Methylation Status Of CpG Sites Research Articles

Methylation patterns at the adjacent CpG sites within enhancers are a part of cell identity

BackgroundIt is generally accepted that methylation status of CpG sites spaced up to 50 bp apart is correlated, and accumulation of locally disordered methylation at adjacent CpG sites is involved in neoplastic transformation, acting in similar way as stochastic accumulation of mutations.ResultsWe used EPIC microarray data from 596 samples, representing 12 healthy tissue and cell types, as well as 572 blood cancer specimens to analyze methylation status of adjacent CpG sites across human genome, and subsequently validated our findings with NGS and Sanger sequencing. Our analysis showed that there is a subset of the adjacent CpG sites in human genome, with cytosine at one CpG site methylated and the other devoid of methyl group. These loci map to enhancers that are targeted by families of transcription factors involved in cell differentiation. Moreover, our results suggest that the methylation at these loci differ between alleles within a cell, what allows for remarkable level of heterogeneity of methylation patterns. However, different types of specialized cells acquire only one specific and stable pattern of methylation at each of these loci and that pattern is to a large extent lost during neoplastic transformation.ConclusionsWe identified a substantial number of adjacent CpG loci in human genome that display remarkably stable and cell type specific methylation pattern. The methylation pattern at these loci appears to reflect different methylation of alleles in cells. Furthermore, we showed that changes of methylation status at those loci are likely to be involved in regulation of the activity of enhancers and contribute to neoplastic transformation.

AMD1 promotes breast cancer aggressiveness via a spermidine-eIF5A hypusination-TCF4 axis

BackgroundBasal-like breast cancer (BLBC) is the most aggressive subtype of breast cancer due to its aggressive characteristics and lack of effective therapeutics. However, the mechanism underlying its aggressiveness remains largely unclear. S-adenosylmethionine decarboxylase proenzyme (AMD1) overexpression occurs specifically in BLBC. Here, we explored the potential molecular mechanisms and functions of AMD1 promoting the aggressiveness of BLBC.MethodsThe potential effects of AMD1 on breast cancer cells were tested by western blotting, colony formation, cell proliferation assay, migration and invasion assay. The spermidine level was determined by high performance liquid chromatography. The methylation status of CpG sites within the AMD1 promoter was evaluated by bisulfite sequencing PCR. We elucidated the relationship between AMD1 and Sox10 by ChIP assays and quantitative real-time PCR. The effect of AMD1 expression on breast cancer cells was evaluated by in vitro and in vivo tumorigenesis model.ResultsIn this study, we showed that AMD1 expression was remarkably elevated in BLBC. AMD1 copy number amplification, hypomethylation of AMD1 promoter and transcription activity of Sox10 contributed to the overexpression of AMD1 in BLBC. AMD1 overexpression enhanced spermidine production, which enhanced eIF5A hypusination, activating translation of TCF4 with multiple conserved Pro-Pro motifs. Our studies showed that AMD1-mediated metabolic system of polyamine in BLBC cells promoted tumor cell proliferation and tumor growth. Clinically, elevated expression of AMD1 was correlated with high grade, metastasis and poor survival, indicating poor prognosis of breast cancer patients.ConclusionOur work reveals the critical association of AMD1-mediated spermidine-eIF5A hypusination-TCF4 axis with BLBC aggressiveness, indicating potential prognostic indicators and therapeutic targets for BLBC.

