Epigenetic Modifications Of DNA Research Articles

Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy.

Epigenetic modifications of DNA, including methylation, hydroxymethylation, formylation, and carboxylation of cytosines, are proposed to function in gene regulation during reproduction and development. Changes in cytosine methylation are associated with a range of diseases, such as cancer. Immunofluorescence uses specific antibodies to quantitatively detect the global amount of cytosine modifications by fluorescence microscopy. The most critical stage of immunofluorescence is the antigen retrieval to remove the protein content around the DNA, allowing specific antibodies to bind to DNA epitopes. Acid treatments have commonly been used for antigen retrieval. Previously, trypsin was added after acid in the protocol, which increased the amount of detectable DNA methylation. In this study, the protocol was further enhanced by the addition of pepsin, which is able to target charged hydrophobic amino acids in proteins, unlike trypsin, which breaks positive hydrophilic amino acids. The global levels of cytosine modifications in CF-1, HeLa, and AR42J cells were compared using this protocol. In all cells, the sequential treatment of trypsin and pepsin increased the specificity of the staining. With the synergistic effect of the two enzymes, it is possible to target different protein groups packaging DNA molecules and removing them effectively. The findings suggest that this revised protocol can be conveniently used for each cytosine modification in the cells examined, and should be optimized for other cells. These new antigen retrieval conditions may more accurately detect the changes in cytosine modifications during development and in diseases.

Polyurethane foam induces epigenetic modification of mitochondrial DNA during different metamorphic stages of Tenebrio molitor

The present work investigated the changes in DNA methylation pattern of Tenebrio molitor mitochondria genome at different development stages, which was fed with polyurethane foam as a sole diet. Polyurethane foam could influence the global methylation levels in mitochondria DNA of Tenebrio molitor. Different leves of 5-methylcytosine appeared at CpG and non-CpG sites of Tenebrio molitor mtDNA while they were fed with polyurethane foam: 10 CpG and 49 non-CpG sites at larval stage, 4 CpG and 31 non-CpG sites at pupa stage, 7 CpG and 56 non-CpG sites at adult stage in general. Moreover, we observed the decreased levels of ATP generation with the mitochondria DNA methylation variation. The results demonstrated that mitochondria DNA gene could be methylated in response to environmental pollutants to modulate stage-specific functions. Moreover, mtDNA methylation of polyurethane-foam-feeding Tenebrio molitor existed discrepancy in the developmental stage. The tentative methylation mechanism of mtDNA might be that polyurethane foam induced oxidative stress and increased the permeability of mitochondrial membranes, which resulted in transmethylase entry into mitochondria.

Substituted anthraquinones represent a potential scaffold for DNA methyltransferase 1-specific inhibitors.

In humans, the most common epigenetic DNA modification is methylation of the 5-carbon of cytosines, predominantly in CpG dinucleotides. DNA methylation is an important epigenetic mark associated with gene repression. Disruption of the normal DNA methylation pattern is known to play a role in the initiation and progression of many cancers. DNA methyltransferase 1 (DNMT1), the most abundant DNA methyltransferase in humans, is primarily responsible for maintenance of the DNA methylation pattern and is considered an important cancer drug target. Recently, laccaic acid A (LCA), a highly substituted anthraquinone natural product, was identified as a direct, DNA-competitive inhibitor of DNMT1. Here, we have successfully screened a small library of simplified anthraquinone compounds for DNMT1 inhibition. Using an endonuclease-coupled DNA methylation assay, we identified two anthraquinone compounds, each containing an aromatic substituent, that act as direct DNMT1 inhibitors. These simplified anthraquinone compounds retain the DNA-competitive mechanism of action of LCA and exhibit some selectivity for DNMT1 over DNMT3a. The newly identified compounds are at least 40-fold less potent than LCA, but have significantly less complex structures. Collectively, this data indicates that substituted anthraquinone compounds could serve as a novel scaffold for developing DNMT1-specific inhibitors.

