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PubMed2026

Screening and identification of the ncRNA-mRNA regulatory network associated with DNA methylation in goose embryonic myoblasts.

BACKGROUND: Local goose breeds Shitou and Wuzong exhibit distinct growth rates, implying divergent embryonic muscle development. This study used embryonic myoblasts from the Magang goose, an established model with superior growth traits, to explore the underlying common regulatory mechanisms. Extending our previous findings that 5-AZA (DNA methylation inhibitor) and BC339 (DNA hydroxylation inhibitor) oppositely affect myoblast proliferation and differentiation, we performed whole-transcriptome sequencing on inhibitor-treated goose embryonic myoblasts. This aimed to identify DNA methylation-mediated ncRNA-mRNA networks governing myoblast fate, with key interactions being functionally validated. RESULT: 5-AZA significantly promotes cell proliferation and differentiation by inhibiting DNA methyltransferase activity and reducing DNA methylation levels, whereas BC339 significantly suppresses cell proliferation and differentiation by inhibiting demethylation and increasing DNA methylation levels. Specifically, we identified 6,309 mRNAs, 579 lncRNAs, 194 miRNAs, and 825 circRNAs that were differentially expressed in response to 5-AZA and BC339 treatment. Based on GO and KEGG enrichment analyses, differentially expressed genes related to muscle development were selected to construct a ceRNA network. This network comprises 5 differentially expressed lncRNAs (DELs: MSTRG.17572.1, XR_001211738.1, MSTRG.1886.1, XR_001212555.1, MSTRG.8995.2), 2 differentially expressed circRNAs (DECs: novel_circ_029953, novel_circ_017636), 11 differentially expressed miRNAs (DEMs: miR-383-x, miR-10174-y, miR-191-x, miR-24-x, miR-9619-y, novel-m0303-5p, novel-m0105-3p, miR-204-x, miR-211-z, novel-m0075, miR-26-y), 5 differentially expressed genes (DEGs: KIF3A, CCND1, PPM1A, Table 2, TGFBR1), forming a total of 24 interactions. This study identified miR-9619-y as a critical negative regulator of goose embryonic myoblast development through targeted inhibition of CCND1. Dual-luciferase reporter assays confirmed the direct binding of miR-9619-y to the 3'-untranslated region of CCND1. Functional experiments demonstrated that overexpression of miR-9619-y significantly reduced the EdU-positive cell ratio and myotube area percentage, accompanied by cell cycle arrest at the G0/G1 phase. Conversely, inhibition of miR-9619-y promoted myoblast proliferation and differentiation while decreasing the proportion of cells in G0/G1 phase. During the proliferation stage, miR-9619-y overexpression significantly suppressed CCND1 expression at both mRNA and protein levels, down-regulated MyoD expression, and reduced Myf5 mRNA abundance; whereas miR-9619-y inhibition up-regulated these genes and their corresponding proteins. During the differentiation stage, overexpression of miR-9619-y similarly decreased the mRNA levels of CCND1, Myh1, and MyoG, as well as the protein levels of MyHC and CCND1, with inhibition producing the opposite effects. CONCLUSION: In this study, we predicted a ceRNA network based on bioinformatics analysis governing goose embryonic myoblast development, identifying key molecular components including mRNAs, miRNAs, lncRNAs, and circRNAs, along with 24 regulatory axes. Functional experiments further demonstrated that miR-9619-y arrests cell cycle progression and negatively regulates the proliferation and differentiation of goose embryonic myoblasts, as evidenced by its impact on both the mRNA and protein expression of key myogenic factors through targeted inhibition of CCND1. These findings, together with the bioinformatically predicted ceRNA network, suggest potential complex post-transcriptional regulatory mechanisms underlying myogenesis in geese and offer candidate molecular targets for genetic improvement of meat production performance in waterfowl breeding programs.

Animals↗
PubMed2006

Using immunofluorescence to observe methylation changes in mammalian preimplantation embryos.

