Promoter-associated CpG Islands Research Articles

Induced epigenetic changes memorized across generations in mice.

Transgenerational epigenetic inheritance in mammals has long been debatable. In this issue of Cell, Takahashi etal. induce DNA methylation at promoter-associated CpG islands (CGIs) of two metabolism-related genes and show that the acquired epigenetic changes and associated metabolic phenotypes are stably propagated across several generations in transgenic mice.

Transgenerational inheritance of acquired epigenetic signatures at CpG islands in mice

Transgenerational epigenetic inheritance in mammals remains a debated subject. Here, we demonstrate that DNA methylation of promoter-associated CpG islands (CGIs) can be transmitted from parents to their offspring in mice. We generated DNA methylation-edited mouse embryonic stem cells (ESCs), in which CGIs of two metabolism-related genes, the Ankyrin repeat domain 26 and the low-density lipoprotein receptor, were specifically methylated and silenced. DNA methylation-edited mice generated by microinjection of the methylated ESCs exhibited abnormal metabolic phenotypes. Acquired methylation of the targeted CGI and the phenotypic traits were maintained and transmitted across multiple generations. The heritable CGI methylation was subjected to reprogramming in parental PGCs and subsequently reestablished in the next generation at post-implantation stages. These observations provide a concrete step toward demonstrating transgenerational epigenetic inheritance in mammals, which may have implications in our understanding of evolutionary biology as well as the etiology, diagnosis, and prevention of non-genetically inherited human diseases.

Transgenerational Transcriptomic and DNA Methylome Profiling of Mouse Fetal Testicular Germline and Somatic Cells after Exposure of Pregnant Mothers to Tributyltin, a Potent Obesogen.

Obesogens such as tributyltin (TBT) are xenobiotic compounds that promote obesity, in part by distorting the normal balance of lipid metabolism. The obesogenic effects of TBT can be observed in directly exposed (F1 and F2 generations) and also subsequent generations (F3 and beyond) that were never exposed. To address the effects of TBT exposure on germ cells, we exposed pregnant transgenic OG2 mouse dams (F0), which specifically express EGFP in germline cells, to an environmentally relevant dose of TBT or DMSO throughout gestation through drinking water. When fed with a high-fat diet, F3 male offspring of TBT-exposed F0 dams (TBT-F3) accumulated much more body fat than did DMSO-F3 males. TBT-F3 males also lost more body fluid and lean compositions than did DMSO-F3 males. Expression of genes involved in transcriptional regulation or mesenchymal differentiation was up-regulated in somatic cells of TBT-F1 (but not TBT-F3) E18.5 fetal testes, and promoter-associated CpG islands were hyper-methylated in TBT-F1 somatic cells. Global mRNA expression of protein-coding genes in F1 or F3 fetal testicular cells was unaffected by F0 exposure to TBT; however, expression of a subset of endogenous retroviruses was significantly affected in F1 and F3. We infer that TBT may directly target testicular somatic cells in F1 testes to irreversibly affect epigenetic suppression of endogenous retroviruses in both germline and somatic cells.

Epigenetic changes during tumour development – functional epigenetics study

DNA methylation plays a key role in demarcation of regulatory regions, including promoter-associated CpG islands. While CpG islands are typically maintained in an unmethylated state, a proportion of islands are subject to hypermethylation during tumorigenesis. A key question is to understand what triggers the aberrant changes to the DNA methylation landscape in cancer. Using methylome sequencing, we have identified a subset of distinct CpG islands that display partial DNA methylation encroachment across 5ʹ or 3ʹ CpG island borders.

DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells.

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between –225 and –56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

H3.3-K27M drives neural stem cell-specific gliomagenesis in a human iPSC-derived model.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive childhood tumor of the brainstem with currently no curative treatment available. The vast majority of DIPGs carry a histone H3 mutation leading to a lysine 27-to-methionine exchange (H3K27M). We engineered human induced pluripotent stem cells (iPSCs) to carry an inducible H3.3-K27M allele in the endogenous locus and studied the effects of the mutation in different disease-relevant neural cell types. H3.3-K27M upregulated bivalent promoter-associated developmental genes, producing diverse outcomes in different cell types. While being fatal for iPSCs, H3.3-K27M increased proliferation in neural stem cells (NSCs) and to a lesser extent in oligodendrocyte progenitor cells (OPCs). Only NSCs gave rise to tumors upon induction of H3.3-K27M and TP53 inactivation in an orthotopic xenograft model recapitulating human DIPGs. In NSCs, H3.3-K27M leads to maintained expression of stemness and proliferative genes and a premature activation of OPC programs that together may cause tumor initiation.