Abstract LB197: Methylation patterns at adjacent CpG sites within enhancers are part of cell identity and are disrupted during malignant transformation

Abstract Background: Following the principles of physiology of DNA methylation, it is generally accepted that methylation status of CpG sites spaced up to 50 bp is correlated and accumulation of locally disordered methylation at CpG sites is a part of neoplastic transformation, acting in similar way as stochastic accumulation of mutations during malignant transformation. Methods: We developed a new algorithm allowing to analyze methylation status at the adjacent CpG sites. We then, used EPIC microarray data to analyze genome wide methylation patterns at the adjacent CpG sites in almost 600 samples, representing 12 healthy tissues and cell types as well as 572 cancer specimens. We also used bisulfite next generation sequencing data and Sanger sequencing to validate our findings. Results: Our analysis, identified in human genome a subset of loci with adjacent CpG sites spaced less than 50bp (average 28bp), harboring one cytosine methylated and the other devoid of methyl group. These loci are enriched in enhancers that are targeted by families of transcription factors involved in cell differentiation. The methylation patterns at these loci can differ between alleles within a cell, what allows for remarkable level of heterogeneity of methylation patterns. However, our analysis also showed that, different types of specialized cells, acquire during differentiation only one specific and remarkably stable pattern of methylation at each of these loci and that pattern is to a large extent lost during neoplastic transformation. Conclusions: Contrary to generally accepted principle of identical methylation status of the adjacent CpG sites being essential for regulatory function of DNA methylation. We have shown that methylation at a subset of adjacent CpG sites in human genome located within important functional genomic elements differs in healthy cells. These CpG sites acquire remarkably stable and tissue specific patterns of methylation during cell differentiation. Moreover, the methylation patterns at these CpG sites, are lost during malignant transformation suggesting the involvement of identified phenomenon in cancer pathology. Funding: This work was funded by grant OPUS22 from Polish National Science Centre (grant number: 2021/43/B/NZ2/02979) and Polish National Agency for Academic Exchange (grant number: PPN/PPO/2018/1/00088/U). Citation Format: Olga Taryma-Leśniak, Jan Bińkowski, Patrycja K. Przybylowicz, Katarzyna E. Sokolowska, Konrad Borowski, Tomasz K. Wojdacz. Methylation patterns at adjacent CpG sites within enhancers are part of cell identity and are disrupted during malignant transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB197.

Unveiling the prognostic significance of SOX5 in esophageal squamous cell carcinoma: a comprehensive bioinformatic and experimental analysis.

This study aimed to investigate the expression and prognostic significance of SOX5 in esophageal squamous cell carcinoma (ESCC). Gene Expression Omnibus (GEO) data were analyzed to assess SOX5 expression in ESCC and normal tissues. Survival analysis was performed to evaluate its prognostic significance. Pathway enrichment analysis was conducted to identify pathways associated with low SOX5 expression. Methylation status of CpG sites in ESCC cases was examined, and SOX5 expression was evaluated. Differential expression and ChIP-seq data analyses were used to identify genes significantly correlated with SOX5 and to obtain target genes. A protein-protein interaction (PPI) network was constructed using hub genes, and their association with immune cell infiltration was determined. In vitro ESCC cell experiments validated the findings. SOX5 was significantly downregulated in ESCC samples compared to normal samples. Its downregulation was associated with shorter survival in ESCC patients. Pathway enrichment analysis revealed enrichment in regulated necrosis, NLRP3 inflammasome, formation of the cornified envelope, and PD-1 signaling. Methylation status of two CpG sites negatively correlated with SOX5 expression. Differential expression analysis identified 122 genes significantly correlated with SOX5, and 28 target genes were obtained from ChIP-seq analysis. Target genes were enriched in DNA replication, cell cycle, spindle, and ATPase activity. Five hub genes were identified, and the PPI network showed significant associations with immune cell infiltration. In vitro experiments confirmed SOX5 downregulation, upregulation of hub genes, and their functional effects on ESCC cell apoptosis and proliferation. These findings enhance understanding of SOX5 in ESCC and potential therapeutic strategies.

The Effect of DNA Methylation in the Development and Progression of Chronic Kidney Disease in the General Population: An Epigenome-Wide Association Study Using the Korean Genome and Epidemiology Study Database.

