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Epigenetic modifications, including DNA methylation and histone modifications, are involved in regulation of gene expression, and alterations in these modifications are implicated in cancer onset and progression. The specific pattern of DNA methylation depends on the balance between methylation and demethylation processes. Recent studies have shown that TET proteins play a key role in DNA demethylation. TET proteins (TET1, TET2, TET3) are iron(II) and α-ketoglutarate dependent dioxygenases, and their enzymatic activity involves hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine and further to 5-formylcytosine and 5-carboxylcytosine. These modified cytosines are removed by enzymes involved in DNA repair. However, the role of TETs in gene expression regulation is not limited to their catalytic activity. TETs can interact with proteins of complexes involved in the modification of histones (i.e. EZH2, OGT, Sin3a or HCF1) and by affecting their activity and, chromatin binding ability, they can cause changes in patterns of histone methylation, acetylation and O-GlcNAcylation. There is growing evidence that decreased expression of TET proteins and mutation in TET genes are associated with cancer onset and progression.

Toll-like receptor 4 (TLR4) is important in promoting the immune response in various cancers. Recently, TLR4 is highly expressed in a stage-dependent manner in gastric cancer, but the regulatory mechanism of TLR4 expression has been not elucidated it. Here, we investigated the mechanism underlying regulation of TLR4 expression through promoter methylation and histone modification between transcriptional regulation and silencing of the TLR4 gene in gastric cancer cells. Chromatin immunoprecipitation was carried out to screen for factors related to TLR4 methylation such as MeCP2, HDAC1, and Sp1 on the TLR4 promoter. Moreover, DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC) induced demethylation of the TLR4 promoter and increased H3K4 trimethylation and Sp1 binding to reactivate silenced TLR4. In contrast, although the silence of TLR4 activated H3K9 trimethylation and MeCP2 complex, combined treatment with TLR4 agonist and 5-aza-dC upregulated H3K4 trimethylation and activated with transcription factors as Sp1 and NF-κB. This study demonstrates that recruitment of the MeCP2/HDAC1 repressor complex increases the low levels of TLR4 expression through epigenetic modification of DNA and histones on the TLR4 promoter, but Sp1 activates TLR4 high expression by hypomethylation and NF-κB signaling in gastric cancer cells.

Carcinogenesis as well as cancer progression result from genetic and epigenetic changes of the genome that leads to dysregulation of transcriptional activity of genes. Epigenetic mechanisms in cancer cells comprise (i) post-translation histone modification (i.e., deacetylation and methylation); (ii) DNA global hypomethylation; (iii) promoter hypermethylation of tumour suppressor genes and genes important for cell cycle regulation, cell differentiation and apoptosis; and (iv) posttranscriptional regulation of gene expression by noncoding microRNA. These epigenetic aberrations can be readily reversible and responsive to both synthetic agents and natural components of diet. A source of one of such diet components are cruciferous vegetables, which contain high levels of a number of glucosinolates and deliver, after enzymatic hydrolysis, sulforaphane and other bioactive isothiocyanates, that are involved in effective up-regulation of transcriptional activity of certain genes and also in restoration of active chromatin structure. Thus a consumption of cruciferous vegetables, treated as a source of isothiocyanates, seems to be potentially useful as an effective cancer preventive factor or as a source of nutrients improving efficacy of standard chemotherapies. In this review an attempt is made to elucidate the role of sulforaphane in regulation of gene promoter activity through a direct down-regulation of histone deacetylase activity and alteration of gene promoter methylation in indirect ways, but the sulforaphane influence on non-coding micro-RNA will not be a subject of this review.

DNA methylation, an epigenetic control mechanism in mammals, is widely present in the intestinal tract during the differentiation and proliferation of epithelial cells. Cells in stem cell pools or villi have different patterns of DNA methylation. The process of DNA methylation is dynamic and occurs at many relevant regulatory elements during the rapid transition of stem cells into fully mature, differentiated epithelial cells. Changes in DNA methylation patterns most often take place in enhancer and promoter regions and are associated with transcription factor binding. During differentiation, enhancer regions associated with genes important to enterocyte differentiation are demethylated, activating gene expression. Abnormal patterns of DNA methylation during differentiation and proliferation in the intestinal tract can lead to the formation of aberrant crypt foci and destroy the barrier and absorptive functions of the intestinal epithelium. Accumulation of these epigenetic changes may even result in tumorigenesis. In the current review, we discuss recent findings on the association between DNA methylation and cell differentiation and proliferation in the small intestine and highlight the possible links between dysregulation of this process and tumorigenesis.

Successful therapeutic protein production in vitro and in vivo requires efficient and long-term transgene expression supported by optimized vector and transgene cis-regulatory sequence elements. This study provides a comparative analysis of CpG-rich, highly expressed, versus CpG-depleted, poorly expressed green fluorescent protein (GFP) reporter transgenes, transcribed by various promoters in two different cell systems. Long-term GFP expression from a defined locus in stable Chinese hamster ovary cells was clearly influenced by the combination of transgene CpG content and promoter usage, as shown by differential silencing effects on selection pressure removal among the cytomegalovirus (CMV) promoter and elongation factor (EF)-1α promoter. Whereas a high intragenic CpG content promoted local DNA methylation, CpG depletion rather accelerated transgene loss and increased the local chromatin density. On lentiviral transfer of various expression modules into epigenetically sensitive P19 embryonic pluripotent carcinoma cells, CMV promoter usage led to rapid gene silencing irrespective of the intragenic CpG content. In contrast, EF-1α promoter-controlled constructs showed delayed silencing activity and high-level transgene expression, in particular when the CpG-rich GFP reporter was used. Notably, GFP silencing in P19 cells could be prevented completely by the bidirectional, dual divergently transcribed A2UCOE (ubiquitously acting chromatin-opening element derived from the human HNRPA2B1-CBX3 locus) promoter. Because the level of GFP expression by the A2UCOE promoter was entirely unaffected by the intragenic CpG level, we suggest that A2UCOE can overcome chromatin compaction resulting from intragenic CpG depletion due to its ascribed chromatin-opening abilities. Our analyses provide insights into the interplay of the intragenic CpG content with promoter sequences and regulatory sequence elements, thus contributing toward the design of therapeutic transgene expression cassettes for future gene therapy applications.

Epigenetics is the study of the heritable changes on gene expression that are responsible for the regulation of development and that have an impact on several diseases. However, it is of equal importance to understand how epigenetic machinery works. DNA methylation is the most studied epigenetic mark and is generally associated with the regulation of gene expression through the repression of promoter activity and by affecting genome stability. Therefore, the ability of the cell to interpret correct methylation marks and/or the correct interpretation of methylation plays a role in many diseases. The major family of proteins that bind methylated DNA is the methyl-CpG binding domain proteins, or the MBDs. Here, we discuss the structure that makes these proteins a family, the main functions and interactions of all protein family members and their role in human disease such as psychiatric disorders and cancer.

