these tumors is under further examination. Among the 239 genes that were differentially methylated and the 411 genes that were differentially expressed between ABC and GCB there was an overlap of 16 genes (greater than expected by chance Fisher Exact p=0.005). A predictor based on the methylation profiles of these 16 genes was on its own 92% accurate in identifying ABC vs. GCB cases among our cohort of DLBCLs. Although there was a general trend for inverse correlation in expression between the 239 differentially methylated genes, these 16 overlapping genes displayed marked and extreme inverse correlation. This was validated by single locus quantitative methylation sequencing (MassArray) and QPCR. The 16 genes included genes known to play critical roles in B-cell differentiation, proliferation and metabolism but not previously implicated in DLBCL. Gain and loss of function assays of a subset of these genes in ABC and GCB DLBCL cell lines show that they have tumor suppressor functions in DLBCL. Our results demonstrate for the first time that ABC and GCB DLBCLs are epigenetically distinct diseases; they identify new biological differences and candidate tumor suppressor genes between these tumors and demonstrate that a DNA methylation classifier can be used to distinguish GCB and ABC DLBCL subtypes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of factors regulating the gene expression of alcohol dehydrogenases in hepatocellular carcinoma

2021 ◽  
Author(s):  
Jinghe Xie ◽  
Yaqi Qiu ◽  
Shuai Zhang ◽  
Keqing Ma ◽  
Yimeng Ou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Dna Methylation ◽  
Copy Number ◽  
Gene Copy Number ◽  
Copy Number Variations ◽  
The Cancer Genome Atlas ◽  
Gene Copy ◽  
Excessive Alcohol Consumption ◽  
Alcohol Dehydrogenases

Abstract Background Excessive alcohol consumption has been documented to increase the risk of liver hepatocellular carcinoma (HCC) development. Accordingly, a broad interest pointed to alcohol dehydrogenases (ADHs), which display essential roles in alcohol metabolism. Despite the relevance of ADHs expression and the prognosis of HCC has been estimated, so far, limited research concerning the factors that are responsible for the regulation of ADHs expression has been reported. Methods In this study, using The Cancer Genome Atlas (TCGA) and RegNetwork database, we predicted potential factors consisting of DNA methylation, gene copy number variations, transcription factors (TFs) and microRNAs (miRNAs) that might impact ADHs gene expression in HCC. Results We found that DNA methylation induced the down-regulated expression of ADH1B. Of note, our results implicated that gene copy number variation might not have effects on ADHs expression. Regarding TFs, we speculated that NFYA modulated ADH1C, E2F1 and TFAP2A regulated ADH6 expression based on their expression and prognostic value. Moreover, miR-185 and miR-561 might elicit the repression of ADH4, and miR-105 might impair ADH6 expression. Conclusion This study revealed that multiple factors, including DNA methylation, TFs and microRNAs, affect the expression of ADH family members, which provided new insights into discovering promising HCC-suppressive targets.

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