DNA methylation is an important epigenetic mark involved in gene silencing, X chromosome and transposon inactivation, genomic imprinting, and chromosome stability. Recently, it has been increasingly recognized that DNA methylation plays an essential regulatory function in mammalian development and cancer biology, serving to repress nontranscribed genes stably in differentiated adult somatic cells. DNA methylation is subject to reprogramming during development, involving both demethylation (active and passive) and de novo methylation phases. Recent data show deregulation of genome-wide DNA methylation levels in embryos generated by somatic nuclear transfer, suggesting this epigenetic mark and its regulation are essential to ensure viable embryos. Immunofluorescence using antibodies against 5-methylcytosine is an invaluable method for the large-scale screening of genome-wide methylation. This method has a high degree of reproducibility, essential for the analysis of small numbers of valuable samples, providing information on methylation profiles of individual cells and embryos.

Animals↗
PubMed2005

The chemokine receptor CXCR4 is regulated by DNA methylation in pancreatic cancer.

Despite the biological and clinical importance of the interaction between the chemokine receptor CXCR4 and its ligand CXCL12 (SDF-1alpha) in human cancers, little is known about transcriptional regulation of the CXCR4 gene. Although aberrant hypermethylation in cancer has been described typically in genes with tumor-suppressor properties, this epigenetic alteration has also been observed to affect potential cancer-promoting genes. We now demonstrate that DNA methylation influences CXCR4 expression in human pancreatic cancer. Gene expression profiling and reverse transcription-PCR identified a significant proportion of pancreatic cancer cell lines that displayed little or no CXCR4 mRNA expression. Using methylation-specific PCR, combined bisulfite restriction analysis, and bisulfite sequencing, we found the 5' CpG islands of the CXCR4 gene to be unmethylated in normal pancreas, whereas promoter hypermethylation was detected in 45% (9 of 20) of pancreatic cancer cell lines and in 46% (46 of 100) of primary pancreatic adenocarcinomas. There was a significant inverse correlation between methylation and mRNA expression level of CXCR4 (P=0.008) in a large panel of pancreatic cancer cell lines. Constitutive as well as inducible expression of CXCR4 could be restored in methylated cell lines pharmacologically using epigenetic modifying drugs. These findings demonstrate the first evidence for epigenetic regulation of CXCR4 in human cancers, providing new insights into the role of CXCR4/CXCL12 interactions in tumor progression.

Chemokine CXCL12↗
PubMed2025

Methylome profiling of cell-free DNA during the early life course in (un)complicated pregnancies using MeD-seq: Protocol for a cohort study embedded in the prospective Rotterdam periconception cohort.

INTRODUCTION: Placental DNA methylation differences have been associated with timing in gestation and pregnancy complications. Maternal cell-free DNA (cfDNA) partly originates from the placenta and could enable the minimally invasive study of placental DNA methylation dynamics. We will for the first time longitudinally investigate cfDNA methylation during pregnancy by using Methylated DNA Sequencing (MeD-seq), which is compatible with low cfDNA levels and has an extensive genome-wide coverage. We aim to investigate DNA methylation in placental tissues and cfDNA during different trimesters in uncomplicated pregnancies, and in pregnancies with placental-related complications, including preeclampsia and fetal growth restriction. Identified gestational-age and disease-specific differentially methylated regions (DMRs) could lead to numerous applications including biomarker development. METHODS AND ANALYSIS: Our study design involves three sub-studies. Sub-study 1 is a single-centre prospective, observational subcohort embedded within the Rotterdam Periconception cohort (Predict study). We will longitudinally collect maternal plasma in each trimester and during delivery, and sample postpartum placentas (n = 300). In sub-study 2, we will prospectively collect first and second trimester placental tissues (n = 10 per trimester). In sub-study 3 we will retrospectively collect plasma after non-invasive prenatal testing (NIPT) in an independent validation case-control cohort (n = 30-60). A methylation-dependent restriction enzyme (LpnPI) will be used to generate DNA fragments followed by sequencing on the Illumina NextSeq2000 platform. DMRs will be identified in placental tissues and cell types, and in cfDNA related to gestational-age or placental-related complications. (Paired) placental methylation profiles will be correlated to DMRs in cfDNA to aid tissue-of-origin analysis. We will establish a methylation score to predict associated diseases. DISCUSSION: This study will provide insights in placental DNA methylation dynamics in health and disease, and could lead to clinical relevant biomarkers.