Erasure and subsequent reinstatement of DNA methylation in the germline, especially at imprinted CpG islands (CGIs), is crucial to embryogenesis in mammals. The mechanisms underlying DNA methylation establishment remain poorly understood, but a number of post-translational modifications of histones are implicated in antagonizing or recruiting the de novo DNA methylation complex. In mouse oogenesis, DNA methylation establishment occurs on a largely unmethylated genome and in nondividing cells, making it a highly informative model for examining how histone modifications can shape the DNA methylome. Using a chromatin immunoprecipitation (ChIP) and genome-wide sequencing (ChIP-seq) protocol optimized for low cell numbers and novel techniques for isolating primary and growing oocytes, profiles were generated for histone modifications implicated in promoting or inhibiting DNA methylation. CGIs destined for DNA methylation show reduced protective H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3) in both primary and growing oocytes, while permissive H3K36me3 increases specifically at these CGIs in growing oocytes. Methylome profiling of oocytes deficient in H3K4 demethylase KDM1A or KDM1B indicated that removal of H3K4 methylation is necessary for proper methylation establishment at CGIs. This work represents the first systematic study performing ChIP-seq in oocytes and shows that histone remodeling in the mammalian oocyte helps direct de novo DNA methylation events.

Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

Aberrant epigenetic modification in the form of regional hypermethylation and global hypomethylation has been implicated in the dysregulation of gene expression in various cancers, including prostate cancer (PCa). Recently discovered 5-hydroxymethylated marks (5hmC), considered to be key intermediates in the process of DNA demethylation, have also been shown to exhibit significant global loss in solid tumors as compared to normal tissue. Similar to cytosine base methylation (5mC), 5hmC may also play a key role in the regulation of gene expression and, consequently, gene function in PCa. We hypothesize that dynamic interplay between 5mC and 5hmC enrichment in promoter, CpG island, and intragenic regions shows specific association patterns with gene expression. A systematic investigation of these methylation marks may provide novel insights into epigenomic architecture by elucidating novel pathways and candidate genes in prostate tumorigenesis. Using Next Generation Sequencing (NGS), we characterized genome-wide 5mC and 5hmC marks and RNA expression data in a normal prostate tissue-derived representative cell line (RWPE-1). We have completed NGS following methyl-binding protein capture (MBD-seq) and hydroxymethyl-selective chemical labeling (hMe-Seal), and have performed integrative analysis correlating enriched genes stratified by region (intergenic regions proximal to DNase I hypersensitivity sites, as well as promoter, CpG island, and intragenic regions) to expression data obtained from RNA-seq and also validated using publicly available microarray expression data (GEO Accession Number: GSM375783) We divided expression data into equal tiers representing genes with no expression, low expression, and high expression, and have performed pathway-based analysis on genes significantly enriched for either 5mC or 5hmC marks for each tier stratified by gene region. We found gene expression to exhibit a strong positive correlation with core promoter and CpG island hydroxymethylation, with a corresponding negative correlation to methylation of these regions. In contrast, gene expression was found to be positively correlated with both methylation and hydroxymethylation in both intragenic regions and intergenic regions proximal to ENCODE DNase I hypersensitivity sites (GEO Accession Number: GSM1008595), which are associated with open chromatin and may play a role in downstream gene regulation. Interestingly, pathway-based analysis of these stratified tiers, grouped via the Molecular Complex Detection algorithm (MCODE) to identify pathway clusters with high biological significance, showed 5hmC and 5mC enrichment of the same pathways in each expression tier, suggesting a possible co-operative role for intragenic co-expression of these marks in regulating biological pathways. Clustered pathways co-enriched in intragenic 5mC and 5hmC showed strong enrichment in Gene Ontology terms related to core cellular functions within each expression tier, such as ion channel and transport regulation, primary metabolic processes, and protein and RNA binding. Currently, we are investigating these pathways to identify key candidate genes regulated by 5hmC and 5mC marks in normal prostate. This is the first study to correlate locus-specific global 5hmC enrichment to expression in normal prostate cells. Our preliminary analysis has shown correlation between 5hmC and 5mC enrichment in genic, promoter, CpG island, and upstream DNase I hypersensitivity site-proximal regions and expression in RWPE-1 cells. Insights from this model will subsequently be explored via whole-genome differential analysis between normal prostate and PCa to determine the effect of epigenetic alterations in cancer on gene expression. Subsequently, our characterization of key cellular pathways exhibiting dynamic enrichment patterns for 5hmC or 5mC marks will potentially allow us to identify differentially epigenetically modified target genes implicated in prostate cancer tumorigenesis. Citation Format: Shivani N. Kamdar, Linh T. Ho, Ruth Isserlin, Ken J. Kron, Theodorus van der Kwast, Alexandre R. Zlotta, Neil E. Fleshner, Gary Bader, Bharati Bapat. Genome-wide integrative analysis correlating locus-specific DNA methylation and hydroxymethylation to gene expression in normal prostate cells. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-32.

Levels of γ-aminobutyric acid (GABA) and glutamic acid decarboxylase 1 (GAD1), the enzyme that synthesizes GABA, are significantly increased in neoplastic tissues. However, the mechanism underlying this increase remains elusive. Instead of silencing gene transcription, we showed that the GAD1 promoter was hypermethylated in both colon and liver cancer cells, leading to the production of high levels of GAD1. GAD1 is a target gene that is silenced by H3K27me3. The key locus responsible for GAD1 reactivation was mapped to a DNA methylation-sensitive CTCF-binding site (CTCF-BS3) within the third intron of GAD1. Chromosome configuration capture (3C) analysis indicated that an intrachromosomal loop was formed by CTCF self-dimerisation in normal cells (CTCF binds to both unmethylated CTCF-BS3 and CTCF-BS2). The CTCF dimer then interacted with suppressor of zeste 12 homologue (SUZ12), which is a domain of Polycomb repressive complex 2 (PRC2), promoting the methylation of H3K27 and the silencing of GAD1 expression. This silencing was shown to be inhibited by DNA methylation in cancer cells. These findings strongly suggest that GAD1 is reactivated by DNA methylation, which provided a model for DNA methylation and the active orchestration of oncogenic gene expression by CTCF in cancer cells.

Summary Chondrocytes play an essential role in endochondral bone development, which is requisite for mammalian skeletal development. During endochondral bone development, chondrocytes undergo well-organized stages of sequential differentiation, including proliferation and hypertrophy, a process harmoniously modulated by various transcription factors. Epigenetics, including DNA methylation and histone modification, has recently emerged as an essential regulatory system for gene expression, not only in physiological conditions, but also in human disease. During chondrocyte differentiation, transcriptional co-regulators, which form transcriptional protein complexes with specific transcription factors, are predominantly involved in this epigenetic process and cooperatively regulate chondrocyte gene expression. Importantly, several studies indicate that epigenetic regulators correlate with cartilage-related diseases, such as osteoarthritis, and are noted as a potential therapeutic target. Here, current studies of epigenetic regulation of chondrocyte differentiation are reviewed and novel aspects for the molecular mechanisms involved in chondrogenesis are introduced.