Although knowledge of the genetic factors influencing kidney disease is increasing, epigenetic profiles, which are associated with chronic kidney disease (CKD), have not been fully elucidated. We sought to identify the DNA methylation status of CpG sites associated with reduced kidney function and examine whether the identified CpG sites are associated with CKD development. We analyzed DNA methylation patterns of 440 participants in the Korean Genome and Epidemiology Study (KoGES) with estimated glomerular filtration rates (eGFRs) ≥ 60 mL/min/1.73 m2 at baseline. CKD development was defined as a decrease in the eGFR of <60 at any time during an 8-year follow-up period ("CKD prediction" analysis). In addition, among the 440 participants, 49 participants who underwent a second methylation profiling were assessed for an association between a decline in kidney function and changes in the degree of methylation of CpG sites during the 8 years ("kidney function slope" analysis). In the CKD prediction analysis, methylation profiles of a total of 403,129 CpG sites were evaluated at baseline in 440 participants, and increased and decreased methylation of 268 and 189 CpG sites, respectively, were significantly correlated with the development of CKD in multivariable logistic regression. During kidney function slope analysis using follow-up methylation profiles of 49 participants, the percent methylation changes in 913 CpG sites showed a linear relationship with the percent change in eGFR during 8 years. During functional enrichment analyses for significant CpG sites found in the CKD prediction and kidney function slope analyses, we found that those CpG sites represented MAPK, PI3K/Akt, and Rap1 pathways. In addition, three CpG sites from three genes, NPHS2, CHCHD4, and AHR, were found to be significant in the CKD prediction analysis and related to a decline in kidney function. It is suggested that DNA methylation on specific genes is associated with the development of CKD and the deterioration of kidney function.

The correlation between CpG island methylation of hTERT promoter and human age prediction

DNA methylation is an epigenetic modification that occurs during the life cycle of individuals. Its degree is closely associated with the methylation status of CpG sites in its promoter region. Based on the previous screening that the hTERT methylation is both related to tumors and age, we suspected that the age inference based on hTERT methylation would be disturbed by the disease of the tested person. Herein, eight CpG sites in the hTERT promoter region were analyzed by real-time methylation-specific PCR, and we found that CpG2, CpG5, and CpG8 were closely related to the tumor (P < 0.05). The remaining five CpG sites had a large error in predicting age alone. Combining them to establish a model yielded better results, with an average age error of 4.35 years. This study provides a reliable and accurate detection method for the DNA methylation status of multiple CpG sites on the hTERT gene promoter, which can be used for the prediction of forensic age and assistant diagnosis of clinical diseases.

MHapTk: a comprehensive toolkit for the analysis of DNA methylation haplotypes.

Bisulfite sequencing remains the gold standard technique to detect DNA methylation profiles at single-nucleotide resolution. The DNA methylation status of CpG sites on the same fragment represents a discrete methylation haplotype (mHap). The mHap-level metrics were demonstrated to be promising cancer biomarkers and explain more gene expression variation than average methylation. However, most existing tools focus on average methylation and neglect mHap patterns. Here, we present mHapTk, a comprehensive python toolkit for the analysis of DNA mHap. It calculates eight mHap-level summary statistics in predefined regions or across individual CpG in a genome-wide manner. It identifies methylation haplotype blocks, in which methylations of pairwise CpGs are tightly correlated. Furthermore, mHap patterns can be visualized with the built-in functions in mHapTk or external tools such as IGV and deepTools. https://jiantaoshi.github.io/mhaptk/index.html. Supplementary data are available at Bioinformatics online.

Whole human genome 5’-mC methylation analysis using long read nanopore sequencing