Abstract 4102: DNA methylation and repressive histones in the promoters of immune checkpoints in tumor tissues and peripheral blood of breast and colorectal cancer patients

Abstract Higher expression of immune checkpoints in tumor microenvironment plays significant roles in inhibiting anti-tumor immunity, which is associated with poor prognosis and progression of cancer patients. Major epigenetic modifications in both DNA and histone could be involved in upregulation of immune checkpoints (ICs) in cancer. We investigated transcriptomic expression of different ICs and some ligands including PD-L1 and galectin-9 in the tumor microenvironment (TME) and in circulation of primary breast cancer (PBC) and colorectal cancer (CRC) patients. We found that transcriptomic expressions of PD-1, CTLA-4, TIM-3 and LAG-3 in PBC and PD-1, CTLA-4, TIM-3, TIGIT, PD-L1 and galectin-9 in CRC were significantly upregulated in tumor tissues (TT), compared with normal tissues (NT). Additionally, expressions of TIM-3, TIGIT, and PD-L1 were significantly upregulated, while LAG-3 was downregulated in peripheral blood of both PBC and CRC patients. We then investigated the epigenetic modifications beyond this upregulation of immune checkpoint genes. Interestingly, we found that CpG islands in the promoters of PD-1, CTLA-4 and TIM-3 in PBC, and CTLA-4 and TIGIT in CRC patients were significantly hypomethylated in tumor compared with normal tissues. Notably, CpGs in PD-L1 promoter were completely hypomethylated in PBC and CRC in both TT and NT. In addition, PD-L1 was hypomethylated in in peripheral blood of both PBC and CRC patients, while TIGIT was hypomethylated only in circulation of CRC patients, compared with healthy donors. Next, we checked the abundance of repressive histones (H3K9me3 and H3K27me3) in the promoter regions of ICs/ligands. We found that in PBC TT, distribution of both H3K9me3 and H3K27me3 was lower in the promoters of PD-1, CTLA-4 and LAG-3, while distribution of only H3K27me3 was lower in TIM-3 promoter, compared with NT. Furthermore, in CRC TT, the distribution of H3K9me3 was lower in PD-1 and TIGIT promoters, and H3K27me3 was lower in CTLA-4 promoter, while both H3K9me3 and H3K27me3 were lower in TIM-3 promoter, compared with NT. Our data demonstrate that both DNA methylation and repressive histones are involved in upregulation of ICs and their ligands in the tumor microenvironment and peripheral blood of breast and colorectal cancer patients. These epigenetic modifications could be useful as diagnostic/prognostic biomarkers and/or therapeutic targets in breast and colorectal cancers. Citation Format: Eyad Elkord, Varun Sasidharan Nair, Salman M. Toor. DNA methylation and repressive histones in the promoters of immune checkpoints in tumor tissues and peripheral blood of breast and colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4102.

Recent Advances in Use of Topoisomerase Inhibitors in Combination Cancer Therapy.

Inhibitors targeting human topoisomerase I and topoisomerase II alpha have provided a useful chemotherapy option for the treatment of many patients suffering from a variety of cancers. While the treatment can be effective in many patient cases, use of these human topoisomerase inhibitors is limited by side-effects that can be severe. A strategy of employing the topoisomerase inhibitors in combination with other treatments can potentially sensitize the cancer to increase the therapeutic efficacy and reduce resistance or adverse side effects. The combination strategies reviewed here include inhibitors of DNA repair, epigenetic modifications, signaling modulators and immunotherapy. The ongoing investigations on cellular response to topoisomerase inhibitors and newly initiated clinical trials may lead to adoption of novel cancer therapy regimens that can effectively stop the proliferation of cancer cells while limiting the development of resistance.

Integrative Analysis of Genome-Wide Association Studies and DNA Methylation Profile Identified Genetic Control Genes of DNA Methylation for Kashin-Beck Disease.

