Epigenetic and Molecular Alterations in Obesity: Linking CRP and DNA Methylation to Systemic Inflammation.

Obesity is marked by excessive fat accumulation in the adipose tissue, which disrupts metabolic processes and causes chronic systemic inflammation. Commonly, body mass index (BMI) is used to assess obesity-related risks, predicting potential metabolic disorders. However, for a better clustering of obese patients, we must consider molecular and epigenetic changes which may be responsible for inflammation and metabolic changes. Our study involved two groups of patients, obese and healthy donors, on which routine analysis were performed, focused on BMI, leukocytes count, and C-reactive protein (CRP) and completed with global DNA methylation and gene expression analysis for genes involved in inflammation and adipogenesis. Our results indicate that obese patients exhibited elevated leukocytes levels, along with increased BMI and CRP. The obese group revealed a global hypomethylation and upregulation of proinflammatory genes, with adipogenesis genes following the same trend of being overexpressed. The study confirms that obesity is linked to systematic inflammation and metabolic dysfunction through epigenetic and molecular alterations. The CRP was correlated with the hypomethylation status in obese patients, and this fact may contribute to a better understanding of the roles of specific genes in adipogenesis and inflammation, leading to a better personalized therapy.

Locus-level L1 DNA methylation profiling reveals the epigenetic and transcriptional interplay between L1s and their integration sites.

Long interspersed element 1 (L1) retrotransposons are implicated in human disease and evolution. Their global activity is repressed by DNA methylation, but deciphering the regulation of individual copies has been challenging. Here, we combine short- and long-read sequencing to unveil L1 methylation heterogeneity across cell types, families, and individual loci and elucidate key principles involved. We find that the youngest primate L1 families are specifically hypomethylated in pluripotent stem cells and the placenta but not in most tumors. Locally, intronic L1 methylation is intimately associated with gene transcription. Conversely, the L1 methylation state can propagate to the proximal region up to 300 bp. This phenomenon is accompanied by the binding of specific transcription factors, which drive the expression of L1 and chimeric transcripts. Finally, L1 hypomethylation alone is typically insufficient to trigger L1 expression due to redundant silencing pathways. Our results illuminate the epigenetic and transcriptional interplay between retrotransposons and their host genome.

Changes in DNA methylation associated with a specific mode of delivery: a pilot study.

Background: The mode of delivery represents an epigenetic factor with potential to affect further development of the individual by multiple mechanisms. DNA methylation may be one of them, representing a major epigenetic mechanism involving direct chemical modification of the individual's DNA. This pilot study aims to examine whether a specific mode of delivery induces changes of DNA methylation by comparing the umbilical cord blood and peripheral blood of the newborns. Methods: Blood samples from infants born by vaginal delivery and caesarean section were analysed to prepare the Methylseq library according to NEBNext enzymatic Methyl-seq Methylation Library Preparation Kit with further generation of target-enriched DNA libraries using the Twist Human Methylome Panel. DNA methylation status was determined using Illumina next-generation sequencing (NGS). Results: We identified 168 differentially methylated regions in umbilical cord blood samples and 157 regions in peripheral blood samples. These were associated with 59 common biological, metabolic and signalling pathways for umbilical cord and peripheral blood samples. Conclusion: Caesarean section is likely to represent an important epigenetic factor with the potential to induce changes in the genome that could play an important role in development of a broad spectrum of disorders. Our results could contribute to the elucidation of how epigenetic factors, such as a specific mode of delivery, could have adverse impact on health of an individual later in their life.

Simultaneous voltammetric determination of 7-methyl-guanine and 5-methyl-cytosine using a cathodically pre-treated boron-doped diamond electrode.