Humans↗
PubMed1994

Hypomethylation of the rat aryl sulfotransferase IV gene and amplification of a DNA sequence during multistage 2-acetylaminofluorene hepatocarcinogenesis.

Rat hepatic aryl sulfotransferase IV (AST IV), which catalyses sulfuric acid esterification of N-hydroxy-2-acetylaminofluorene to its ultimate carcinogenic form, is differentially expressed during multistep 2-acetylaminofluorene (AAF) hepatocarcinogenesis. Two molecular mechanisms associated with this effect involve modulation of mRNA translational capacity at the early stages, and gene transcription at the late stages of the carcinogenic process. To characterize further the molecular mechanisms that may be involved in the transient regulation of the enzyme expression, an AST IV cDNA was used to assess the change in methylation profile and restriction fragment length polymorphism (RFLP) in the gene domain of genomic DNA derived from rats at different stages of carcinogenesis. The onset of hypomethylation of the AST IV gene domain and amplification of a 5.3-kb DNA sequence was found to correlate with the stage in AAF hepatocarcinogenesis, where rats begin to exhibit irreversible loss in hepatic enzyme expression and the liver becomes committed to hepatoma formation. This represents the first observation of both altered methylation status of AST IV gene domain and amplification of a DNA sequence whose expression may play a role in the genesis and/or progression of neoplastic transformation of initiated cells during AAF hepatocarcinogenesis.

2-Acetylaminofluorene↗
PubMed2005

Integrative genomic analysis identifies NDRG2 as a candidate tumor suppressor gene frequently inactivated in clinically aggressive meningioma.

Although meningiomas are common central nervous system tumors, little is known about the genetic events responsible for malignant progression. In this study, we employed gene expression profiling to identify transcripts whose expression was lost in anaplastic (WHO grade III) versus benign (WHO grade I) meningioma. Approximately 40% of genes down-regulated in anaplastic meningioma were localized to chromosomes 1p and 14q. One specific gene located at 14q11.2, NDRG2, was consistently down-regulated in grade III meningioma, a finding which we validated at both the transcript and protein levels in independent sets of clinically and pathologically diverse meningiomas. Loss of NDRG2 expression was also seen in a subset of lower-grade meningiomas, including atypical meningiomas (WHO grade II) with clinically aggressive behavior. Furthermore, we found that the loss of NDRG2 expression was significantly associated with hypermethylation of the NDRG2 promoter. Collectively, these data identify NDRG2 as the first specific candidate tumor suppressor gene on chromosome 14q that is inactivated during meningioma progression. In addition, these findings highlight the utility of combining genomic, epigenetic, and expression data to identify clinically significant tumor biomarkers, and suggest that NDRG2 expression will be a useful and functionally relevant biomarker to predict aggressive behavior in patients with meningioma.

Base Sequence↗
PubMed2005

Two-part permutation tests for DNA methylation and microarray data.

BACKGROUND: One important application of microarray experiments is to identify differentially expressed genes. Often, small and negative expression levels were clipped-off to be equal to an arbitrarily chosen cutoff value before a statistical test is carried out. Then, there are two types of data: truncated values and original observations. The truncated values are not just another point on the continuum of possible values and, therefore, it is appropriate to combine two statistical tests in a two-part model rather than using standard statistical methods. A similar situation occurs when DNA methylation data are investigated. In that case, there are null values (undetectable methylation) and observed positive values. For these data, we propose a two-part permutation test. RESULTS: The proposed permutation test leads to smaller p-values in comparison to the original two-part test. We found this for both DNA methylation data and microarray data. With a simulation study we confirmed this result and could show that the two-part permutation test is, on average, more powerful. The new test also reduces, without any loss of power, to a standard test when there are no null or truncated values. CONCLUSION: The two-part permutation test can be used in routine analyses since it reduces to a standard test when there are positive values only. Further advantages of the new test are that it opens the possibility to use other test statistics to construct the two-part test and that it avoids the use of any asymptotic distribution. The latter advantage is particularly important for the analysis of microarrays since sample sizes are usually small.

Algorithms↗
PubMed2025

A Protocol for Detecting DNA Methylation Changes at CpG Sites of Stemness-Related Genes in Aging Stem Cells.