Epigenetic modifications of DNA are regarded as a crucial factor for understanding the molecular basis of complex phenotypes. This study aims to uncover insight into the epigenetic modifications for Kashin-Beck disease (KBD) by integrating genome-wide association studies (GWAS), methylation quantitative trait loci (meQTLs), and DNA methylation profiles data. The knee articular cartilages of 5 KBD patients and 5 healthy controls were collected for DNA methylation profiling, using Illumina Infinium HumanMethylation450 BeadChip. Mass spectrograph validation of identified differently methylated genes was conducted using independent samples of 4 KBD patients and 3 healthy controls, together with a previous sample of 2743 Han Chinese individuals of GWAS study for KBD and a study of 697 normal subjects for meQTLs annotation datasets. KBD GWAS single nucleotide polymorphisms (SNPs) and normal meQTLs SNPs were integrated with DNA methylation profiles of KBD articular cartilage to identify genetic control (GC) genes of DNA methylation for KBD. Quantitative polymerase chain reaction (qPCR) was performed to validate the mRNA expression of several identified candidate genes. A total of 162 CpG sites, 253 SNPs, and 123 GC genes for KBD were identified. Enrichment analysis detected 642 marked GO terms and 19 KEGG pathways (P < 0.05). Six potential key GC genes were conducted for qPCR experiment (ERG, MN1, MITF, WISP1, TRIO, and NOSTRIN). The results suggest that GC genes of DNA methylation may lead to the erosion of cartilage in KBD, which may help us in understanding the epigenetic alteration of KBD.

Drugs Targeting Epigenetic Modifications and Plausible Therapeutic Strategies Against Colorectal Cancer.

Genetic variations along with epigenetic modifications of DNA are involved in colorectal cancer (CRC) development and progression. CRC is the fourth leading cause of cancer-related deaths worldwide. Initiation and progression of CRC is the cumulation of a variety of genetic and epigenetic changes in colonic epithelial cells. Colorectal carcinogenesis is associated with epigenetic aberrations including DNA methylation, histone modifications, chromatin remodeling, and non-coding RNAs. Recently, epigenetic modifications have been identified like association of hypermethylated gene Claudin11 (CLDN11) with metastasis and prognosis of poor survival of CRC. DNA methylation of genes CMTM3, SSTR2, MDF1, NDRG4 and TGFB2 are potential epigenetic biomarkers for the early detection of CRC. Tumor suppressor candidate 3 (TUSC3) mRNA expression is silenced by promoter methylation, which promotes epidermal growth factor receptor (EGFR) signaling and rescues the CRC cells from apoptosis and hence leading to poor survival rate. Previous scientific evidences strongly suggest epigenetic modifications that contribute to anticancer drug resistance. Recent research studies emphasize development of drugs targeting histone deacetylases (HDACs) and DNA methyltransferase inhibitors as an emerging anticancer strategy. This review covers potential epigenetic modification targeting chemotherapeutic drugs and probable implementation for the treatment of CRC, which offers a strong rationale to explore therapeutic strategies and provides a basis to develop potent antitumor drugs.

Enzymatic Synthesis of Base-Functionalized Nucleic Acids for Sensing, Cross-linking, and Modulation of Protein-DNA Binding and Transcription.

Protein-DNA interactions are important in replication, transcription, repair, as well as epigenetic modifications of DNA, which involve methylation and demethylation of DNA resulting in regulation of gene expression. Understanding of these processes and chemical tools for studying and perhaps even modulating them could be of great relevance and importance not only in chemical biology but also in real diagnostics and treatment of diseases. In the past decade, we have been working on development of synthesis of base-modified 2'-deoxyribo- or ribonucleoside triphosphates (dNTPs or NTPs) and their use in enzymatic synthesis of modified nucleic acids using DNA or RNA polymerases. These synthetic and enzymatic methods are briefly summarized with focus on recent development and outlining of scope, limitations, and further challenges. The main focus of this Account is on applications of base-modified nucleic acids in sensing of protein-DNA interactions, in covalent cross-linking to DNA-binding proteins ,and in modulation of protein-DNA binding and transcription. Several environment-sensitive fluorescent nucleotides were incorporated to DNA probes which responded to protein binding by light-up, changing of color, or lifetime of fluorescence. Using a cyclodextrin-peptide transporter, fluorescent nucleotides can be transported through the cell membrane and incorporated to genomic DNA. Several dNTPs bearing reactive groups (i.e., vinylsulfonamide or chloroacetamide) were used for polymerase synthesis of DNA reactive probes which cross-link to Cys, His, or Lys in peptides or proteins. An attractive challenge is to use DNA modifications and bioorthogonal reactions in the major groove of DNA for modulation and switching of protein-DNA interactions. We have systematically explored the influence of major-groove modifications on recognition and cleavage of DNA by restriction endonucleases and constructed simple chemical switches of DNA cleavage. Systematic study of the influence of major-groove modifications on transcription with bacterial RNA polymerases revealed not only that some modified bases are tolerated, but also that the presence of 5-hydroxymethyluracil or -cytosine can even enhance the transcription (350 or 250% compared to native DNA). Based on these results, we have constructed the first chemical switch of transcription based on photocaging of hydroxymethylpyrimidines in DNA by 2-nitrobenzyl protection (transcription off), photochemical deprotection of the DNA (transcription on), and enzymatic phosphorylation (only for 5-hydroxymethyluracil, transcription off). Although it has been so far demonstrated only in vitro, it is the proof-of-principle first step toward chemical epigenetics.