Multi-layered epigenetic regulation of IRS2 expression in the liver of obese individuals with type 2 diabetes

Aims/hypothesisIRS2 is an important molecular switch that mediates insulin signalling in the liver. IRS2 dysregulation is responsible for the phenomenon of selective insulin resistance that is observed in type 2 diabetes. We hypothesise that epigenetic mechanisms are involved in the regulation of IRS2 in the liver of obese and type 2 diabetic individuals.MethodsDNA methylation of seven CpG sites was studied by bisulphite pyrosequencing and mRNA and microRNA (miRNA) expression was assessed by quantitative real-time PCR in liver biopsies of 50 obese non-diabetic and 31 obese type 2 diabetic participants, in a cross-sectional setting. Methylation-sensitive luciferase assays and electrophoretic mobility shift assays were performed. Furthermore, HepG2 cells were treated with insulin and high glucose concentrations to induce miRNA expression and IRS2 downregulation.ResultsWe found a significant downregulation of IRS2 expression in the liver of obese individuals with type 2 diabetes (0.84 ± 0.08-fold change; p = 0.0833; adjusted p value [pa] = 0.0417; n = 31) in comparison with non-diabetic obese participants (n = 50). This downregulation correlated with hepatic IRS2 DNA methylation at CpG5. Additionally, CpG6, which is located in intron 1 of IRS2, was hypomethylated in type 2 diabetes; this site spans the sterol regulatory element binding transcription factor 1 (SREBF1) recognition motif, which likely acts as transcriptional repressor. The adjacent polymorphism rs4547213 (G>A) was significantly associated with DNA methylation at a specificity-protein-1 (SP1) binding site (CpG3). Moreover, DNA methylation of cg25924746, a CpG site located in the shore region of the IRS2 promoter-associated CpG island, was increased in the liver of individuals with type 2 diabetes, as compared with those without diabetes. A second epigenetic mechanism, upregulation of hepatic miRNA hsa-let-7e-5p (let-7e-5p) in obese individuals with type 2 diabetes (n = 29) vs non-diabetic obese individuals (n = 49) (1.2 ± 0.08-fold change; p = 0.0332; pa = 0.0450), is likely to act synergistically with altered IRS2 DNA methylation to decrease IRS2 expression. Mechanistic in vitro experiments demonstrated an acute upregulation of let-7e-5p expression and simultaneous IRS2 downregulation in a liver (HepG2) cell line upon hyperinsulinaemic and hyperglycaemic conditions.Conclusions/interpretationOur study highlights a new multi-layered epigenetic network that could be involved in subtle dysregulation of IRS2 in the liver of individuals with type 2 diabetes. This might lead to fine-tuning of IRS2 expression and is likely to be supplementary to the already known factors regulating IRS2 expression. Thereby, our findings could support the discovery of new diagnostic and therapeutic strategies for type 2 diabetes.Graphical abstract

Analysis of Promoter Hypermethylation of DAPK and BAX Apoptotic Genes in Iranian Gastric Cancer Patients Undergoing Chemotherapy