The P-element induced wimpy testis (Piwi) protein family, a subfamily of the Argonaute protein family, is involved in gene silencing and shows specific expression in spermatogenic cells. To reveal the transcriptional regulatory mechanisms of Piwil1 in chickens, we cloned sequences of the chicken Piwil1 promoter region and performed luciferase reporter and electrophoretic mobility shift assays to analyze the transcriptional activity and identify important transcriptional regulatory elements. The results showed that the region from -90 to -43 in the 5'-flanking region of Piwil1 contains a transcriptional regulatory CCAAT box that was necessary for the transcriptional activity of the Piwil1 promoter. Moreover, the transcription factor nuclear factor Y (NF-Y) was bound to the Piwil1 promoter CCAAT box specifically in germ cells. In addition, bisulfite sequencing to determine the methylation profile of the Piwil1 promoter CpG island in different spermatogenic and non-germ cell populations was performed. Compared with germ cells, non-germ cells showed increased methylation of the promoter region containing the CCAAT box, loss of NF-Y binding, and silencing of the Piwil1 locus. It is demonstrated that the specific expression of Piwil1 in chicken germ cells is regulated by the transcription factor NF-Y and differential CpG island methylation.

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

Transcription factors (TFs) are sequence-specific DNA-binding proteins acting as critical regulators of gene expression. The Plant Promoter Analysis Navigator (PlantPAN; http://PlantPAN2.itps.ncku.edu.tw) provides an informative resource for detecting transcription factor binding sites (TFBSs), corresponding TFs, and other important regulatory elements (CpG islands and tandem repeats) in a promoter or a set of plant promoters. Additionally, TFBSs, CpG islands, and tandem repeats in the conserve regions between similar gene promoters are also identified. The current PlantPAN release (version 2.0) contains 16 960 TFs and 1143 TF binding site matrices among 76 plant species. In addition to updating of the annotation information, adding experimentally verified TF matrices, and making improvements in the visualization of transcriptional regulatory networks, several new features and functions are incorporated. These features include: (i) comprehensive curation of TF information (response conditions, target genes, and sequence logos of binding motifs, etc.), (ii) co-expression profiles of TFs and their target genes under various conditions, (iii) protein-protein interactions among TFs and their co-factors, (iv) TF-target networks, and (v) downstream promoter elements. Furthermore, a dynamic transcriptional regulatory network under various conditions is provided in PlantPAN 2.0. The PlantPAN 2.0 is a systematic platform for plant promoter analysis and reconstructing transcriptional regulatory networks.

Environment-sensitive plant development follows a program inscribed in the DNA, and differential expression of the genes is the prerequisite of this program. In the last decades, it was possible to unravel how genes are correctly transcribed in time and space by interaction of trans-acting factors with cis elements often located in the promoter of a gene. However recently, we learned that, in addition, another level of control, called epigenetic regulation, acting via alterations in chromatin structure is involved. In this review, we will first summarise the basic mechanisms underlying epigenetic control of gene expression in plants. In the following article, we will focus on some selected recent results showing epigenetic control of major steps in plant development. This review cannot cover all new results in the field and we want to apologise to all who are not mentioned. Key Concepts Plant development involves coordinated expression of genes. Recent findings show that this is also under epigenetic control. Epigenetic control is exerted by different mechanisms, including DNA methylation, histone modifications and chromatin remodelling. These modifications alter chromatin state, and by this, it can induce or repress expression of genes. Major developmental steps, including seed development, flowering, sexual reproduction and leaf senescence are controlled by epigenetic mechanisms. Stress responses of plants are also regulated by epigenetic mechanisms. Keywords: chromatin; DNA methylation; epigenetic control; histone modifications

The potential use of human embryonic stem cells (hESCs) in cell-based therapies points out the critical importance of epigenomic evaluation for cell-based therapies. Specifically, DNA methylation appears to be a crucial player in establishing cell fate commitment and lineage choices. In this study, we report the global changes observed on the CpG islands distributed in promoters, gene bodies, and intergenic regions and the major biochemical pathways and genes involved in methylation changes as H9-hESCs turn into a neuronal culture containing medium-sized spiny striatal neurons (MSNs). Using an ontogeny-recapitulating protocol of striatal neuron differentiation, we analyzed DNA methylation profiles during the conversion from pluripotency to neuropotency up to the acquisition of a mature neuronal phenotype. H9-hESCs changed the methylation pattern both through de novo methylation and hypomethylation of specific gene promoters. Bioinformatic analysis allowed us to identify a panel of striatal-associated genes, which were regulated by DNA methylation and differentially expressed during striatal commitment. Importantly, DNA methylation analysis revealed that H9-hESCs did not acquire methylation-based oncogenic properties after differentiation. Indeed, hypermethylation of cancer-associated genes that characterize transformed cells, such as Polycomb repressive complex-associated genes, was not detected in the neuronal cultures. However, the oncosuppressor gene, BCL2L11, became hypermethylated in H9-hESC-derived mature neurons. Whole-genome DNA methylation profiling could become a technological platform to predict the differentiative potential of hESC-derived cultures and establish further biosafety assessment quality control tools of the cell-based products.

Recent evidence has shown that the level of 5-hydroxymethylcytosine (5 hmC) in chromosomal DNA is aberrantly decreased in a variety of cancers, but whether this decrease is a cause or a consequence of tumorigenesis is unclear. Here we show that, in gastric cancers, the 5 hmC decrease correlates with a decrease in ten-eleven translocation 1 (TET1) expression, which is strongly associated with metastasis and poor survival in patients with gastric cancer. In gastric cancer cells, TET1-targeted siRNA induced a decrease in 5 hmC, whereas TET1 overexpression induced an increase in 5 hmC and reduced cell proliferation, thus correlating decreased 5 hmC with gastric carcinogenesis. We also report the epigenetic signatures responsible for regulating TET1 transcription. Methyl-CpG Binding Domain Sequencing and Reduced Representation Bisulfite Sequencing identified unique CpG methylation signatures at the CpG island 3'-shore region located 1.3 kb from the transcription start site of TET1 in gastric tumor cells but not in normal mucosa. The luciferase activity of constructs with a methylated 3'-shore sequence was greatly decreased compared with that of an unmethylated sequence in transformed gastric cancer cells. In gastric cancer cells, dense CpG methylation in the 3'-shore was strongly associated with TET1 silencing and bivalent histone marks. Thus, a decrease in 5 hmC may be a cause of gastric tumorigenesis owing to a decrease in TET1 expression through DNA methylation coupled with bivalent marks in the 3'-shore of TET1.