ABSTRACT Methylation microarray and bisulphite sequencing are often used to study 5’-methylcytosine (5’-mC) modification of CpG dinucleotides in the human genome. Although both technologies produce trustworthy results, the evaluation of the methylation status of CpG sites suffers from the potential side effects of DNA modification by bisulphite and/or the ambiguity of mapping short reads in repetitive and highly homologous genomic regions, respectively. Nanopore sequencing is an attractive alternative for the study of 5’-mC since it allows sequencing of native DNA molecules, whereas the long reads produced by this technology help to increase the resolution of those genomic regions. In this work, we show that nanopore sequencing with 10X coverage depth, using DNA from a human cell line, produces 5’-mC methylation frequencies consistent with those obtained by 450k microarray, digital restriction enzyme analysis of methylation, and reduced representation bisulphite sequencing. High correlation between methylation frequencies obtained by nanopore sequencing and the other methodologies was also noticeable in either low or high GC content regions, including CpG islands and transcription start sites. We also showed that a minimum of five reads per CpG yields strong correlations (>0.89) in replicate nanopore sequencing runs and an almost uniform linearity of the methylation frequency variation between zero and one. Furthermore, nanopore sequencing was able to correctly display methylation frequency patterns based on genomic annotations of CpG regions. These results demonstrate that nanopore sequencing is a fast, robust, and reliable approach to the study of 5’-mC in the human genome with low coverage depth.

Lack of association between methylation status of CpG in the MCP-1 promoter and type 2 diabetes mellitus and its complications in a Moroccan population

BackgroundMonocyte chemoattractant protein −1 (MCP-1) plays an important role in the development of diseases related to metabolic syndromes, such as type 2 diabetes mellitus (T2DM) and its complications. The present study investigates the relationship between the methylation status of CpG sites in the MCP-1 promoter and T2DM and its complications in Morocco. Material and methodsWe conducted a population-based case-control study of 108 T2DM cases and 101 controls. Methylation Specific Polymerase chain reaction (MSP) was used to analyze the MCP-1 methylation profile in leucocyte's DNA. ResultsThis study did not indicate a significant association between the methylation status of CpG sites in the MCP1 promoter and T2DM and its complications (p ˃ 0.05). The hypomethylation profile is significantly associated with higher LDL-C levels (142.37 ± 58.37; p = 0.041) and treatment with antidepressants (18.8%; p = 0.043) in patients with T2DM. The hypermethylation profile is most frequently observed in type 2 diabetic patients with complications and cigarettes smokers (31.6%; p = 0.052). ConclusionResults of our study showed no association between the methylation status of CpG in the MCP-1 promoter and T2DM and its complications in the Moroccan population. However, the methylation status might be related to LDL-C levels, treatment with antidepressants, and smoking status.

Association between heat stress during intrauterine development and the expression and regulation of ovarian steroid hormone receptors in adult Holstein cows.

Dairy cattle experience stressful environmental situations that affect production. Heat stress during gestation can influence the intrauterine development of offspring, resulting in long-term damage that can affect the reproductive life of the adult offspring. The aim of the present study was to evaluate changes in the expression and regulation of steroid hormone receptors in the ovary of Holstein cows gestated under different temperature-humidity index (THI) during their in utero development. Animals were classified by their exposure to temperature-humidity index (THI) ≥72 during their development in utero according to date of birth or date of effective service of their mother. This study was not carried out under controlled conditions, but the conditions to which the cows were naturally exposed during their development were considered retrospectively, controlling the variables in the statistical analyses (age as a covariate, dairy farm as a random factor). Gestation was divided into two periods (P1=days 0-150; and P2=day 151 to calving) and three trimesters (T1=days 0-90; T2=days 91-180; and T3=day 181 to calving), and the exposure to THI ≥72 was calculated in each one. The following characteristics were evaluated: gene expression of estrogen receptor (ESR) 1, ESR2 and progesterone receptor (PGR), CpG methylation in the 5'UTR of ESR1 and ESR2, and protein expression of ESR1, ESR2, PGR and coregulatory proteins in the dominant follicles of daughter cows in adulthood. We found associations between heat stress variables during gestation and the methylation status of CpG sites in the 5'UTR of ESR1 and ESR2 in dominant follicles. Results also showed association between exposure to high THI values during intrauterine development and expression of ESR1, ESR2 and PGR and coregulatory proteins in dominant follicles of adult cows. These results provide novel information about the impact of prenatal heat stress on molecular aspects at the ovary level in the offspring, during their adult life, which probably impacts the reproductive aspects of the herd.