Background: The apoptotic route is mostly damaged in gastric cancer tumor cells. DNA methylation of promoter associated CpG islands inactivates tumor suppressor genes. The objective of the present study was to analyze the hypermethylation of death-associated protein kinase (DAPK) and Bcl-2-associated X protein (BAX) genes in individuals suffering from gastric cancer and undergoing chemotherapy. Methods: Genomic DNA was extracted from blood samples and the tissue fixed in the paraffin of 30 patients and normal individuals. Hypermethylation investigation of DAPK and BAX genes was conducted via methylation specific PCR technique, the outcomes of which were analyzed through electrophoresis and SPSS software version 20. Results: Methylation of both BAX and DAPK genes with a frequency of (28.3%, 21.7%) in blood and (23.3%, 23.3%) in tissue, respectively, had a significant relationship with gastric cancer (P˂0.01). A significant relationship was also observed between the methylation of BAX gene in tissue and tumor type (12, 35.3% and P˂0.01). No relationship was found between methylation and grade, stage, node, age, sex, and other pathologic and clinical data of the patients (P>0.05). There was a significant association between simultaneous methylation of DAPK and BAX genes in tumor and typical tissues with methylation a frequency of 40% and 95.83%, respectively (P˂ 0.01). Conclusion: Methylation of the BAX and DAPK genes can be used as a biomarker in bloodand an approach in the early detection of malignity and illness management. Methylation inhibitors with the potential for drug targeting of DAPK and BAX can further be employed in pharmacotherapy.

Bisphenol a Exposure, DNA Methylation, and Asthma in Children.

Epidemiological studies have reported the relationship between bisphenol A (BPA) exposure and increased prevalence of asthma, but the mechanisms remain unclear. Here, we investigated whether BPA exposure and DNA methylation related to asthma in children. We collected urinary and blood samples from 228 children (Childhood Environment and Allergic Diseases Study cohort) aged 3 years. Thirty-three candidate genes potentially interacting with BPA exposure were selected from a toxicogenomics database. DNA methylation was measured in 22 blood samples with top-high and bottom-low exposures of BPA. Candidate genes with differential methylation levels were validated by qPCR and promoter associated CpG islands have been investigated. Correlations between the methylation percentage and BPA exposure and asthma were analyzed. According to our findings, MAPK1 showed differential methylation and was further investigated in 228 children. Adjusting for confounders, urinary BPA glucuronide (BPAG) level inversely correlated with MAPK1 promoter methylation (β = −0.539, p = 0.010). For the logistic regression analysis, MAPK1 methylation status was dichotomized into higher methylated and lower methylated groups with cut off continuous variable of median of promoter methylation percentage (50%) while performing the analysis. MAPK1 methylation was lower in children with asthma than in children without asthma (mean ± SD; 69.82 ± 5.88% vs. 79.82 ± 5.56%) (p = 0.001). Mediation analysis suggested that MAPK1 methylation acts as a mediation variable between BPA exposure and asthma. The mechanism of BPA exposure on childhood asthma might, therefore, be through the alteration of MAPK1 methylation. The mechanism of BPA exposure on childhood asthma might, therefore, be through the alteration of MAPK1 methylation.

Chromatin dysregulation and DNA methylation at transcription start sites associated with transcriptional repression in cancers

Although promoter-associated CpG islands have been established as targets of DNA methylation changes in cancer, previous studies suggest that epigenetic dysregulation outside the promoter region may be more closely associated with transcriptional changes. Here we examine DNA methylation, chromatin marks, and transcriptional alterations to define the relationship between transcriptional modulation and spatial changes in chromatin structure. Using human papillomavirus-related oropharyngeal carcinoma as a model, we show aberrant enrichment of repressive H3K9me3 at the transcriptional start site (TSS) with methylation-associated, tumor-specific gene silencing. Further analysis identifies a hypermethylated subtype which shows a functional convergence on MYC targets and association with CREBBP/EP300 mutation. The tumor-specific shift to transcriptional repression associated with DNA methylation at TSSs was confirmed in multiple tumor types. Our data may show a common underlying epigenetic dysregulation in cancer associated with broad enrichment of repressive chromatin marks and aberrant DNA hypermethylation at TSSs in combination with MYC network activation.

DNA methylome of human neonatal umbilical cord: Enrichment of differentially methylated regions compared to umbilical cord blood DNA at transcription factor genes involved in body patterning and effects of maternal folate deficiency or children's sex.