Slowing Down Ageing: The Role of Nutrients and Microbiota in Modulation of the Epigenome.

The human population is getting ageing. Both ageing and age-related diseases are correlated with an increased number of senescent cells in the organism. Senescent cells do not divide but are metabolically active and influence their environment by secreting many proteins due to a phenomenon known as senescence associated secretory phenotype (SASP). Senescent cells differ from young cells by several features. They possess more damaged DNA, more impaired mitochondria and an increased level of free radicals that cause the oxidation of macromolecules. However, not only biochemical and structural changes are related to senescence. Senescent cells have an altered chromatin structure, and in consequence, altered gene expression. With age, the level of heterochromatin decreases, and less condensed chromatin is more prone to DNA damage. On the one hand, some gene promoters are easily available for the transcriptional machinery; on the other hand, some genes are more protected (locally increased level of heterochromatin). The structure of chromatin is precisely regulated by the epigenetic modification of DNA and posttranslational modification of histones. The methylation of DNA inhibits transcription, histone methylation mostly leads to a more condensed chromatin structure (with some exceptions) and acetylation plays an opposing role. The modification of both DNA and histones is regulated by factors present in the diet. This means that compounds contained in daily food can alter gene expression and protect cells from senescence, and therefore protect the organism from ageing. An opinion prevailed for some time that compounds from the diet do not act through direct regulation of the processes in the organism but through modification of the physiology of the microbiome. In this review we try to explain the role of some food compounds, which by acting on the epigenetic level might protect the organism from age-related diseases and slow down ageing. We also try to shed some light on the role of microbiome in this process.

TDG Gene Polymorphisms and Their Possible Association with Colorectal Cancer: A Case Control Study.

Genetic alterations that might lead to colorectal cancer involve essential genes including those involved in DNA repair, inclusive of base excision repair (BER). Thymine DNA glycosylase (TDG) is one of the most well characterized BER genes that catalyzes the removal of thymine moieties from G/T mismatches and is also involved in many cellular functions, such as the regulation of gene expression, transcriptional coactivation, and the control of epigenetic DNA modification. Mutation of the TDG gene is implicated in carcinogenesis. In the present study, we aimed to investigate the association between TDG gene polymorphisms and their involvement in colon cancer susceptibility. One hundred blood samples were obtained from colorectal cancer patients and healthy controls for the genotyping of seven SNPs in the TDG gene. DNA was extracted from the blood, and the polymorphic sites (SNPs) rs4135113, rs4135050, rs4135066, rs3751209, rs1866074, and rs1882018 were investigated using TaqMan genotyping. One of the six TDG SNPs was associated with an increased risk of colon cancer. The AA genotype of the TDG SNP rs4135113 increased the risk of colon cancer development by more than 3.6-fold, whereas the minor allele A increased the risk by 1.6-fold. It also showed a 5-fold higher risk in patients over the age of 57. SNP rs1866074 showed a significant protective association in CRC patients. The GA genotype of TDG rs3751209 was associated with a decreased risk in males. There is a significant relationship between TDG gene function and colorectal cancer progression.