DNA methylation is an essential epigenetic mechanism involved in many essential cellular processes. During development epigenetic reprograming takes place during gametogenesis and then again in the pre-implantation embryo. These two reprograming windows ensure genome-wide removal of methylation in the primordial germ cells so that sex-specific signatures can be acquired in the sperm and oocyte. Following fertilization the majority of this epigenetic information is erased to give the developing embryo an epigenetic profile coherent with pluripotency. It is estimated that ∼65% of the genome is differentially methylated between the gametes, however following embryonic reprogramming only parent-of-origin methylation at known imprinted loci remains. This suggests that trans-acting factors such as Zfp57 can discriminate imprinted differentially methylated regions (DMRs) from the thousands of CpG rich regions that are differentially marked in the gametes. Recently transient imprinted DMRs have been identified suggesting that these loci are also protected from pre-implantation reprograming but succumb to de novo remethylation at the implantation stage. This highlights that "ubiquitous" imprinted loci are also resilient to gaining methylation by protecting their unmethylated alleles. In this review I examine the processes involved in epigenetic reprograming and the mechanisms that ensure allelic methylation at imprinted loci is retained throughout the life of the organism, discussing the critical differences between mouse and humans. This article is part of a Directed Issue entitled: Epigenetics Dynamics in development and disease.

CpG islands (CGIs) are one of the most widely studied regulatory features of the human genome, with critical roles in development and disease. Despite such significance and the original epigenetic definition, currently used CGI sets are typically predicted from DNA sequence characteristics. Although CGIs are deeply implicated in practical analyses of DNA methylation, recent studies have shown that such computational annotations suffer from inaccuracies. Here we used whole-genome bisulfite sequencing from 10 diverse human tissues to identify a comprehensive, experimentally obtained, single-base resolution CGI catalog. In addition to the unparalleled annotation precision, our method is free from potential bias due to arbitrary sequence features or probe affinity differences. In addition to clarifying substantial false positives in the widely used University of California Santa Cruz (UCSC) annotations, our study identifies numerous novel epigenetic loci. In particular, we reveal significant impact of transposable elements on the epigenetic regulatory landscape of the human genome and demonstrate ubiquitous presence of transcription initiation at CGIs, including alternative promoters in gene bodies and non-coding RNAs in intergenic regions. Moreover, coordinated DNA methylation and chromatin modifications mark tissue-specific enhancers at novel CGIs. Enrichment of specific transcription factor binding from ChIP-seq supports mechanistic roles of CGIs on the regulation of tissue-specific transcription. The new CGI catalog provides a comprehensive and integrated list of genomic hotspots of epigenetic regulation.

The levels of allopregnanolone (ALLO), a neurosteroid, in brain and serum are related to severity of depression and anxiety. Steroid 5α-reductase type I is the rate-limiting enzyme in ALLO biosynthesis and plays an important role in control of the ALLO level in mammalian brain. In this study, we examined an epigenetic mechanism for transcriptional regulation of srd5a1, which codes for steroid 5α-reductase type I, using isolation-reared mice. The mRNA level of srd5a1 was decreased in the prefrontal cortex (PFC) in isolation-reared mice. Rearing in social isolation increased methylation of cytosines at -82 and -12 bp downstream of the transcription start site, which are located in a GC box element in the promoter region of srd5a1. Binding of Sp1, a ubiquitous transcription factor, to the GC box was decreased in the promoter region of srd5a1 in the PFC in isolation-reared mice. Site-specific methylation at cytosine -12 of a srd5a1 promoter-luciferase reporter construct, but not that of cytosine -82, downregulated the promoter activity of srd5a1. These findings suggest that transcription of srd5a1 in brain is regulated by environmental factor-induced cytosine methylation in the promoter region. This finding could contribute to development of antidepressant and anxiolytic agents.

We have previously demonstrated that the expression of human ribosomal RNA genes (rDNA) in normal and cancer cells is differentially regulated by methylation of the promoter CpG islands. Furthermore, we showed that the methyl CpG-binding protein MBD2 plays a selective role in the methylation-mediated block in rDNA expression. Here, we analyzed the role of three functional mammalian DNA methyltransferases (DNMTs) in regulating the rDNA promoters activity. Immunofluorescence analysis and biochemical fractionation showed that all three DNMTs (DNMT1, DNMT3A, and DNMT3B) are associated with the inactive rDNA in the nucleolus. Although DNMTs associate with both methylated and unmethylated rDNA promoters, DNMT1 preferentially associates with the methylated genes. The rDNA primary transcript level was significantly elevated in DNMT1−/− or DNMT3B−/− human colon carcinoma (HCT116) cells. Southern blot analysis demonstrated a moderate level of rDNA promoter hypomethylation in DNMT1−/− cells and a dramatic loss of rDNA promoter methylation in double knockout cells. Transient overexpression of DNMT1 or DNMT3B suppressed the luciferase expression from both methylated and unmethylated pHrD-IRES-Luc, a reporter plasmid where the rDNA promoter drives luciferase expression. DNMT1-mediated suppression of the unmethylated promoter involves de novo methylation of the promoter, whereas histone deacetylase 2 cooperates with DNMT1 to inhibit the methylated rDNA promoter. Unlike DNMT1, both the wild type and catalytically inactive DNMT3B mutant can suppress rDNA promoter irrespective of its methylation status. DNMT3B-mediated suppression of the rDNA promoter also involves histone deacetylation. Treatment of HCT116 cells with Decitabine (a DNMT inhibitor) or trichostatin A (a histone deacetylase inhibitor) up-regulated endogenous rDNA expression. These inhibitors synergistically activated methylated pHrD-IRES-Luc, whereas they exhibited additive effects on the unmethylated promoter. These results demonstrate localization of DNMTs with the inactive rDNA in the nucleolus, the specific role of DNMT1 and DNMT3B in rDNA expression and the differential regulation of rDNA expression from the methylated and unmethylated rDNA promoters.

DNA is not the only information passed from generation to generation. There are heritable modifications which do not involve changes in DNA sequence. The study of these modifications is called epigenetics. Many epigenetic mechanisms have been discovered including DNA methylation and histone alterations (methylation, acetylation, phosphorylation, ubiquitination). RNA molecules are also involved and important to proper processing or regulation of the DNA-encoded information. Diet, environment and lifestyle play the key roles in initiating epigenetic programming. Somatic health and especially male reproduction are shaped by epigenetics.

The process of aging culminates in a multitude of chronological irreversible changes at the cellular and molecular levels that push the organism toward a progressive loss of physiological integrity and increased susceptibility to disease. Mounting evidence indicates that aging trajectories can be closely tracked down by changes in the DNA methylation patterns over the entire lifespan. The tracing of underlying mechanisms is a promising research path to gain a deeper understanding of the molecular nature of aging. Here we discuss current knowledge about dynamics of DNA methylation in mammals and describe changes in DNA methylation occurring during aging. We explore mechanisms that could underpin aging-related DNA methylation changes and highlight models in which those changes can be understood in the context of the complex interplay between DNA methylation, chromatin states, and their modulators.