The DOHaD (developmental origins of health and disease) hypothesis claims that fetal malnutrition or exposure to environmental pollutants may affect their lifelong health. Epigenetic changes may play significant roles in DOHaD; however, access to human fetuses for research has ethical and technical hurdles. Umbilical cord blood (CB) has been commonly used as an epigenetic surrogate of fetuses, but it does not provide direct evidence of fetal exposure to pollutants. Here, we propose umbilical cord tissue (UC), which accumulates substances delivered to fetuses during gestation, as an alternative surrogate for epigenetic studies on fetuses. To explore the feasibility to examine UC epigenome by deep sequencing, we determined CpG methylation profiles of human postnatal UC by reduced representation bisulfite sequencing. Principal component analysis clearly separated the DNA methylomes of UC and CB pairs isolated from the same newborn (n = 10). Although all UC chromosomes were modestly hypomethylated compared to CB chromosomes, GO analysis revealed strong enrichment of differentially methylated regions (DMRs) at promoter-associated CpG islands in the HOX gene clusters and other genes encoding transcription factors involved in determination of the body pattern. DNA methylomes of UC autosomes were largely comparable between males and females. Deficiency of folate during pregnancy has been suggested to affect fetal DNA methylation to cause congenital anomalies. Whereas DNA methylome of UC was not significantly affected by early-gestational (12 weeks) low levels of maternal plasma folate (< 8 ng/ml, n = 10) compared to controls (>19 ng/mL, n = 10), two specific loci of LTR12C endogenous retroviruses in chromosome 12 were significantly hypermethylated in the low-folate group. Our study suggests that UC is useful as an alternative surrogate for studying environmental effects on DNA methylation in human fetuses, compensating CB by providing additional information about epigenetic regulation of genes involved in developmental body patterning and endogenous retroviruses.

Serotonin transporter gene methylation predicts long-term cortisol concentrations in hair

Epigenetic signatures, such as DNA methylation (DNAM), have been implicated in long-term dysregulation of the hypothalamus–pituitary–adrenal (HPA) axis and related health risks. Based on a wealth of neuroendocrine studies on genetic polymorphisms in the serotonin transporter gene (SLC6A4), this locus constitutes a key candidate to explore associations of DNAM patterns and HPA-axis functioning. The few studies addressing this link so far exclusively relied on spot measurements of HPA-axis activity, which may not adequately reflect cortisol output over prolonged periods of time. To address this gap, hair cortisol concentrations (HCC), a valid measure of integrated long-term cortisol levels, were utilized to investigate endocrine correlates of SLC6A4 DNAM in 183 adults. Whole blood samples were drawn for DNAM analyses of 83 CpG sites within a 799-bp promoter-associated CpG island of SLC6A4 via bisulfite pyrosequencing. In addition, all participants were genotyped for the serotonin transporter polymorphism (5-HTTLPR). First, results revealed a significant negative association of SLC6A4 DNAM and HCC. Second, there was no significant main effect of 5-HTTLPR genotype on HCC when analyses were conducted on the basis of both bi-allelic classification and the 5-HTTLPR/rs25531 mini-haplotype. Third, the current data revealed a significant interaction of SLC6A4 DNAM and 5-HTTLPR genotype on HCC. Comparable to the pattern we had previously observed concerning cortisol stress reactivity, the S allele relates to increased HCC in individuals displaying low levels of SLC6A4 DNAM. By contrast, no such effect occurred under conditions of high SLC6A4 DNAM, indicating that epigenetic changes may compensate for genotype-dependent differences in long-term cortisol output. Together, respective findings support the idea of an epigenetic contribution to long-term HPA-axis activity and further highlight the usefulness of combining genetic and epigenetic information in future neuroendocrine studies.

Epigenetic and transcriptional dysregulation of VWA2 associated with a MYC-driven oncogenic program in colorectal cancer

VWA2 encodes AMACO, a secreted protein up-regulated in most colorectal carcinomas (CRC), constituting a promising biomarker. The mechanism responsible for its aberrant up-regulation has not been previously described. In this work, we analyzed VWA2 DNA methylation in over 400 primary CRCs. No epigenetic alterations were found in its promoter-associated CpG island. However, the region located downstream of the transcriptional start site was hypomethylated in most CRCs. ChIP-Seq revealed increased levels of the active mark H3K4me3 and reduction of the repressive mark H3K27me3. In contrast, several CRC cell lines exhibited hypermethylation of VWA2. 5-AZA-2-deoxycitidine treatment led to transcriptional activation of VWA2, supporting a functional link between DNA methylation and transcription. VWA2 expression in primary CRCs correlated with that of Myc and Myc-target genes. Transcriptional up-regulation of VWA2 is extremely frequent (78%) and strong (average fold change >15) in CRC, but not in other types of cancer. VWA2 undergoes hypomethylation in the majority of CRCs. This alteration could partly underlie the previously reported over-expression of AMACO. Co-expression profiling suggests that VWA2 might be a constituent of a larger oncogenic transcriptional program regulated by c-Myc. Up-regulation of VWA2 is virtually exclusive of CRC, reinforcing its potential as a specific biomarker.