Advances in the Profiling of Single‐Cell DNA Modifications

AbstractEpigenetic modifications on DNA include canonical 5‐methylcytosine (5mC) and its iteratively oxidized derivatives mediated by TET dioxygenase: 5‐hydroxymethylcytosine (5hmC), 5‐formylcytosine (5fC), and 5‐carboxycytosine (5caC). All of them have been recognized to play important roles in gene expression regulation and to be indispensable for normal mammalian reproduction and development. In order to understand the mechanisms underlying these roles, over the past few decades, technical advances have been made to comprehensively profile the distribution pattern of bulk epigenetic modifications in genomic DNA. Recently, single‐cell methods have identified the epigenetic patterns even within an individual cell. This review focuses on single‐cell DNA epigenetic modifications sequencing technologies and their applications, and gives prospects on the development of single‐cell methods based on bisulfite‐free strategy.

Label-Free Raman Observation of TET1 Protein-Mediated Epigenetic Alterations in DNA.

Epigenetic modifications of DNA are known to modulate gene activity and expression and are believed to result in genetic diseases, such as cancer. Four modified cytosines were discovered in mammalian genomes: 5-methycytoine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC). They are regarded as DNA epigenetic markers and play key roles in the regulation of the dynamic balance between DNA methylation and demethylation. Although detection approaches toward 5mC are ubiquitous, few assays have reported the simultaneous determination of all four modified cytosines as well as monitoring of their dynamic alterations. Here, we developed a label-free surface enhanced Raman spectroscopy (SERS)-based method for directly sensing the four DNA modifications by using a plasmonic gold nanohole array (PGNA) with well-controlled hot spots and an open surface as the substrate. This method is based on identifying SERS spectral features resulting from DNA base modifications. Our study shows that 5mC, 5hmC, 5fC, and 5caC exhibit distinct Raman spectroscopic signatures at 785, 660, 1450, and 1680 cm-1, respectively. Moreover, the developed method can be used for tracking of the dynamic alterations among these four modified cytosines in DNA mediated by the ten-eleven translocation (TET) protein. The dynamic stepwise conversion from 5mC into 5hmC, 5fC, and 5caC is further demonstrated to be a typical three-step consecutive reaction with rate constants of 0.6, 0.25, and 0.15 min-1, respectively, which has not been achieved before via a SERS-based method.

Role of ten-eleven translocation proteins and 5-hydroxymethylcytosine in hepatocellular carcinoma.

In mammals, methylation of the 5th position of cytosine (5mC) seems to be a major epigenetic modification of DNA. This process can be reversed (resulting in cytosine) with high efficiency by dioxygenases of the ten‐eleven translocation (TET) family, which perform oxidation of 5mC to 5‐hydroxymethylcytosine (5hmC), 5‐formylcytosine and 5‐carboxylcytosine. It has been demonstrated that these 5mC oxidation derivatives are in a dynamic state and have pivotal regulatory functions. Here, we comprehensively summarized the recent research progress in the understanding of the physiological functions of the TET proteins and their mechanisms of regulation of DNA methylation and transcription. Among the three TET genes, TET1 and TET2 expression levels have frequently been shown to be low in hepatocellular carcinoma (HCC) tissues and received most attention. The modulation of TET1 also correlates with microRNAs in a post‐transcriptional regulatory process. Additionally, recent studies revealed that global genomic 5hmC levels are down‐regulated in HCC tissues and cell lines. Combined with the reported results, identification of 5hmC signatures in HCC tissues and in circulating cell‐free DNA will certainly contribute to early detection and should help to design therapeutic strategies against HCC. 5hmC might also be a novel prognostic biomarker of HCC. Thus, a detailed understanding of the molecular mechanisms resulting in the premalignant and aggressive transformation of TET proteins and cells with 5hmC disruption might help to develop novel epigenetic therapies for HCC.

Research advances in TET enzyme and its intermediate product 5hmC

DNA methylation is a significant epigenetic modification mode, which plays an important role in embryo reprogramming, stem cell differentiation and tumor occurrence. The ten-eleven translocation (TET) enzyme is a crucial demethylation enzyme, which can catalyze 5-methylcytosine(5mC) to 5-hydroxymethylcytosine(5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine(5caC). These bases represent the epigenetic modifications of DNA and regulate the process of DNA methylation. Understanding the role of TET enzyme in regulating the DNA methylation modification and gene expression can help us to gain the knowledge for the normal growth development and epigenetic regulation in human diseases.