In eukaryotes, the packaging of DNA and proteins, termed chromatin, is an essential determinant of transcription. The basic unit of chromatin, the nucleosome, is composed of ∼146 base pairs of DNA spooled around a dense protein core containing two copies each of histones H2A, H2B, H3, and H4. These histone proteins are highly conserved and possess tails, which extend and interact with DNA. Covalent modifications that alter the charge of the histone tail are believed to influence their electrostatic interaction with DNA and act as interaction surfaces for epigenetic effectors that influence the level of chromatin compaction and accessibility to the transcriptional apparatus. Less-compacted chromosomal regions, euchromatin, tend to be gene rich and transcriptionally active. In contrast, condensed constitutive heterochromatin plays an essential function in chromosomal integrity and structural regions such as the centromere and telomere. Facultative heterochromatin plays an essential role by imposing the differentiation-specific transcriptional regulation required to underpin cellular identity. Long noncoding RNAs (lncRNAs) are emerging as key transcriptional regulators, which cooperate with the polycomb repressive complexes and the heterochromatin apparatus in differentiation. Unsurprisingly, disruption of this key pathway is now recognized to play important roles in carcinogenesis and metastases. In this chapter, we review current understanding of the normal and oncogenic functions of lncRNAs, polycomb, and heterochromatin.

Epigenetic regulation of gene expression and genome function is essential to establish and maintain diverse cellular functions during mammalian development—alteration of which leads to cancer. Coordinated by chromatin and DNA modifier proteins and noncoding RNAs, epigenetic mechanisms impact chromatin structure and control the accessibility of genes and genomic elements and thereby influence their targeting by protein machinery. Epigenetic processes are extensively reprogrammed in cancer cells resulting in altered histone modifications, DNA methylation, and expression of long noncoding RNAs (lncRNAs), disrupting normal patterns of gene expression. These findings bring a new perspective to our understanding on the underlying causes of cancers in that epigenomic abnormalities are equally important as the genetic alterations. Accumulating evidence suggests that lncRNA-mediated processes that control locus and cell-type specific gene expression to significantly impact cellular transformation. Here, we discuss the molecular characteristics of lncRNAs, and review the recent advances in how lncRNA-mediated processes that are linked to cancer epigenetics.

DNA methylation is a critical epigenetic marker involved in development, differentiation, and cell fate diversity. Here we discuss the possible role of adaptive and stochastic changes in DNA methylation in aging. We describe recent progress in studying stochastic changes in DNA methylation, most notably alterations at the level of the single cell. We show that fidelity of individual CpG sites is several orders of magnitude lower than the fidelity of DNA sequence integrity. At this high level of instability, age-related methylation changes could well be a cause of the gradual loss of specific cell fate or differentiation status and, therefore contribute to loss of function and increased disease risk at old age. However, the current lack of a complete understanding of the relationship between DNA methylation patterns and gene expression essentially constrains definite conclusions about the impact of DNA methylation changes on the aging process.

Epigenetic alterations, layered onto the genome, determine gene expression patterns and include histone modifications, chromatin remodeling, and DNA methylation, with implications for detection, prognostication, and therapy in lung cancer. Distinct cellular phenotypes are based on differential gene expression. Epigenetic mechanisms include DNA methylation, histone modifications, regulatory RNAs, genome-organizing proteins, and chromatin remodeling complexes. Both genetic and epigenetic alterations can contribute to lung cancer and they can interact. Numerous DNA methylation changes are seen in lung cancer, most commonly hypermethylation of promoter CpG-dense regions and loss of methylation in gene bodies. DNA methylation alterations in a variety of bodily fluids can be used as biomarkers for the presence of lung cancer. Epigenetic alternations are in principle reversible; epigenetic therapies thus offer opportunities for treatment by undoing cancer-driving epigenetic changes or by activating targets for therapy. Epigenetics has given new breath to the fight against lung cancer, including in early detection and treatment.

DNA methylation is a prevalent epigenetic modification involved in transcriptional regulation and essential for mammalian development. While the genome‐wide distribution of this mark has been studied to great detail, the mechanisms responsible for its correct deposition, as well as the cause for its aberrant localization in cancers, have not been fully elucidated. Here, we have compared the activity of individual DNMT3A isoforms in mouse embryonic stem and neuronal progenitor cells and report that these isoforms differ in their genomic binding and DNA methylation activity at regulatory sites. We identify that the longer isoform DNMT3A1 preferentially localizes to the methylated shores of bivalent CpG island promoters in a tissue‐specific manner. The isoform‐specific targeting of DNMT3A1 coincides with elevated hydroxymethylcytosine (5‐hmC) deposition, suggesting an involvement of this isoform in mediating turnover of DNA methylation at these sites. Through genetic deletion and rescue experiments, we demonstrate that this isoform‐specific recruitment plays a role in de novo DNA methylation at CpG island shores, with potential implications on H3K27me3‐mediated regulation of developmental genes. ISSN:0261-4189 ISSN:1460-2075

Epigenetic regulation of activity-induced gene expression involves multiple levels of molecular interaction, including histone and DNA modifications, as well as mechanisms of DNA repair. Here we demonstrate that the genome-wide deposition of Inhibitor of growth family member 1 (ING1), which is a central epigenetic regulatory protein, is dynamically regulated in response to activity in primary cortical neurons. ING1 knockdown leads to decreased expression of genes related to synaptic plasticity, including the regulatory subunit of calcineurin, Ppp3r1. In addition, ING1 binding at a site upstream of the transcription start site (TSS) of Ppp3r1 depends on yet another group of neuroepigenetic regulatory proteins, the Piwi-like family, which are also involved in DNA repair. These findings provide new insight into a novel mode of activity-induced gene expression, which involves the interaction between different epigenetic regulatory mechanisms traditionally associated with gene repression and DNA repair.Author contributionsL.J.L., Q.Z., T.W.B and W.W. designed the experiments. N.K., A.K., X.L., C.D., S.L. and W.W. designed and assembled shRNA constructs. L.J.L., W.W., X.L., C.D., P.R.M., E.Z., and S.L. conducted experiments. Q.Z. and Y.W. analysed ChIP-seq data. L.J.L., Q.Z., and W.W. wrote the paper. All authors reviewed and edited the manuscript.Conflicts of interestNone.

Functionally different chromatin domains display distinct chemical marks. Constitutive heterochromatin is commonly associated with trimethylation of lysine 9 on histone H3 (H3K9me3), hypoacetylated histones, and DNA methylation, but the contributions of and interplay among these features are not fully understood. To dissect the establishment of heterochromatin, we investigated the relationships among these features using an in vivo tethering system in Neurospora crassa Artificial recruitment of the H3K9 methyltransferase DIM-5 (defective in methylation-5) induced H3K9me3 and DNA methylation at a normally active, euchromatic locus but did not bypass the requirement of DIM-7, previously implicated in the localization of DIM-5, indicating additional DIM-7 functionality. Tethered heterochromatin protein 1 (HP1) induced H3K9me3, DNA methylation, and gene silencing. The induced heterochromatin required histone deacetylase 1 (HDA-1), with an intact catalytic domain, but HDA-1 was not essential for de novo heterochromatin formation at native heterochromatic regions. Silencing did not require H3K9me3 or DNA methylation. However, DNA methylation contributed to establishment of H3K9me3 induced by tethered HP1. Our analyses also revealed evidence of regulatory mechanisms, dependent on HDA-1 and DIM-5, to control the localization and catalytic activity of the DNA methyltransferase DIM-2. Our study clarifies the interrelationships among canonical aspects of heterochromatin and supports a central role of HDA-1-mediated histone deacetylation in heterochromatin spreading and gene silencing.