Current Smoking Alters Gene Expression and DNA Methylation in the Nasal Epithelium of Patients with Asthma.

Current smoking contributes to worsened asthma prognosis and more severe symptoms and limits the beneficial effects of corticosteroids. As the nasal epithelium can reflect smoking-induced changes in the lower airways, it is a relevant source to investigate changes in gene expression and DNA methylation. This study explores gene expression and DNA methylation changes in current and ex-smokers with asthma. Matched gene expression and epigenome-wide DNA methylation samples collected from nasal brushings of 55 patients enrolled in a clinical trial investigation of current and ex-smoker patients with asthma were analyzed. Differential gene expression and DNA methylation analyses were conducted comparing current smokers with ex-smokers. Expression quantitative trait methylation (eQTM) analysis was completed to explore smoking-relevant genes by CpG sites that differ between current and ex-smokers. To investigate the relevance of the smoking-associated DNA methylation changes for the lower airways, significant CpG sites were explored in bronchial biopsies from patients who had stopped smoking. A total of 809 genes and 18,814 CpG sites were differentially associated with current smoking in the nose. The cis-eQTM analysis uncovered 171 CpG sites with a methylation status associated with smoking-related gene expression, including AHRR, ALDH3A1, CYP1A1, and CYP1B1. The methylation status of CpG sites altered by current smoking reversed with 1 year of smoking cessation. We confirm that current smoking alters epigenetic patterns and affects gene expression in the nasal epithelium of patients with asthma, which is partially reversible in bronchial biopsies after smoking cessation. We demonstrate the ability to discern molecular changes in the nasal epithelium, presenting this as a tool in future investigations into disease-relevant effects of tobacco smoke.

DNA methylation landscape of 16 canine somatic tissues by methylation-sensitive restriction enzyme-based next generation sequencing

DNA methylation plays important functions in gene expression regulation that is involved in individual development and various diseases. DNA methylation has been well studied in human and model organisms, but only limited data exist in companion animals like dog. Using methylation-sensitive restriction enzyme-based next generation sequencing (Canine DREAM), we obtained canine DNA methylation maps of 16 somatic tissues from two dogs. In total, we evaluated 130,861 CpG sites. The majority of CpG sites were either highly methylated (> 70%, 52.5–64.6% of all CpG sites analyzed) or unmethylated (< 30%, 22.5–28.0% of all CpG sites analyzed) which are methylation patterns similar to other species. The overall methylation status of CpG sites across the 32 methylomes were remarkably similar. However, the tissue types were clearly defined by principle component analysis and hierarchical clustering analysis with DNA methylome. We found 6416 CpG sites located closely at promoter region of genes and inverse correlation between DNA methylation and gene expression of these genes. Our study provides basic dataset for DNA methylation profiles in dogs.

Whole Genomic DNA Methylation Profiling of CpG Sites in Promoter Regions of Dorsal Root Ganglion in Diabetic Neuropathic Pain Mice.

DNA methylation and demethylation play an important role in neuropathic pain. In general, DNA methylation of CpG sites in the promoter region impedes gene expression, whereas DNA demethylation contributes to gene expression. Here, we evaluated the methylation status of CpG sites in genomic DNA promoter regions in dorsal root ganglions (DRGs) of diabetic neuropathic pain (DNP) mice. In our research, streptozotocin (STZ) was intraperitoneally injected into mice to construct DNP models. The DNP mice showed higher fasting blood glucose (above 11.1mmol/L), lower body weight, and mechanical allodynia than control mice. Whole-genome bisulfite sequencing (WGBS) revealed an altered methylation pattern in CpG sites in the DNA promoter regions in DRGs of DNP mice. The results showed 376 promoter regions with hypermethylated CpG sites and 336 promoter regions with hypomethylated CpG sites. In addition, our data indicated that altered DNA methylation occurs primarily on CpG sites in DNA promoter regions. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that differentially methylated CpG sites annotated genes were involved in activities of the nervous and sensory systems. Enrichment analysis indicated that genes in these pathways contributed to diabetes or pain. In conclusion, our study enriched the role of DNA methylation in DNP.