Given the importance of identifying the presence of biomarkers of human diseases in DNA samples, the main objective of this work was to investigate, for the first time, the electro-catalytic oxidation of 7-methyl-guanine (7-mGua) and 5-methyl-cytosine (5-mCyt) on a boron doped diamond electrode pre-treated cathodically (red-BDDE), using differential pulse voltammetry (DPV) and cyclic voltammetry (CV). The anodic peak potentials of 7-mGua and 5-mCyt by DPV were at E = 1.04 V and E = 1.37 V at pH = 4.5, indicating excellent peak separation of approximately 330 mV between species. Using DPV, experimental conditions such as supporting electrolyte, pH and influence of interferents were also investigated to develop a sensitive and selective method for individual and simultaneous quantification of these biomarkers. The analytical curves for the simultaneous quantification of 7-mGua and 5-mCyt in the acid medium (pH = 4.5) were: concentration range of 0.50-5.00 µmol L-1 (r = 0.999), detection limit of 0.27 µmol L-1 for 7-mGua; from 3.00 to 25.00 µmol L-1 (r = 0.998), with a detection limit of 1.69 µmol L-1 for 5-mCyt. A new DP voltammetric method for the simultaneous detection and quantification of biomarkers 7-mGua and 5-mCyt using a red-BDDE is proposed.

Deciphering the role of aberrant DNA methylation in NAFLD and NASH.

The global incidence of nonalcoholic fatty liver disease (NAFLD) is mounting incessantly, and it is emerging as the most frequent cause of chronic and end stage liver disorders. It is the starting point for a range of conditions from simple steatosis to more progressive nonalcoholic steatohepatitis (NASH) and associated hepatocellular carcinoma (HCC). Dysregulation of insulin secretion and dyslipidemia due to obesity and other lifestyle variables are the primary contributors to establishment of NAFLD. Onset and progression of NAFLD is orchestrated by an interplay of metabolic environment with genetic and epigenetic factors. An incompletely understood mechanism of NAFLD progression has greatly hampered the progress in identification of novel prognostic and therapeutic strategies. Emerging evidence suggests altered DNA methylation pattern as a key determinant of NAFLD pathogenesis. Environmental and lifestyle factors can manipulate DNA methylation patterns in a reversible manner, which manifests as changes in gene expression. In this review we attempt to highlight the importance of DNA methylation in establishment and progression of NAFLD. Development of novel diagnostic, prognostic and therapeutic strategies centered around DNA methylation signatures and modifiers has also been explored.

A study on DNA methylation modifying natural compounds identified EGCG for induction of IFI16 gene expression related to the innate immune response in cancer cells.

Innate immune sensor IFN-induced protein 16 (IFI16) exhibits anti-inflammatory effects via IFNß and IFN-stimulated gene (ISG)15 induction in cancer cells. Epigallocatechin gallate (EGCG) is a potent natural DNA methyltransferase inhibitor (DNMTi). Previous studies revealed that conventional DNMTis, such as 5-azacytidine (5-aza-dc), induce IFI16 expression and EGCG decreases DNMT mRNA expression and global methylation (5mC) level via promoter demethylation of tumor suppressor genes in cancer cell lines. To the best of our knowledge, however, EGCG-mediated IFI16 promoter methylation status has been overlooked. Here, initial screening was performed to determine IFI16 expression and its correlation with DNMTs in cancer cell lines from various databases. Following treatment of breast cancer cell lines with 5-aza-dc, vitamin C and EGCG, expression levels of IFI16 and its downstream transcription targets IFNß1 and ISG15 were assessed using RT-qPCR, and the 5mC level was assessed using ELISA. In silico molecular docking simulation was performed for all DNMTs to predict the mode of ligands binding with proteins. Finally, promoter methylation level in IFI16 gene was assessed following EGCG treatment. EGCG treatment induced IFI16 expression, interacted with certain amino acids residues in DNMT proteins and decreased 5mC level and promoter methylation of IFI16. The present results may provide a basis for targeting IFI16 expression as a therapeutic option in breast cancer cell lines.

Biomimetic Iron Complex Achieves TET Enzyme Reactivity*.