1. The aim of this study was to identify the active control area of the duck retinoic acid-inducible gene I (duRIG-I) core promoter, to predict the binding sites of transcription factors and to provide a theoretical basis for the study of duRIG-I function and mechanism of regulation. 2. The promoter region of duRIG-I was obtained from Ensembl; the CpG island in the duRIG-I promoter was predicted online; and the methylation status of the duRIG-I promoter was detected by the bisulphite sequencing PCR method. 3. There was an obvious CpG island in the duRIG-I promoter, with a total of 44 CG dinucleotides. However, the level of methylation was hypomethylation (0.2%). 4. The core transcriptional regulatory region was identified, localised between -301 and ~+14 bp, where multiple transcription factor binding sites including IRF1, RXRβ, AP-2αA, RAP1, NF-1 and SP1 motifs were identified that significantly enhanced RIG-I promoter activity.

Although only 1.5% of the human genome appears to code for proteins, much effort in cancer research has been devoted to this minimal fraction of our DNA. However, the last few years have witnessed the realization that a large class of non-coding RNAs (ncRNAs), named microRNAs, contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies have also shown that epigenetic silencing of microRNAs with tumor suppressor features by CpG island hypermethylation is a common hallmark of human tumors. Thus, we wondered whether there were other ncRNAs undergoing aberrant DNA methylation-associated silencing in transformed cells. We focused on the transcribed-ultraconserved regions (T-UCRs), a subset of DNA sequences that are absolutely conserved between orthologous regions of the human, rat and mouse genomes and that are located in both intra- and intergenic regions. We used a pharmacological and genomic approach to reveal the possible existence of an aberrant epigenetic silencing pattern of T-UCRs by treating cancer cells with a DNA-demethylating agent followed by hybridization to an expression microarray containing these sequences. We observed that DNA hypomethylation induces release of T-UCR silencing in cancer cells. Among the T-UCRs that were reactivated upon drug treatment, Uc.160+, Uc283+A and Uc.346+ were found to undergo specific CpG island hypermethylation-associated silencing in cancer cells compared with normal tissues. The analysis of a large set of primary human tumors (n=283) demonstrated that hypermethylation of the described T-UCR CpG islands was a common event among the various tumor types. Our finding that, in addition to microRNAs, another class of ncRNAs (T-UCRs) undergoes DNA methylation-associated inactivation in transformed cells supports a model in which epigenetic and genetic alterations in coding and non-coding sequences cooperate in human tumorigenesis.

Epigenetics is the study of changes in gene function that cannot be explained by changes in DNA sequence. A mammalian body contains more than two hundred types of cells. Since all of them are derived from a single fertilized egg, their genotypes are identical. However, the gene expression patterns are different between the cell types, indicating that each cell type has unique own “epigenotype”. Epigenetic gene regulation mechanisms essentially contribute to various processes of mammalian development. The essence of epigenetic regulation is the structural change of chromatin to modulate gene activity in a spatiotemporal manner. DNA methylation and histone modifications are the major epigenetic mechanisms. Sex determination is the process for gender establishment. There are two types of sex‐determining mechanisms in animals, environmental sex determination (ESD) and genotypic sex determination (GSD). Recent studies have provided some evidence that epigenetic mechanisms play indispensable roles in ESD and GSD. Some fishes undergo ESD, in which DNA methylation is essentially involved. GSD is employed in therian mammals, where Sry (sex‐determining region on the Y chromosome) triggers testis differentiation from undifferentiated gonads. Sry expression is tightly regulated in a spatiotemporal manner. A recent study demonstrated that histone modification is involved in Sry regulation. In this review, we discuss the role of epigenetic mechanisms for sex determination in mammals and other vertebrates.

We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery. We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.

DNA methylation in eukaryotes invokes heritable alterations of the of the cytosine base in DNA without changing the underlying genomic DNA sequence. DNA methylation may be modified by environmental exposures as well as gene polymorphisms and may be a mechanistic link between environmental risk factors and the development of disease. In this review, we consider the role of DNA methylation in bone cells (osteoclasts/osteoblasts/osteocytes) and their progenitors with special focus on in vitro and ex vivo analyses. The number of studies on DNA methylation in bone cells is still somewhat limited, nevertheless it is getting increasingly clear that this type of the epigenetic changes is a critical regulator of gene expression. DNA methylation is necessary for proper development and function of bone cells and is accompanied by disease characteristic functional alterations as presently reviewed including postmenopausal osteoporosis and mechanical strain.

It is well known that phenotype of animals may be modified by the nutritional modulations through epigenetic mechanisms. As a key and central component of epigenetic network, DNA methylation is labile in response to nutritional influences. Alterations in DNA methylation profiles can lead to changes in gene expression, resulting in diverse phenotypes with the potential for decreased growth and health. Here, I reviewed the biological process of DNA methylation that results in the addition of methyl groups to DNA; the possible ways including methyl donors, DNA methyltransferase (DNMT) activity and other cofactors, the critical periods including prenatal, postnatal and dietary transition periods, and tissue specific of epigenetic modulation of DNA methylation by nutrition and its mechanisms in animals. Keywords: Critical period, DNA methylation, Epigenetics, Nutrition, Tissue specific

Epigenetic mechanisms play critical roles in oogenesis and early embryo development in mammals. One of these epigenetic mechanisms, DNA methylation is accomplished through the activities of DNA methyltransferases (DNMTs), which are responsible for adding a methyl group to the fifth carbon atom of the cytosine residues within cytosine-phosphate-guanine (CpG) and non-CpG dinuclotide sites. Five DNMT enzymes have been identified in mammals including DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L. They function in two different methylation processes: maintenance and de novo. For maintenance methylation, DNMT1 preferentially transfers methyl groups to the hemi-methylated DNA strands following DNA replication. However, for de novo methylation activities both DNMT3A and DNMT3B function in the methylation of the unmodified cytosine residues. Although DNMT3L indirectly contributes to de novo methylation process, DNMT2 enables the methylation of the cytosine 38 in the anticodon loop of aspartic acid transfer RNA and does not methylate DNA. In this review article, we have evaluated and discussed the existing published studies to characterize the spatial and temporal expression patterns of the DNMTs in mouse, bovine and human oocytes and early embryos. We have also reviewed the effects of in vitro culture conditions (serum abundance and glucose concentration), aging, superovulation, vitrification, and somatic cell nuclear transfer technology on the dynamics of DNMTs.