Methylation of TGM-3 Promoter and Its Association with Oral Squamous Cell Carcinoma (OSCC).

Background:Oral Squamous Cell Carcinoma (OSCC) is among the ten most common cancers worldwide. Hypermethylation of CpG sites in the promoter region and subsequent down-regulation of a tumor suppressor gene, TGM-3 has been proposed to be linked to different types of human cancers including OSCC. In this study, methylation status of CpG sites in the promoter region of TGM-3 has been evaluated in a cohort of patients with OSCC compared to normal controls.Methods:Forty fresh tissue samples were obtained from newly diagnosed OSCC patients and normal individuals referred to dentistry clinic for tooth extraction. DNA was extracted, bisulfite conversion was performed and it was subjected to PCR using bisulfite-sequencing PCR (BSP) primers. Prepared samples were sequenced on a DNA analyzer with both forward and reverse primers of the region of interest. The peak height values of cytosine and thymine were calculated and methylation levels for each CpG site within the DNA sequence was quantified.Results:Quantitative DNA methylation analyses in CpG islands revealed that it was significantly higher in OSCC patients compared to controls. DNA methylation at CpG1/CpG3/CpG5 (p=0.004–0.01) and CpG1/CpG3 (p=0.001–0.019) sites was associated with tumor stage and grade, respectively. Male OSCC patients had higher methylation rate at CpG3 (p=0.032), while smoker patients showed higher methylation rate at CpG6 (p=0.045).Conclusion:These results manifested the contribution of DNA methylation of TGM-3 in OSCC and its potential association with clinico-pathologic parameters in OSCC.

PP255 Epigenetic Profile Predicts Response To Immunotherapy In Patients With Non-Small-Cell Lung Cancer: An Early Assessment

IntroductionEpigenetics is an innovative discipline that aims to provide biomarkers to aid in early diagnosis, patient risk classification, or outcome prediction. The identification of therapeutic targets is of particular interest in cancer therapy for selecting groups of patients who may benefit most from an intervention. Understanding the relationships between the immune system and tumor cells has led to new immunotherapy-based therapies that provide a promising alternative to conventional cancer therapies. The aim of this study was to conduct an early assessment of a novel epigenetic signature (EPIMMUNE) that could predict response to programmed cell death protein 1 (PD-1) inhibitor immunotherapy in patients with non-small cell lung cancer (NSCLC).MethodsWe identified the novel epigenetic signature EPIMMUNE through the Early Awareness and Alert System, “SINTESIS-new technologies” of the Agencia de Evaluación de Tecnologías Sanitarias in Spain (AETS-ISCIII). A literature search of PubMed, Embase, the Web of Science, the Trip database, the International Clinical Trials Registry Platform, ClinicalTrials.gov, The Cochrane Library, and the Centre for Reviews and Dissemination databases was conducted. Clinical studies on EPIMMUNE published in English or Spanish up to August 2019 were reviewed.ResultsOnly one retrospective study was found. Identification of EPIMMUNE was accomplished through interrogation of the DNA methylation status of CpG sites in 142 samples from adult patients with NSCLC who were treated with PD-1 inhibitors. EPIMMUNE was defined by 301 CpG sites whose methylation status was significantly associated with clinical response (progression-free and overall survival). No studies assessing the long-term clinical utility, impact on therapeutic decision making, or economic implications of EPIMMUNE were found.ConclusionsThe EPIMMUNE signature could provide an accurate and valid biomarker for identifying patients with NSCLC who may benefit from treatment with PD-1 inhibitors. However, the technology is under development, and there is only a single study on detecting the EPIMMUNE epigenetic profile and identifying the DNA methylation profiles associated with increased survival after PD-1 inhibitor therapy. More diagnostic accuracy studies and prospective, long-term trials are needed to evaluate the clinical impact this technology may have on therapeutic decision making. Given the limited evidence available, further research is needed before the technology can be disseminated.