Functional role of DNMT3A mutation in acute myeloid leukemia

A current broad spectrum of genomic studies on acute myeloid leukemia (AML) has demonstrated that a gene encoding for DNA methyltransferase, specifically DNA methyltransferase 3 alpha (DNMT3A) is frequently mutated. However, DNMT3A variants are present in elderly healthy individuals and patients with AML in complete remission, which suggests that DNMT3A mutations may contribute to pre-leukemic clonal hematopoiesis. Although DNMT3A mutation has been thought to play a pivotal role in AML pathogenesis through the loss of DNA methylation functionality, other potential disease-related mechanisms are poorly understood. We identified that DNMT3A Arg882 mutation has two distinct mechanisms for developing clonal hematopoiesis and leukemia: (1) DNA methylation-dependent effect, which caused up-regulation of the anterior Hoxb cluster and many hematopoietic stem cell (HSC) -related genes, with hypo-methylation of the promoter-associated CpG island; and (2) DNA methylation-independent effect with enhanced recruitment of polycomb repressive complex 1 (PRC1) that subsequently suppressed the expression of an array of differentiation-associated genes. Through these mechanisms, DNMT3A mutations were shown to inhibit the differentiation of HSCs and leukemic cells. These results identified PRC1 as a promising candidate for the development of therapeutic strategies in mutant DNMT3A-associated AML. Here, we review recent studies on AML with a focus on the clinical features and functional roles of DNMT3A mutations, and discuss future challenges to effectively cure DNMT3A mutation-associated hematopoietic disorders.

Aberrant DNA methylation in lymphocytes of children with neurodevelopmental disorders

Recent research in the field of genomics and epigenetics has provided evidence that alterations in the system of epigenetic regulation are highly involved in the molecular etiology of neurodegenerative and neuropathic disorders. However, there is a gap in knowledge on the epigenetic perturbations that may accompany the CNS impairments during the development in the prenatal period and their manifestation as a congenital encephalopathy in the early postnatal period of child development. The present study is one of the first attempts aimed at addressing this gap. Here, we present data on genome-wide profiles of DNA methylation obtained using the Illumina HumanMethylation450 microarray in peripheral blood cells in a sample of young children (up to four years of age) diagnosed with congenital encephalopathy. We provide evidence on systematic alterations in the epigenome—predominant hypermethylation of gene promoter associated CpG islands—related to the CNS impairment in children. Specifically, we found significant DNA methylation changes in genes involved in DNA-dependent transcription regulation and transcription factor binding, with a key role of the transcription factor JUN; in genes controlling cellular response to hypoxia; and in genes involved in the control of neuronal development, functioning, and death.