Human papillomavirus (HPV) oncoproteins drive distinctive promoter methylation patterns in cancer. However, the underlying mechanism remains to be elucidated. Cyclin A1 (CCNA1) promoter methylation is strongly associated with HPV-associated cancer. CCNA1 methylation is found in HPV-associated cervical cancers, as well as in head and neck squamous cell cancer. Numerous pieces of evidence suggest that E7 may drive CCNA1 methylation. First, the CCNA1 promoter is methylated in HPV-positive epithelial lesions after transformation. Second, the CCNA1 promoter is methylated at a high level when HPV is integrated into the human genome. Finally, E7 has been shown to interact with DNA methyltransferase 1 (Dnmt1). Here, we sought to determine the mechanism by which E7 increases methylation in cervical cancer by using CCNA1 as a gene model. We investigated whether E7 induces CCNA1 promoter methylation, resulting in the loss of expression. Using both E7 knockdown and overexpression approaches in SiHa and C33a cells, our data showed that CCNA1 promoter methylation decreases with a corresponding increase in expression in E7 siRNA-transfected cells. By contrast, CCNA1 promoter methylation was augmented with a corresponding reduction in expression in E7-overexpressing cells. To confirm whether the binding of the E7–Dnmt1 complex to the CCNA1 promoter induced methylation and loss of expression, ChIP assays were carried out in E7-, del CR3-E7 and vector control-overexpressing C33a cells. The data showed that E7 induced CCNA1 methylation by forming a complex with Dnmt1 at the CCNA1 promoter, resulting in the subsequent reduction of expression in cancers. It is interesting to further explore the genome-wide mechanism of E7 oncoprotein-mediated DNA methylation.

Mammalian DNA methylation plays an essential role in development. To date, only snapshots of different mouse and human cell types have been generated, providing a static view on DNA methylation. To enable monitoring of methylation status as it changes over time, we establish a reporter of genomic methylation (RGM) that relies on a minimal imprinted gene promoter driving a fluorescent protein. We show that insertion of RGM proximal to promoter-associated CpG islands reports the gain or loss of DNA methylation. We further utilized RGM to report endogenous methylation dynamics of non-coding regulatory elements, such as the pluripotency-specific super enhancers of Sox2 and miR290. Loci-specific DNA methylation changes and their correlation with transcription were visualized during cell-state transition following differentiation of mouse embryonic stem cells and during reprogramming of somatic cells to pluripotency. RGM will allow the investigation of dynamic methylation changes during development and disease at single-cell resolution., National Institutes of Health (U.S.) (Grant HD 045022)

The ubiquitin-proteasome system plays an essential regulatory role in various cellular processes. Besides its involvement in normal cellular functions, the alteration of proteasomal activity contributes to the pathological states of several clinical disorders, including cancer. Aberrant methylation of the CpG islands has been reported as an alternative way to inactivate gene expression involved in the ubiquitination process and thus protein degradation in tumor tissues. In this study, we aimed to determine the CpG methylation pattern of the UCHL1 promoter, as well as the mutation spectrum and the expression pattern of P53 in sporadic colorectal cancer (CRC) from Tunisian patients. We found that UCHL1 was methylated in 68.57 % and correlated significantly with lymph node metastasis (P = 0.029) and transcriptional silencing in tumor tissues (P = 0.013). Mutation screening of exons 5-9 of P53 showed that 42.85 % of cases harbor somatic mutation and are positively correlated with the methylated pattern of UCHL1 (P = 0.001). Furthermore, cytoplasmic accumulation of P53 was strongly associated with the unmethylated UCHL1 profile (P = 0.006), supporting the relationship between these two proteins in CRC.

We recently identified a compensatory survival role for protein kinase D1 (PKD1) in protecting dopaminergic neurons from oxidative insult. To investigate the molecular mechanism of Prkd1 gene expression, we cloned the 5'-flanking region (1620-bp) of the mouse Prkd1 gene. Deletion analyses revealed that the -250/+113 promoter region contains full promoter activity in MN9D dopaminergic neuronal cells. In silico analysis of the Prkd1 promoter uncovered binding sites for key redox transcription factors including Sp1 and NF-κB. Over-expression of Sp1, Sp3, and NF-κB-p65 proteins stimulated Prkd1 promoter activity. Binding of Sp3 and NF-κB-p65 to the Prkd1 promoter was confirmed using chromatin immunoprecipitation. Treatment with the Sp inhibitor mithramycin A significantly attenuated Prkd1 promoter activity and PKD1 mRNA and protein expression. Further mechanistic studies revealed that inhibition of histone deacetylation and DNA methylation up-regulated PKD1 mRNA expression. Importantly, negative modulation of PKD1 signaling by pharmacological inhibition or shRNA knockdown increased dopaminergic neuronal sensitivity to oxidative damage in a human mesencephalic neuronal cell model. Collectively, our findings demonstrate that Sp1, Sp3, and NF-κB-p65 can transactivate the mouse Prkd1 promoter and that epigenetic mechanisms, such as DNA methylation and histone modification, are key regulatory events controlling the expression of pro-survival kinase PKD1 in dopaminergic neuronal cells. Previously, we demonstrated that protein kinase D1 (PKD1) plays a survival role during the early stage of oxidative stress in dopaminergic neuronal cells.

Epigenetic modifiers have fundamental roles in defining unique cellular identity through the establishment and maintenance of lineage-specific chromatin and methylation status. Several DNA modifications such as 5-hydroxymethylcytosine (5hmC) are catalysed by the ten eleven translocation (Tet) methylcytosine dioxygenase family members, and the roles of Tet proteins in regulating chromatin architecture and gene transcription independently of DNA methylation have been gradually uncovered. However, the regulation of immunity and inflammation by Tet proteins independent of their role in modulating DNA methylation remains largely unknown. Here we show that Tet2 selectively mediates active repression of interleukin-6 (IL-6) transcription during inflammation resolution in innate myeloid cells, including dendritic cells and macrophages. Loss of Tet2 resulted in the upregulation of several inflammatory mediators, including IL-6, at late phase during the response to lipopolysaccharide challenge. Tet2-deficient mice were more susceptible to endotoxin shock and dextran-sulfate-sodium-induced colitis, displaying a more severe inflammatory phenotype and increased IL-6 production compared to wild-type mice. IκBζ, an IL-6-specific transcription factor, mediated specific targeting of Tet2 to the Il6 promoter, further indicating opposite regulatory roles of IκBζ at initial and resolution phases of inflammation. For the repression mechanism, independent of DNA methylation and hydroxymethylation, Tet2 recruited Hdac2 and repressed transcription of Il6 via histone deacetylation. We provide mechanistic evidence for the gene-specific transcription repression activity of Tet2 via histone deacetylation and for the prevention of constant transcription activation at the chromatin level for resolving inflammation.

The barley gene HvS40, encoding a putative regulator of leaf senescence, is strongly induced during leaf senescence. As shown by chromatin immunoprecipitation, euchromatic histone modification H3K9ac is added at promoter close to ATG and coding sequence of HvS40 after onset of senescence. In parallel, level of heterochromatic H3K9me2 decreases at this gene. Bisulfite sequencing revealed no DNA-methylation in this region, but a heavily methylated DNA-island, starting 664 bp upstream from translational start site in both, mature and senescent leaves. A decrease in DNA methylation in senescing leaves could be shown at one specific CpG motif at the end of this methylation island. In addition, global changes in chromatin structure during senescence were analyzed via immunocytology, revealing senescence-associated changes in spatial distribution of heterochromatic H3K9me2 patterns in the nuclei. Our results prove a senescence-specific mechanism, altering histone modification marks at distinct sequences of the senescence-associated gene HvS40 and altering distribution of heterochromatic areas in the nuclei.