The epigenetic marker 5-methyl-2'-deoxycytidine (5mdC) is the most prevalent modification to DNA. It is removed inter alia via an active demethylation pathway: oxidation by Ten-Eleven Translocation 5-methyl cytosine dioxygenase (TET) and subsequent removal via base excision repair or direct demodification. Recently, we have shown that the synthetic iron(IV)-oxo complex [FeIV (O)(Py5 Me2 H)]2+ (1) can serve as a biomimetic model for TET by oxidizing the nucleobase 5-methyl cytosine (5mC) to its natural metabolites. In this work, we demonstrate that nucleosides and even short oligonucleotide strands can also serve as substrates, using a range of HPLC and MS techniques. We found that the 5-position of 5mC is oxidized preferably by 1, with side reactions occurring only at the strand ends of the used oligonucleotides. A detailed study of the reactivity of 1 towards nucleosides confirms our results; that oxidation of the anomeric center (1') is the most common side reaction.

Bisulphite miRNA-seq reveals widespread CpG and non-CpG 5-(hydroxy)methyl-Cytosine in human microRNAs.

In the last decade, the field of epitranscriptomics highlighted a wide array of post-transcriptional modifications in human RNAs, including microRNAs (miRNAs). Recent reports showed that human miRNAs undergo cytosine methylation. We describe the first high-throughput NGS-based method (BS-miRNA-seq) and an analysis pipeline (MAmBA) to attain high-resolution mapping of (hydroxy)-methyl-5-cytosine ((h)m5C) modifications in human miRNAs. Our method uncovers that miRNAs undergo widespread cytosine modification in various sequence contexts.Furthermore, validation of our data with specific antibodies reveals both m5C and hm5C residues in human mature miRNAs. BS-miRNA-seq and MAmBA may contribute to the precise mapping of (h)m5C on miRNAs in various cell types and tissues, a key achievement towards the understanding of the functional implications of this modification in miRNAs. MAmBA is available for download at https://github.com/flcvlr/MAmBA.

Direct evidence for (G)O6···H2-N4(C)+ hydrogen bonding in transient G(syn)-C+ and G(syn)-m5C+ Hoogsteen base pairs in duplex DNA from cytosine amino nitrogen off-resonance R1ρ relaxation dispersion measurements.

NMR relaxation dispersion studies have shown that Watson-Crick G-C and A-T base pairs in duplex DNA exist in dynamic equilibrium with their Hoogsteen counterparts. Hoogsteen base pairs form through concurrent rotation of the purine base about the glycosidic bond from an anti to a syn conformation and constriction of the C1'-C1' distance across the base pair by ∼2 Što allow Hoogsteen type hydrogen bonding. Owing to their unique structure, Hoogsteen base pairs can play important roles in DNA recognition, the accommodation, recognition, and repair of DNA damage, and in DNA replication. NMR relaxation dispersion experiments targeting imino nitrogen and protonated base and sugar carbons have provided insights into many structural features of transient Hoogsteen base pairs, including one of two predicted hydrogen bonds involving (G)N7···H-N3(C)+ and (A)N7···H-N3(T). Here, through measurement of cytosine amino (N4) R1ρ relaxation dispersion, we provide direct evidence for the second (G)O6···H2-N4(C)+ hydrogen bond in G(syn)-C+ transient Hoogsteen base pairs. The utility of cytosine N4 R1ρ relaxation dispersion as a new sensitive probe of transient Hoogsteen base pairs, and cytosine dynamics in general, is further demonstrated by measuring G(syn)-C+ Hoogsteen exchange near neutral pH and in the context of the naturally occurring DNA modification 5-methyl cytosine (m5C), in DNA samples prepared using chemical synthesis and a 15N labeled m5C phosphoramidite.

Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression.

How the covalent modification of mRNA ribonucleotides, termed epitranscriptomic modifications, alters mRNA function remains unclear. One issue has been the difficulty of quantifying these modifications. Using purified HIV-1 genomic RNA, we show that this RNA bears more epitranscriptomic modifications than the average cellular mRNA, with 5-methylcytosine (m5C) and 2'O-methyl modifications being particularly prevalent. The methyltransferase NSUN2 serves as the primary writer for m5C on HIV-1 RNAs. NSUN2 inactivation inhibits not only m5C addition to HIV-1 transcripts but also viral replication. This inhibition results from reduced HIV-1 protein, but not mRNA, expression, which in turn correlates with reduced ribosome binding to viral mRNAs. In addition, loss of m5C dysregulates the alternative splicing of viral RNAs. These data identify m5C as a post-transcriptional regulator of both splicing and function of HIV-1 mRNA, thereby affecting directly viral gene expression.