Aging adversely affects the self-renewal and differentiation capabilities of stem cells, which impairs tissue regeneration as well as the homeostasis. Epigenetic mechanisms, specifically DNA methylation, play a key role in the maintenance of pluripotency in stem cells and regulation of pluripotency-related gene expression. Age-related modifications in methylation patterns could influence the expression of genes critical for stem cell potency maintenance, including transcription factors Nanog and Sox2. The following chapter describes a step-by-step bisulfite sequencing protocol for detection of methylation changes in the aging stem cells and provides valuable insights into the stem cells epigenetic profile. Further, the methodology describes the steps of genomic DNA extraction, bisulfite conversion, real-time PCR amplification, and sequencing for an in-depth view of the epigenetic profile derived from aging stem cells.

DNA Methylation↗
PubMed2006

Differential and epigenetic gene expression profiling identifies frequent disruption of the RELN pathway in pancreatic cancers.

BACKGROUND & AIMS: Recently described genome-wide approaches robustly detect many candidate genes that are regulated by DNA methylation, but many of these genes do not represent important targets for functional inactivation. Here we used a microarray-based strategy to identify biologically relevant genes associated with epigenetic silencing in pancreatic cancer. METHODS: We compared information from differential gene expression analysis with the transcriptional responses to epigenetic modifiers. RESULTS: Using this approach, we identified 7 novel targets for aberrant methylation in pancreatic cancer. One of the genes identified, RELN (Reelin), a key regulator of neuronal migration, is frequently silenced in pancreatic cancers, as are several of its downstream mediators. Importantly, small interfering RNA-mediated knockdown of RELN in pancreatic cancer cells that retain RELN expression resulted in greatly enhanced cell motility, invasiveness, and colony-forming ability. Increased cell motility was also induced by knockdown of downstream components of the RELN pathway, including ApoER2, VLDLR, and DAB1. Treatment of pancreatic cancer cells with histone deacetylase inhibitors, valproic acid and suberoylanilide hydroxamic acid, restored the expression of RELN and DAB1 and markedly inhibited their migration. CONCLUSIONS: The high prevalence of the silencing of RELN pathway components and its reversal by histone deacetylase inhibitors suggest the importance of this pathway as a diagnostic and therapeutic target for pancreatic cancer.

Cell Adhesion Molecules, Neuronal↗
PubMed2005

p16(INK4a) promoter methylation and protein expression in breast fibroadenoma and carcinoma.

The potential role of p16(INK4a) methylation in breast cancer is controversial whereas there are no data on fibroadenoma. To assess if inactivation of p16(INK4a) by promoter hypermethylation occurs in this hyperproliferative benign breast lesion or, on the contrary, it is strictly related to the carcinogenic process, we have tested the different histological components of 15 cases of fibroadenoma and the intraductal and infiltrating components of 15 cases of carcinoma and their adjacent non-tumoral epithelium. All samples were obtained by laser-assisted microdissection. The relationship between promoter methylation status, immunohistochemical protein expression and ki67 proliferative activity was evaluated for each lesion. Our data demonstrate that hypermethylation of p16(INK4a) promoter is a common event occurring at similar frequency in all the different histological areas of the benign and malignant breast lesions taken into exam. Conversely, protein p16 expression, although heterogeneously distributed within the section, is considerably higher in breast carcinoma as compared to fibroadenoma in both tumoral and non-tumoral epithelia and stroma. The protein localization was almost exclusively nuclear in fibroadenoma and non-tumoral epithelia whereas, in carcinoma, the staining was both nuclear and cytoplasmic or cytoplasmic alone. Furthermore, in a subset of fibroadenoma with higher proliferative activity, p16 protein expression was substantially decreased as compared to those showing lower proliferation. We did not observe this association in carcinomas. Our data demonstrate that the hypermethylation of the p16(INK4a) promoter is not specifically associated with malignancy and that, on the contrary, the overexpression of p16 and its cytoplasmic sequestration is a feature of breast carcinoma.

Breast Neoplasms↗
PubMed2006

Expression of multiple epigenetically regulated cancer/germline genes in nonsmall cell lung cancer.