Detection and manipulation of methylation in blood cancer DNA using terahertz radiation

DNA methylation is a pivotal epigenetic modification of DNA that regulates gene expression. Abnormal regulation of gene expression is closely related to carcinogenesis, which is why the assessment of DNA methylation is a key factor in cancer research. Terahertz radiation may play an important role in active demethylation for cancer therapy because the characteristic frequency of the methylated DNA exists in the terahertz region. Here, we present a novel technique for the detection and manipulation of DNA methylation using terahertz radiation in blood cancer cell lines. We observed the degree of DNA methylation in blood cancer at the characteristic resonance of approximately 1.7 THz using terahertz time-domain spectroscopy. The terahertz results were cross-checked with global DNA methylation quantification using an enzyme-linked immunosorbent assay. We also achieved the demethylation of cancer DNA using high-power terahertz radiation at the 1.7-THz resonance. The demethylation degrees ranged from 10% to 70%, depending on the type of cancer cell line. Our results show the detection of DNA methylation based on the terahertz molecular resonance and the manipulation of global DNA methylation using high-power terahertz radiation. Terahertz radiation may have potential applications as an epigenetic inhibitor in cancer treatment, by virtue of its ability to induce DNA demethylation, similarly to decitabine.

Epigenetic DNA Modifications Are Correlated With B Chromosomes and Sex in the Cichlid Astatotilapia latifasciata.

Supernumerary B chromosomes are dispensable elements found in several groups of eukaryotes, and their impacts in host organisms are not clear. The cichlid fish Astatotilapia latifasciata presents one or two large metacentric B chromosomes. These elements affect the transcription of several classes of RNAs. Here, we evaluated the epigenetic DNA modification status of B chromosomes using immunocytogenetics and assessed the impact of B chromosome presence on the global contents of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) and the molecular mechanisms underlying these variations. We found that the B chromosome of A. latifasciata has an active pattern of DNA epimarks, and its presence promotes the loss of 5mC in gonads of females with B chromosome (FB+) and promotes the loss of 5hmC in the muscle of males with the B element (MB+). Based on the transcriptional quantification of DNA modification genes (dnmt, tet, and tdg) and their candidate regulators (idh genes, microRNAs, and long non-coding RNAs) and on RNA-protein interaction prediction, we suggest the occurrence of passive demethylation in gonads of FB+ and 5hmC loss by Tet inhibition or by 5hmC oxidation in MB+ muscle. We suggest that these results can also explain the previously reported variations in the transcription levels of several classes of RNA depending on B chromosome presence. The DNA modifications detected here are also influenced by sex. Although the correlation between B chromosomes and sex has been previously reported, it remains unexplained. The B chromosome of A. latifasciata seems to be active and impacts cell physiology in a very complex way, including at the epigenetic level.

Common cancer genetic analysis methods and application study based on TCGA database

The development of second-generation sequencing (NGS) technology is providing numerous data which shifts the focus of cancer research from the sequencing of multi-species to the analysis and comparison of select data via high-throughput sequencing. The NGS also facilitates the diversity of available genetic data analysis methods, the constant optimization and innovation of analytical approaches for high-throughput genomics as well as the rapid development of genetic data mining and analysis models. The Cancer Genome Atlas (TCGA) database is a direct result of this work. The TCGA database provides a comprehensive record of genetic data collected from a tumor patient's sample, including its DNA sequence, transcriptional information, epigenetic modification and related. This review elaborates the latest progress in both the mining algorithm and analysis methods for tumor genomics. Specially, we introduce and review the TCGA database and data analysis approaches while demonstrating its applicability using representative cases. This review may shed light on new tumor-related targets discovery for researchers by means of bid data.