The role of DNA methylation in epigenetics of aging.

Recent research suggests that epigenetics, especially DNA methylation, plays a mechanistic role in aging. Epigenetic clocks, which measure changes in a few hundred specific CpG sites, can accurately predict chronological age in a variety of species, including humans. These clocks are currently the best biomarkers for predicting mortality in humans. Additionally, several studies have characterized the effects of aging across the methylome in a wide variety of tissues from humans and mice. A small fraction (~2%) of the CpG sites show age-related changes, either hypermethylation or hypomethylation with aging. Evaluation of non-CpG site methylation has only been examined in a few studies, with about ~0.5% of these sites showing a change with age. Therefore, while only a small fraction of cytosines in the genome show changes in DNA methylation with age, this represents 2 to 3 million cytosines in the genome. Importantly, the only study to compare the effect of aging on DNA methylation in male and female mice and humans found that >95% of the age-related changes in DNA methylation in the hippocampus were sexually divergent, i.e., the methylation did not differ between males and females at young age but age-related changes occurred in one sex but not the other. The age-related changes in DNA methylation tend to be enriched and under-represented in specific genomic contexts, with some commonalities between tissues and species that require further investigation. The strongest evidence that the age-related changes in DNA methylation play a role in aging comes from studies of anti-aging interventions (e.g., caloric restriction, dwarfism, and rapamycin treatment) in mice. These anti-aging interventions deaccelerate the epigenetic clocks and reverse/prevent 20 to 40% of the age-related changes in DNA methylation. It will be important in the future to demonstrate that at least some of the age-related changes in DNA methylation directly lead to alterations in the transcriptome of cells/tissues that could potentially contribute to aging.

Determination of Chromatin Accessibility in Drosophila MidgutEnterocytes by in situ 5mC Labeling.

Regulation of gene expression involves dynamic changes in chromatin organization, where in many cases open chromatin structure correlates with gene activation. Several methods enable monitoring changes in chromatin accessibility, including ATAC-seq, FAIRE-seq, MNase-seq and DNAse-seq methods, which involve Next-generation-sequencing (NGS). Focusing on the adult Drosophila differentiated gut enterocytes (ECs) we used a sequencing-free method that enables visualizing and semi-quantifying large-scale changes in chromatin structure using in vitro methylation assay with the bacterial CpG Methyltransferase, M. Sssl, that determine chromatin accessibility. In brief, as CpG methylation is minimal in differentiated somatic Drosophila cells, we used the bacterial M. SssI enzyme to methylate CpG dinucleotides in situ depending on their chromatin accessibility. The methylated dinucleotides are detected using 5mCytosine monoclonal antibody and nuclei are visualized microscopically. Thus, the 5mC method enables to monitor large-scale chromatin changes in heterogenic cellular tissue focusing on the cell type of interest and without the need for cell purification or NGS.

Diesel exhaust and house dust mite allergen lead to common changes in the airway methylome and hydroxymethylome.

Exposures to diesel exhaust particles (DEP) from traffic and house dust mite (HDM) allergens significantly increase risks of airway diseases, including asthma. This negative impact of DEP and HDM may in part be mediated by epigenetic mechanisms. Beyond functioning as a mechanical barrier, airway epithelial cells provide the first line of immune defense towards DEP and HDM exposures. To understand the epigenetic responses of airway epithelial cells to these exposures, we exposed human bronchial epithelial cells to DEP and HDM and studied genome-wide 5-methyl-cytosine (5mC) and 5-hydroxy-methylcytosine (5hmC) at base resolution. We found that exposures to DEP and HDM result in elevated TET1 and DNMT1 expression, associated with 5mC and 5hmC changes. Interestingly, over 20% of CpG sites are responsive to both exposures and changes in 5mC at these sites negatively correlated with gene expression differences. These 5mC and 5hmC changes are located in genes and pathways related to oxidative stress responses, epithelial function and immune cell responses and are enriched for binding sites of transcription factors (TFs) involved in these pathways. Histone marks associated with promoters, enhancers and actively transcribed gene bodies were associated with exposure-induced DNA methylation changes. Collectively, our data suggest that exposures to DEP and HDM alter 5mC and 5hmC levels at regulatory regions bound by TFs, which coordinate with histone marks to regulate gene networks of oxidative stress responses, epithelial function and immune cell responses. These observations provide novel insights into the epigenetic mechanisms that mediate the epithelial responses to DEP and HDM in airways.