Methylation status of CpG sites in the NOTCH4 promoter region regulates NOTCH4 expression in patients with tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease (CHD). Although a lower methylation level of whole genome has been demonstrated in TOF patients, little is known regarding the DNA methylation changes in specific gene and its associations with TOF development. NOTCH4 is a mediator of the Notch signalling pathway that plays an important role in normal cardiac development. However, the role of epigenetic regulation of the NOTCH4 gene in the pathogenesis of TOF remains unclear. Considering the NOTCH4 low mutation frequency and reduced expression in the TOF patients, we hypothesized that abnormal DNA methylation change of NOTCH4 gene may influence its expression and responsible for TOF development. In this study, we measured the promoter methylation status of NOTCH4 and was measured and its regulation mechanism was explored, which may be related to TOF disease. Additionally, the promoter methylation statuses of NOTCH4 was measured in order to further understand epigenetic mechanisms that may serve a role in the development of TOF. Immunohistochemical analysis was used to examine NOTCH4 expression in right ventricular outflow tract myocardial tissues in patients with TOF. Compared with healthy controls, patients with TOF displayed significantly reduced in NOTCH4 expression (P=0.0055). Moreover, bisulphite sequencing suggested that the methylation levels of CpG site 2 in the NOTCH4 promoter was significantly higher in the patients than in the controls (P=0.0459). NOTCH4 expression was negatively associated with CpG site 2 methylation levels (r=−0.51; P=0.01). ETS1 transcription factor can serve as transcriptional activators by binding to specific DNA sequences of target genes, such as DLL4 and NOTCH4, which serves an important role in normal heart development. Dual-luciferase reporter and electrophoretic mobility shift assays indicated that the ETS1 transcription factor could bind to the NOTCH4 promoter region. However, binding of ETS1 to the NOTCH4 promoter was abrogated by methylation at the putative ETS1 binding sites. These findings suggested that decreased NOTCH4 expression in patients with TOF may be associated with hypermethylation of CpG site 2 in the NOTCH4 promoter region, due to impaired binding of ETS1.

Gene expression profiling identifies the role of Zac1 in cervical cancer metastasis

The zinc-finger protein which regulates apoptosis and cell cycle arrest 1 (Zac1), encoded by Plagl1 gene, is a seven-zinc-finger containing transcription factor belonging to the imprinted genome and is expressed in diverse types of embryonic and adult human tissues. Zac1 is postulated to be a tumor suppressor by inducing cell cycle arrest and apoptosis through interacting and modulating transcriptional activity of p53 as it was named. Correspondingly, the reduction or loss of Zac1 expression is associated with the incidence and progression of several human tumors, including cervical cancer, breast cancer, ovarian cancer, pituitary tumors, and basal cell carcinoma, implying the rationality of utilizing Zac1 expression as novel a biomarker for the evaluation of cervical cancer prognosis. However, to date, it has not been elucidated whether Zac1 expression is related to the prognosis of patients in clinical cervical cancer tumor samples. To address the questions outlined above, we report here a comprehensive investigation of Zac1 expression in biopsies of clinical cervical carcinoma. By analyzing Zac1 expression in various gene expression profiling of cervical cancer databases, we show the association between high Zac1 expression and poor prognosis of cervical cancer. Functional enrichment analysis showed that high Zac1 expression was associated with epithelial-mesenchymal transition (EMT), which was further observed in clinical characteristics and metastatic carcinoma samples using immunohistochemical staining. Correspondingly, hypomethylation of CpG island on Zac1 promoter was observed in samples with high Zac1 expression in cervical carcinoma. Finally, overexpression of Zac1 in a variety of cervical cancer cell lines increase their mesenchymal biomarker expression and migration, strengthening the correlation between cervical cancers with high Zac1 expression and metastasis in clinical. In summary, this research firstly revealed that identifying Zac1 expression or the methylation status of CpG site on Zac1 promoter may provide us with novel indicators for the evaluation of cervical cancer metastasis.