Abstract 15: Glucose-Mediated Remodeling of Cardiac DNA Methylation

To identify the role of glucose in the development of diabetic cardiomyopathy, we had directly assessed glucose delivery to the intact heart on alterations of DNA methylation and gene expression using both an inducible heart-specific transgene (glucose transporter 4; mG4H) and streptozotocin-induced diabetes (STZ) mouse models. We aimed to determine whether long-lasting diabetic complications arise from prior transient exposure to hyperglycemia via a process termed “glycemic memory.” We had identified DNA methylation changes associated with significant gene expression regulation. Comparing our results from STZ, mG4H, and the modifications which persist following transgene silencing, we now provide evidence for cardiac DNA methylation as a persistent epigenetic mark contributing to glycemic memory. To begin to determine which changes contribute to human heart failure, we measured both RNA transcript levels and whole-genome DNA methylation in heart failure biopsy samples (n = 12) from male patients collected at left ventricular assist device placement using RNA-sequencing and Methylation450 assay, respectively. We hypothesized that epigenetic changes such as DNA methylation distinguish between heart failure etiologies. Our findings demonstrated that type 2 diabetic heart failure patients (n = 6) had an overall signature of hypomethylation, whereas patients listed as ischemic (n = 5) had a distinct hypermethylation signature for regulated transcripts. The focus of this initial analysis was on promoter-associated CpG islands with inverse changes in gene transcript levels, from which diabetes (14 genes; e.g. IGFBP4) and ischemic (12 genes; e.g. PFKFB3) specific targets emerged with significant regulation of both measures. By combining our mouse and human molecular analyses, we provide evidence that diabetes mellitus governs direct regulation of cellular function by DNA methylation and the corresponding gene expression in diabetic mouse and human hearts. Importantly, many of the changes seen in either mouse type 1 diabetes or human type 2 diabetes were similar supporting a consistent mechanism of regulation. These studies are some of the first steps at defining mechanisms of epigenetic regulation in diabetic cardiomyopathy.

Gene promoter-associated CpG island hypermethylation in squamous cell carcinoma of the tongue.

The present study was undertaken to explore and validate novel hypermethylated DNA regions in squamous cell carcinoma of the tongue (SCCT). Genome-wide methylation changes were identified by differential methylation hybridization (DMH) microarray and validated by bisulfite genome sequencing (BGS). The results were compared against datasets from The Cancer Genome Atlas head and neck squamous cell carcinoma (TCGA-HNSCC), Gene Expression Omnibus (GSE26549), and ArrayExpress (E-MTAB-1328). DMH identified 116 hypomethylated and 241 hypermethylated regions. Of the latter, 24 were localized to promoter or 5'-UTR regions. By BGS, promoter sequences of DAPK1, LRPPRC, RAB6C, and ZNF471 were significantly hypermethylated in tumors when compared with matched normal tissues (P<0.0001). A TCGA-HNSCC dataset (516 cases of cancer and 50 normal tissue samples) further confirmed hypermethylation of DAPK1, RAB6C, and ZNF471. Sensitivity and specificity of methylation markers for a diagnosis of cancer were in the range of 70-100% in our study and from TCGA-HNSCC datasets, with an area under curve (AUC) of 0.83 and above. Kaplan-Meier survival analysis of TCGA-HNSCC expression data revealed that patients with low expressions of DAPK1, RAB6C, and ZNF471 showed poorer survival than patients with high expression (P=0.02). Human papillomavirus (HPV) was found in 55% of cases, HPV16 being the predominant genotype. DAPK1 immunohistochemical staining was lower in SCCT than in normal buccal epithelial cells. This is the first study to report hypermethylation of LRPPRC, RAB6C, and ZNF471 in SCCT and its diagnostic and prognostic potentials in a specific head and neck squamous cell carcinoma.

A microwell-based near-infrared fluorescence assay of DNA methylation

Background: DNA methylation is one of the most common epigenetic modifications, and has profound effects on the mammalian genome. The epigenetic silencing of a variety of genes by hypermethylation of promoter-associated CpG islands is often associated with diseases. This paper describes a method for the detection of DNA methylation. Methods: This method realized methylation detection in two steps, including capturing the sheared genomic DNA (gDNA) on probe-coupled microplate and detecting methylation by using a simple immunological procedure based on near-infrared fluorescence (NIRF). Results: This method was validated by detecting the methylation of synthesized oligonucleotides and nine genomic loci in the promoters of three genes, KIR3DL1, p14 ARF and TP53BP2 in three cell lines. The detection results of the gDNA were verified by the bisulfite sequencing polymerase chain reaction (BSP) performed in this study. This study also demonstrated that the detected methylation of these promoters reduced the transcriptions of these genes in the detected cells. Conclusions: This study provides a method for DNA methylation detection that is independent of bisulfite treatment, PCR amplification and immunoprecipitation.

A microwell-based near-infrared fluorescence assay of DNA methylation

Background: DNA methylation is one of the most common epigenetic modifications, and has profound effects on the mammalian genome. The epigenetic silencing of a variety of genes by hypermethylation of promoter-associated CpG islands is often associated with diseases. This paper describes a method for the detection of DNA methylation.

Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters.

Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.