Transcriptional activity of epigenetic remodeling genes declines in keratinocytes after in vitro expansion

PurposeIn vitro expansion is an invaluable method to obtain keratinocytes in amounts necessary for effective transplantation therapies. In vitro cell culturing provokes questions concerning potential epigenetic alterations occurring in expanded cells in the context of usefulness for transplantation and safety. The purpose of this study was to investigate as to whether keratinocyte expansion is associated with changes in the activity of genes responsible for the maintenance of epigenetic stability. Materials and methodsWe focussed on the transcriptional activity of genes involved in different epigenetic mechanisms including DNA methylation and histone modifications. We used quantitative real-time PCR to determine transcript levels of 16 epigenetic remodelling markers in 14 patients in the epidermal cells directly after collection and after in vitro expansion. ResultsWe observed a remarkable decline in the transcriptional activity of the epigenetic remodelling genes following in vitro expansion, while no further fall of expression with passaging. In whole skin, we found even higher expression levels of the epigenetic markers. ConclusionsTransmission to in vitro environment challenges cellular signalling and metabolism. The regulation of epigenetic remodelling maintains the balance between cellular plasticity and phenotype deviation. This preliminary research demonstrated reduced activity of genes responsible for epigenetic modifications of DNA and histones in in vitro expanded epidermal cells. This observation indicates that epigenome re-patterning in cultured epidermal cells is significantly less intensive than in the skin. Also, this observation may imply that after adaptation to in vitro conditions, the epigenome does not undergo extensive transformation during further cultivation.

Tight control of genomic phosphorothioate modification by the ATP-modulated autoregulation and reusability of DndB.

DNA phosphorothioate (PT) modification was recently identified to occur naturally in diverse bacteria and to be governed by DndABCDE proteins. The nuclease resistance as well as the redox and nucleophilic properties of PT sulfur make PT modification a versatile player in restriction-modification (R-M) defense, epigenetic regulation, environmental fitness and the maintenance of cellular redox homeostasis. In this study, we discovered that tight control of PT levels is mediated by the ATPase activity of DndB. The ATP-binding activity of DndB stimulates the dissociation of the DndB-DNA complex, allowing transcriptional initiation, whereas its ATP hydrolysis activity promotes the conversion of DndB-ATP to free DndB that is capable of rebinding to promoter DNA for transcriptional inhibition. Since sulfur incorporation is an ATP-consuming process, these activities provide an economical way to fine-tune PT modification in an ATP-sensing manner. To our knowledge, this ATP-mediated regulation is a rare example among DNA epigenetic modification systems; the features of autoregulation and the repeated usage of DndB allow the dedicated regulation of PT levels in response to cellular ATP concentrations, providing insight into PT function and its role in physiology.

MicroRNA-373 promotes the development of esophageal squamous cell carcinoma by targeting LATS2 and OXR1.

MicroRNA373 was highly expressed in many tumors including esophageal cancer. However, its molecular mechanism is still unclear, especially epigenetic modification, in esophageal squamous cell carcinoma (ESCC). In this study, we investigated serum levels of the miR-371-373 cluster in ESCC patients before and after surgical removal, and further focused on the expression level of miR-373-3p in tumor tissues of ESCC patients and its target genes. In addition, the epigenetic alterations of miR-373-3p promoter was analyzed. The expression levels of miR-371-5p and miR-373-3p were significantly increased in preoperative serum of ESCC patients compared with that of healthy volunteers (P<0.01); however, they dropped significantly after surgical removal (P<0.01). Compared with adjacent normal tissues, miR-373-3p also showed significant up-regulation in cancer tissues (P<0.05). The methylation levels of miR-373-3p promoter were 42.86% in ESCC cancer tissue and 66.67% in adjacent normal tissues. The low methylation of the miR-373-3p promoter may promote the expression of miR-373-3p. Large tumor suppressor 2 (LATS2) and oxidation resistance 1(OXR1) are predicted to be targets of miR-373-3p by the bioinformatics method. They are the genes in the Hippo and the p53 signaling pathway, respectively. Their respective upstream genes, neurofibromatosis type 2 (NF2) and Jun Kinase, and the downstream genes, transcriptional co-activator with PDZ-binding motif (TAZ) and caspase 9, were also detected. The expression of all these genes were significantly decreased in ESCC cancer tissues compared with adjacent normal tissues. This study shows that DNA epigenetic modification in the miR-373-3p promoter region and the Hippo and p53 signaling pathways play important roles during the miR-373-3p mediating ESCC development process.