Genome-wide identification of methylated CpG sites in nongenital cutaneous warts

BackgroundLow-risk HPV infection has not been the subject of epigenetic investigation. The present study was carried out in order to investigate the methylation status of CpG sites in non-genital cutaneous warts.MethodsGenomic DNA was extracted from 24 paired epidermal samples of warts and normal skin. DNA samples were bisulfite converted and underwent genome-wide methylation profiling using the Infinium MethylationEPIC BeadChip Kit.ResultsFrom a total of 844,234 CpG sites, 56,960 and 43,040 CpG sites were found to be hypo- and hypermethylated, respectively, in non-genital cutaneous warts. The most differentially methylated CpG sites in warts were located within the C10orf26, FAM83H-AS1, ZNF644, LINC00702, GSAP, STAT5A, HDAC4, NCALD, and EXOC4 genes.ConclusionNon-genital cutaneous warts exhibit a unique CpG methylation signature.

Aberrant DNA methylation in the PAX2 promoter is associated with Müllerian duct anomalies.

Abnormalities during Müllerian duct and female reproductive tract formation during embryonic development result in Müllerian duct anomalies (MDA). Previous studies have identified a role for mutations in related genes and DNA copy number variation (CNV). However, the correlation between gene methylation and MDA remains to be understood. Endometrial tissues were collected from patients with septate (n = 23) or normal uterus (n = 28). We detected the methylation status of CpG sites and mRNA levels of nine candidate genes, including HOXA10, EMX2, TP63, ITGB3, PAX2, LHX1, GSC, WNT4, and H19, using MethyTarget and quantitative real-time polynucleotide chain reaction (qRT-PCR), respectively RESULTS: Compared with healthy controls, we detected three hypomethylated CpG sites (P < 0.05) and increased mRNA levels of PAX2 (P < 0.05) in individuals with MDA. HOXA10, EMX2, TP63, ITGB3, LHX1, and GSC had 1, 1, 2, 1, 5, and 2 differentially methylated CpG sites (P < 0.05), respectively, but there were no significant differences in their mRNA levels (P > 0.05). WNT4 and H19 did not show differences in methylation (P > 0.05) and mRNA levels (P > 0.05). Aberrant DNA methylation within the promoter of PAX2 may contribute to the development of MDA by regulating its gene expression. However, the methylation status of HOXA10, EMX2, TP63, ITGB3, LHX1, GSC, WNT4, and H19, may not contribute to the development of MDA.

DNA Methylation Analysis of AKT1 Promoter and HTR2A EXON-I of Malaysian Schizophrenia Multiplex Families with Lower Cognitive Performance

Dysfunction of cognitive performance in schizophrenia has been associated with aberrant alteration of DNA methylation of several schizophrenia-risk genes. AKT1 and HTR2A are among the candidate genes for schizophrenia. Their expressions were found reduced in schizophrenia patients. Thus, we aimed to study the methylation status of AKT1 promoter and HTR2A exon-I in Malaysian schizophrenia patients and their affected family members. In this study, each participant was required to perform Trail Making Test (TMT) part A and B to measure their cognitive performance. Genomic DNA extracted from the peripheral blood of 12 Malaysian schizophrenia families and 12 controls families, was subjected to bisulfite conversion. The methylation status of CpG sites of AKT1 promoter at Chr14: 104796054 and HTR2A exon-I at Chr13: 46896918 were identified using methylation-specific polymerase chain reaction (MSP). Our results showed that schizophrenia patients performed worse in both TMT-A and B (p<0.0001) than healthy controls. The patients also displayed significantly (p=0.023) high level of methylation in AKT1 promoter compared to controls. Meanwhile, no significant difference (p=0.248) in methylation status was observed in HTR2A exon-I between schizophrenia and control groups. Therefore, methylation of AKT1 promoter in peripheral bloods of patients may involve in cognitive impairment and schizophrenia pathology. In addition, we were able to demonstrate the heritability of DNA methylation status across family members.