The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius.

DNA methylation is the most common epigenetic modification observed in the genomic DNA (gDNA) of prokaryotes and eukaryotes. Methylated nucleobases, N6-methyl-adenine (m6A), N4-methyl-cytosine (m4C), and 5-methyl-cytosine (m5C), detected on gDNA represent the discrimination mark between self and non-self DNA when they are part of restriction-modification systems in prokaryotes (Bacteria and Archaea). In addition, m5C in Eukaryotes and m6A in Bacteria play an important role in the regulation of key cellular processes. Although archaeal genomes present modified bases as in the two other domains of life, the significance of DNA methylations as regulatory mechanisms remains largely uncharacterized in Archaea. Here, we began by investigating the DNA methylome of Sulfolobus acidocaldarius. The strategy behind this initial study entailed the use of combined digestion assays, dot blots, and genome resequencing, which utilizes specific restriction enzymes, antibodies specifically raised against m6A and m5C and single-molecule real-time (SMRT) sequencing, respectively, to identify DNA methylations occurring in exponentially growing cells. The previously identified restriction-modification system, specific of S. acidocaldarius, was confirmed by digestion assay and SMRT sequencing while, the presence of m6A was revealed by dot blot and identified on the characteristic Dam motif by SMRT sequencing. No m5C was detected by dot blot under the conditions tested. Furthermore, by comparing the distribution of both detected methylations along the genome and, by analyzing DNA methylation profiles in synchronized cells, we investigated in which cellular pathways, in particular the cell cycle, this m6A methylation could be a key player. The analysis of sequencing data rejected a role for m6A methylation in another defense system and also raised new questions about a potential involvement of this modification in the regulation of other biological functions in S. acidocaldarius.

Quantification of Global DNA Methylation Levels by Mass Spectrometry.

Global DNA methylation was classically considered the relative percentage of 5-methylcysine (5mC) with respect to total cytosine (C). Early approaches were based on the use of high-performance separation technologies and UV detection. However, the recent development of protocols using mass spectrometry for the detection has increased sensibility and permitted the precise identification of peak compounds based on their molecular masses. This allows work to be conducted with much less genomic DNA starting material and also to quantify 5-hydroxymethyl-cytosine (5hmC), a recently identified form of methylated cytosine that could play an important role in active DNA demethylation. Here, we describe the protocol that we currently use in our laboratory to analyze 5mC and 5hmC by mass spectrometry. The protocol, which is based on the method originally developed by Le and colleagues using Ultra Performance Liquid Chromatography (UPLC) and mass spectrometry (triple Quadrupole (QqQ)) detection, allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels starting from just 1 µg of genomic DNA, which allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels.

Tagmentation-Based Library Preparation for Low DNA Input Whole Genome Bisulfite Sequencing.

Aberrations of the DNA methylome contribute to onset and progression of diseases. Whole genome bisulfite sequencing (WGBS) is the only analytical method covering the complete methylome. Alternative methods requiring less DNA than WGBS analyze only a minor portion of the methylome and do not cover important regulatory features like enhancers and noncoding RNAs. In tagmentation-based WGBS (TWGBS), several DNA and time-consuming steps of the conventional WGBS library preparation are circumvented by the use of a hyperactive transposase, which simultaneously fragments DNA and appends sequencing adapters. TWGBS requires only nanogram amounts of DNA and, thus, is well suited to study precious biological specimens such as sorted cells or micro-dissected tissue samples.

Quantitative Analysis of the DNA Methylation Sensitivity of Transcription Factor Complexes.