Short-term expression of transgenes is one of the problems frequently associated with non-viral in vivo gene transfer. To obtain experimental evidence for the design of sustainable transgene expression systems, the contribution of epigenetic modifications to the decline in transgene expression needs to be investigated. Bisulfite sequencing and reactivation by hydrodynamic injection of isotonic solution were employed to investigate methylation statues of CpG in transiently expressing plasmid, pCMV-Luc, in mouse liver after hydrodynamic delivery. The cytosines of CpGs in the promoter region of pCMV-Luc were methylated in mouse liver, but the methylation was much later than the decline in the expression. The expression from pre-methylated pCMV-Luc was insensitive to reactivation. Neither an inhibitor of DNA methylation nor an inhibitor of histone deacetylation had significant effects on transgene expression after hydrodynamic injection of pCMV-Luc. Partial hepatectomy, which reduces the transgene expression from the non-integrated vector into the genome, significantly reduced the transgene expression of human interferon γ from a long-term expressing plasmid pCpG-Huγ, suggesting that the CpG-reduced plasmid was not significantly integrated into the genomic DNA. These results indicate that the CpG-reduced plasmids achieve prolonged transgene expression without integration into the host genome, although the methylation status of CpG sequences in plasmids will not be associated with the prolonged expression.

The hallmarks of carcinogenesis are characterized by alterations in the expression of multiple genes that occur via genetic and epigenetic alterations, leading to genome rearrangements and instability. The reversible process of epigenetic regulation, which includes changes in DNA methylation, histone modifications, and alteration in microRNA (miRNA) expression that alter phenotype without any change in the DNA sequence, is recognized as a key mechanism in cancer cell metabolism. Recent advancements in the rapidly evolving field of cancer epigenetics have shown the anticarcinogenic potential of natural compounds targeting epigenetic mechanism as a common molecular approach for cancer treatment. This review summarizes the potential of natural chemopreventive agents to reverse cancer-related epigenetic aberrations by regulating the activity of histone deacetylases, histone acetyltransferases, DNA methyltransferase I, and miRNAs. Furthermore, there is impetus for determining novel and effective chemopreventive strategies, either alone or in combination with other anticancer agents that exhibit similar properties, for improving the therapeutic aspects of cancer.

SummaryAlthough DNA methylation is commonly invoked as a mechanism for transcriptional repression, the extent to which it actively silences transcription factor (TF) occupancy sites in vivo is unknown. To study the role of DNA methylation in the active modulation of TF binding, we quantified the effect of DNA methylation depletion on the genomic occupancy patterns of CTCF, an abundant TF with known methylation sensitivity that is capable of autonomous binding to its target sites in chromatin. Here, we show that the vast majority (>98.5%) of the tens of thousands of unoccupied, methylated CTCF recognition sequences remain unbound upon abrogation of DNA methylation. The small fraction of sites that show methylation-dependent binding in vivo are in turn characterized by highly variable CTCF occupancy across cell types. Our results suggest that DNA methylation is not a primary groundskeeper of genomic TF landscapes, but rather a specialized mechanism for stabilizing intrinsically labile sites.

Methylation of cytosine at the C5 position (5mC) represents an epigenetic modification that plays a fundamental role in embryonic development, transcriptional regulation, and other processes. It can also be a mutational hotspot at CpG dinucleotides as a result of spontaneous hydrolytic deamination of 5mC to thymine. The resulting G · T mismatch pair is recognized by thymine DNA glycosylase (TDG) and revereted to a G · C pair. Recent studies have shown that 5mC is consecutively catalyzed into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by a DNA dioxygenase from the ten-eleven translocation (TET) family. Two oxidative cytosine derivatives, 5fC and 5caC, are eliminated by TDG during active DNA demethylation. Therefore, TDG has versatile roles in epigenetic regulation to control the gene expression as well as the DNA repair pathway to prevent mutagenesis. 5fC and 5caC serve as intermediate products of active DNA demethylation and also behave as DNA damages that threaten genomic integrity. Here, we discuss the potential functions of 5mC oxidative derivatives in epigenetic modification and DNA damage.

Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children under the age of 15. In addition to genetic aberrations, epigenetic modifications such as DNA methylation are altered in cancer and impact gene expression. To identify epigenetic alterations in ALL, genome-wide methylation profiles were generated using the methylated CpG island recovery assay followed by next-generation sequencing. More than 25,000 differentially methylated regions (DMR) were observed in ALL patients with ∼ 90% present within intronic or intergenic regions. To determine the regulatory potential of the DMR, whole-transcriptome analysis was performed and integrated with methylation data. Aberrant promoter methylation was associated with the altered expression of genes involved in transcriptional regulation, apoptosis, and proliferation. Novel enhancer-like sequences were identified within intronic and intergenic DMR. Aberrant methylation in these regions was associated with the altered expression of neighboring genes involved in cell cycle processes, lymphocyte activation and apoptosis. These genes include potential epi-driver genes, such as SYNE1, PTPRS, PAWR, HDAC9, RGCC, MCOLN2, LYN, TRAF3, FLT1, and MELK, which may provide a selective advantage to leukemic cells. In addition, the differential expression of epigenetic modifier genes, pseudogenes, and non-coding RNAs was also observed accentuating the role of erroneous epigenetic gene regulation in ALL.

Aim: We previously reported changes of DNA methylation and transcription patterns in mammalian cells that carry integrated foreign DNA. Experiments were now designed to assess the epigenetic consequences of inserting a 5.6 kbp plasmid into the human genome. Methods: Differential transcription and CpG methylation patterns were compared between transgenomic and nontransgenomic cell clones by using gene chip microarray systems. Results: In 4.7% of the 28.869 gene segments analyzed, transcriptional activities were up- or downregulated in the transgenomic cell clones. Genome-wide profiling revealed differential methylation in 3791 of > 480,000 CpG's examined in transgenomic versus nontransgenomic clones. Conclusion: The data document genome-wide effects of foreign DNA insertions on the epigenetic stability of human cells. Many fields in experimental biology and medicine employ transgenomic or otherwise genome-manipulated cells or organisms without considering the epigenetic consequences for the recipient genomes.

Cytosine methylation at CpG dinucleotides is a central component of epigenetic regulation in vertebrates, and the base excision repair (BER) pathway is important for maintaining both the genetic stability and the methylation status of CpG sites. This perspective focuses on two enzymes that are of particular importance for the genetic and epigenetic integrity of CpG sites, methyl binding domain 4 (MBD4) and thymine DNA glycosylase (TDG). We discuss their capacity for countering C to T mutations at CpG sites, by initiating base excision repair of G · T mismatches generated by deamination of 5-methylcytosine (5mC). We also consider their role in active DNA demethylation, including pathways that are initiated by oxidation and/or deamination of 5mC.