Cancer/germline (CG) antigens represent promising targets for widely applicable mono- and multiantigen cancer vaccines for nonsmall cell lung cancer (NSCLC). Since little is known about their composite expression in this tumor type, we analyzed 7 CG genes (MAGE-A3, NY-ESO-1, LAGE-1, BRDT, HOM-TES-85, TPX-1 and LDHC) in 102 human NSCLC specimens. About 81% of NSCLC express at least 1 and half of the specimen at least 2 CG genes. Activation of most of these genes occurs more frequently in squamous cell cancer than in adenocarcinomas. Even though we found all genes but one to be regulated by genomic methylation, not all of them are co-expressed. In particular, combining CG genes not localized on the X-chromosome may provide effective treatment for an extended number of patients.

Antigens, Neoplasm↗
PubMed2005

Molecular biomarkers for the study of childhood leukemia.

Various specific chromosome rearrangements, including t(8;21), t(15;17), and inv(16), are found in acute myeloid leukemia (AML) and in childhood acute lymphocytic leukemia (ALL), t(12;21) and t(1;19) are common. We sequenced the translocation breakpoints of 56 patients with childhood ALL or AML harboring t(12;21), t(8;21), t(15;17), inv(16), and t(1;19), and demonstrated, with the notable exception of t(1;19), that these rearrangements are commonly detected in the neonatal blood spots (Guthrie cards) of the cases. These findings show that most childhood leukemias begin before birth and that maternal and perinatal exposures such as chemical and infectious agents are likely to be critical. Indeed, we have reported that exposure to indoor pesticides during pregnancy and the first year of life raises leukemia risk, but that later exposures do not. We have also examined aberrant gene methylation in different cytogenetic subgroups and have found striking differences between them, suggesting that epigenetic events are also important in the development of some forms of childhood leukemia. Further, at least two studies now show that the inactivating NAD(P)H:quinone acceptor oxidoreductase (NQO1) C609T polymorphism is positively associated with leukemias arising in the first 1-2 years of life and polymorphisms in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene have been associated with adult and childhood ALL. Thus, low folate intake and compounds that are detoxified by NQO1 may be important in elevating leukemia risk in children. Finally, we are exploring the use of proteomics to subclassify leukemia, because cytogenetic analysis is costly and time-consuming. Several proteins have been identified that may serve as useful biomarkers for rapidly identifying different forms of childhood leukemia.

Biomarkers↗
PubMed2003

RNA interference is required for normal centromere function in fission yeast.

In plants, animals and fungi, active centromeres are associated with arrays of repetitive DNA sequences. The outer repeats at fission yeast (Schizosaccharomyces pombe) centromeres are heterochromatic and are required for the assembly of an active centromere. Components of the RNA interference (RNAi) machinery process transcripts derived from these repeats and mediate the formation of silent chromatin. A subfragment of the repeat (dg) is known to induce silencing of marker genes at euchromatic sites and is required for centromere formation. We show that the RNAi components, Argonaute (Ago1), Dicer (Dcr1) and RNA-dependent RNA polymerase (Rdp1), are required to maintain silencing, lysine 9 methylation of histone H3 and association of Swi6 via this dg ectopic silencer. Deletion of Ago1, Dcr1 or Rdp1 disrupts chromosome segregation leading to a high incidence of lagging chromosomes on late anaphase spindles and sensitivity to a microtubule poison. Analysis of dg transcription indicates that csp mutants, previously shown to abrogate centromere silencing and chromosome segregation, are also defective in the regulation of non-coding centromeric RNAs. In addition, histone H3 lysine 9 methylation at, and recruitment of Swi6 and cohesin to, centromeric repeats is disrupted in these mutants. Thus the formation of silent chromatin on dg repeats and the development of a fully functional centromere is dependent on RNAi.

Centromere↗
PubMed2001

Differentiation of Enterococcus spp. by cell membrane fatty acid methyl ester profiling, biotyping and ribotyping.

AIMS: Gas chromatographic analysis of cell membrane fatty acid methyl esters (FAME), biochemical profiling (biotyping) and EcoRI restriction endonuclease profiling of DNA containing ribosomal RNA sequences (ribotyping) were compared for differentiation of Enterococcus spp. METHODS AND RESULTS: FAME profiling, biotype profiling and ribotyping of 41 strains from retail Swiss-type cheeses and five strains from culture collections resulted in 17, 25 and 26 groups, respectively, with only two pairs of strains having the same FAME group, biotype profile and ribogroup. CONCLUSION: Substantial overlap occurred in groupings assigned by the three methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Differentiation of Enterococcus spp. strains increases if multiple methods are used.