Although DNA modifications play an important role in gene regulation, the underlying mechanisms remain elusive. We developed EpiSELEX-seq to probe the sensitivity of transcription factor binding to DNA modification in vitro using massively parallel sequencing. Feature-based modeling quantifies the effect of cytosine methylation (5mC) on binding free energy in a position-specific manner. Application to the human bZIP proteins ATF4 and C/EBPß and three different Pbx-Hox complexes shows that 5mCpG can both increase and decrease affinity, depending on where the modification occurs within the protein-DNA interface. The TF paralogs tested vary in their methylation sensitivity, for which we provide a structural rationale. We show that 5mCpG can also enhance in vitro p53 binding and provide evidence for increased in vivo p53 occupancy at methylated binding sites, correlating with primed enhancer histone marks. Our results establish a powerful strategy for dissecting the epigenomic modulation of protein-DNA interactions and their role in gene regulation.

Statistical Methods for Identifying Sequence Motifs Affecting Point Mutations.

Mutation processes differ between types of point mutation, genomic locations, cells, and biological species. For some point mutations, specific neighboring bases are known to be mechanistically influential. Beyond these cases, numerous questions remain unresolved, including: what are the sequence motifs that affect point mutations? How large are the motifs? Are they strand symmetric? And, do they vary between samples? We present new log-linear models that allow explicit examination of these questions, along with sequence logo style visualization to enable identifying specific motifs. We demonstrate the performance of these methods by analyzing mutation processes in human germline and malignant melanoma. We recapitulate the known CpG effect, and identify novel motifs, including a highly significant motif associated with A[Formula: see text]G mutations. We show that major effects of neighbors on germline mutation lie within [Formula: see text] of the mutating base. Models are also presented for contrasting the entire mutation spectra (the distribution of the different point mutations). We show the spectra vary significantly between autosomes and X-chromosome, with a difference in T[Formula: see text]C transition dominating. Analyses of malignant melanoma confirmed reported characteristic features of this cancer, including statistically significant strand asymmetry, and markedly different neighboring influences. The methods we present are made freely available as a Python library https://bitbucket.org/pycogent3/mutationmotif.

Epigenetic contribution to successful polyploidizations: variation in global cytosine methylation along an extensive ploidy series in Dianthus broteri (Caryophyllaceae).

Polyploidization is a significant evolutionary force in plants which involves major genomic and genetic changes, frequently regulated by epigenetic factors. We explored whether natural polyploidization in Dianthus broteri complex resulted in substantial changes in global DNA cytosine methylation associated to ploidy. Global cytosine methylation was estimated by high-performance liquid chromatography (HPLC) in 12 monocytotypic populations with different ploidies (2×, 4×, 6×, 12×) broadly distributed within D. broteri distribution range. The effects of ploidy level and local variation on methylation were assessed by generalized linear mixed models (GLMMs). Dianthus broteri exhibited a higher methylation percent (˜33%) than expected by its monoploid genome size and a large variation among study populations (range: 29.3-35.3%). Global methylation tended to increase with ploidy but did not significantly differ across levels due to increased variation within the highest-order polyploidy categories. Methylation varied more among hexaploid and dodecaploid populations, despite such cytotypes showing more restricted geographic location and increased genetic relatedness than diploids and tetraploids. In this study, we demonstrate the usefulness of an HPLC method in providing precise and genome reference-free global measure of DNA cytosine methylation, suitable to advance current knowledge of the roles of this epigenetic mechanism in polyploidization processes.

The Methylated DNA Immunoprecipitation [MeDIP] to Investigate the Epigenetic Remodeling in Cell Fate Determination and Cancer Development.

Epigenetic mechanisms such as DNA methylation, posttranslational modifications of histone proteins, remodeling of nucleosomes, and the expression of noncoding RNAs contribute to the regulation of gene expression for the cell fate determination and tissue development. The disruption of these epigenetic mechanisms, in conjunction with genetic alterations, is a decisive element for cancer development and progression. The cancer phenotype is characterized by global DNA hypomethylation and gene-specific hypermethylation. The methylated DNA immunoprecipitation [MeDIP] is a useful approach currently used to clarify the functional consequences of DNA methylation on cell fate determination and cancer development.