Bacterial Typing Techniques↗
PubMedDate not supplied

Comparative expression analysis of Adh3 during arthropod, urochordate, cephalochordate, and vertebrate development challenges its predicted housekeeping role.

Gene and genome duplications in the vertebrate lineage explain the complexity of extant gene families. Among these, the medium-chain alcohol dehydrogenase (ADH), which expanded by tandem duplications after the cephalochordate-vertebrate split, is a good model with which to analyze the evolution of gene function. Although the ancestral member of this family, ADH3, has been strictly conserved throughout animal evolution, its physiological role is still controversial. Previous evidence indicates that it contributes to formaldehyde cytoprotection, retinoic acid metabolism, and nitric oxide homeostasis. We performed in situ hybridization during Drosophila, ascidian (Ciona intestinalis), and zebrafish (Danio rerio) development. We showed that Adh3 expression was restricted to the fat body in Drosophila embryos at stage 17 and to the anterior endoderm in C. intestinalis tail bud, whereas in the zebrafish 2.5-day larvae the signal appeared widespread. A more comprehensive expression analysis including amphioxus and mice revealed that ancestral Adh3 was tissue specific, whereas a widespread expression was later attained in vertebrates. These variations occurred concomitantly with the expansion of the ADH family and the acquisition of new functions but were unlinked to the genomic changes that led to the transition from fractional to global methylation in vertebrates. Our data challenge the housekeeping role of ADH3 and question its involvement in the prevertebrate retinoic acid pathway.

Alcohol Dehydrogenase↗
PubMed2001

Protein-coding genes are epigenetically regulated in Arabidopsis polyploids.

The fate of redundant genes resulting from genome duplication is poorly understood. Previous studies indicated that ribosomal RNA genes from one parental origin are epigenetically silenced during interspecific hybridization or polyploidization. Regulatory mechanisms for protein-coding genes in polyploid genomes are unknown, partly because of difficulty in studying expression patterns of homologous genes. Here we apply amplified fragment length polymorphism (AFLP)-cDNA display to perform a genome-wide screen for orthologous genes silenced in Arabidopsis suecica, an allotetraploid derived from Arabidopsis thaliana and Cardaminopsis arenosa. We identified ten genes that are silenced from either A. thaliana or C. arenosa origin in A. suecica and located in four of the five A. thaliana chromosomes. These genes represent a variety of RNA and predicted proteins including four transcription factors such as TCP3. The silenced genes in the vicinity of TCP3 are hypermethylated and reactivated by blocking DNA methylation, suggesting epigenetic regulation is involved in the expression of orthologous genes in polyploid genomes. Compared with classic genetic mutations, epigenetic regulation may be advantageous for selection and adaptation of polyploid species during evolution and development.

Arabidopsis↗
PubMed2006

Diverse DNA methylation statuses at alternative promoters of human genes in various tissues.

We characterized the DNA methylation status at 144 tissue-biased and 37 non-tissue-biased alternative promoters of 61 human genes in five normal tissues. Analysis of the collected data revealed that (i) DNA methylation status differed greatly among alternative promoters belonging to the same gene; (ii) DNA methylation status differed between tissues for the majority of the individual promoters, and (iii) 80-90% of CpG-island-containing promoters were not methylated on either allele throughout the five tissues examined. Furthermore, although the statistical significance was not as clear as for the above features, we also found that (iv) the DNA methylation patterns of tissue-biased promoters changed more drastically than those of non-tissue-biased promoters; (v) tissue-biased promoters tended to be less methylated than their respective alternative promoters in the tissues where they were preferentially expressed, and (vi) the 'null' methylation pattern of a given promoter was enriched in the tissues where the transcription was most active. These findings together indicate that there are dynamic physiological changes of DNA methylation. DNA methylation appears to play a significant role in differential usage of alternative promoters and may be related to functional diversification between CpG-island-containing promoters and CpG-island-less promoters.

Brain↗