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Environmental Pollution (Barking, Essex... Jul 2022Lead (Pb) exposure can induce DNA damage and alter DNA methylation but their inter-relationships have not been adequately determined. Our overall aims were to explore...
Lead (Pb) exposure can induce DNA damage and alter DNA methylation but their inter-relationships have not been adequately determined. Our overall aims were to explore such relationships and to evaluate underlying epigenetic mechanisms of Pb-induced genotoxicity in Chinese workers. Blood Pb levels (BLLs) were determined and used as individual's Pb-exposure dose and the Comet assay (i.e., % tail DNA) was conducted to evaluate DNA damage. In the screening assay, 850 K BeadChip sequencing was performed on peripheral blood from 10 controls (BLLs ≤100 μg/L) and 20 exposed workers (i.e., 10 DNA-damaged and 10 DNA-undamaged workers). Using the technique, differentially methylated positions (DMPs) between the controls and the exposed workers were identified. In addition, DMPs were identified between the DNA-undamaged and DNA-damaged workers (% tail DNA >2.14%). In our validation assay, methylation levels of four candidate genes were measured by pyrosequencing in an independent sample set (n = 305), including RRAGC (Ras related GTP binding C), USP1 (Ubiquitin specific protease 1), COPS7B (COP9 signalosome subunit 7 B) and CHEK1 (Checkpoint kinase 1). The result of comparisons between the controls and the Pb-exposed workers show that DMPs were significantly enriched in genes related to nerve conduction and cell cycle. Between DNA-damaged group and DNA-undamaged group, differentially methylated genes were enriched in the pathways related to cell cycle and DNA integrity checkpoints. Additionally, methylation levels of RRAGC and USP1 were negatively associated with BLLs (P < 0.05), and the former mediated 19.40% of the effect of Pb on the % tail DNA. These findings collectively indicated that Pb-induced DNA damage was closely related to methylation of genes in cell cycle regulation, and methylation levels of RRAGC were involved in Pb-induced genotoxicity.
Topics: DNA; DNA Damage; DNA Methylation; Humans; Lead; Occupational Exposure
PubMed: 35385786
DOI: 10.1016/j.envpol.2022.119252 -
Analytical Chemistry Dec 2023N6-methyladenosine (mA) has recently gained much attention due to its diverse biological functions. Currently, the commonly used detection methods for locus-specific mA...
N6-methyladenosine (mA) has recently gained much attention due to its diverse biological functions. Currently, the commonly used detection methods for locus-specific mA marks are complicated to operate, it is difficult to quantify the methylation level, and they have high false-positive levels. Here, we report a new method for locus-specific mA detection based on the methylate-sensitive endonuclease activity of MazF and the simultaneous amplification and testing (SAT) method, termed "mA-MazF-SAT". Mechanically, MazF fails to cleave the A (mA) CA motif; therefore, the undigested template can be SAT-amplified using specific probes targeting the upstream and downstream of sites of interest. Fluorescent signals of SAT amplification can be detected by real-time PCR, and therefore, they achieve the detection of mA existence. After the condition optimization, mA-MazF-SAT can significantly, accurately, and rapidly detect the mA-modified sites in mRNA, rRNA, and lncRNA at the fmol level, as well as 10% mA at the fmol level. In addition, mA-MazF-SAT can quantify the abundance of target mA in biological samples and can be used for the inhibitor selection of mA-related enzymes. Together, we offer a new approach to detect locus-specific mA both qualitatively and quantitatively; it is easy to operate, results can be obtained rapidly, and it has low false-positive levels and high repeatability.
Topics: RNA; RNA, Messenger; Methylation
PubMed: 38049147
DOI: 10.1021/acs.analchem.3c03961 -
Methods in Molecular Biology (Clifton,... 2022Global hypomethylation of genomic DNA is associated with genomic instability and carcinogenic processes. The loss of DNA methylation has been reported in several...
Global hypomethylation of genomic DNA is associated with genomic instability and carcinogenic processes. The loss of DNA methylation has been reported in several cancers; therefore, global methylation levels have been considered as biomarkers for cancer diagnosis. Bisulfite conversion analysis has been widely used as the gold standard method for quantification of DNA methylation levels. However, this method requires cumbersome and time-consuming steps. To quantify global DNA methylation levels in homogeneous solutions, we exemplify a sensing system based on bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (MBD-FLuc) and unmethyl-CpG binding domain (CXXC)-fused firefly luciferase (CXXC-FLuc). MBD-FLuc and CXXC-FLuc bind to methylated and unmethylated CpGs, respectively, in the genomic DNA to excite BOBO-3, an intercalating dye on genomic DNA. These BOBO-3 emission intensities depend on the methylated and unmethylated CpG content. The global DNA methylation levels can be quantified from the BOBO-3 emission intensities. Moreover, we introduce a multicolor BRET assay using MBD-FLuc and CXXC-fused Oplophorus luciferase (CXXC-OLuc) for the simultaneous quantification of methylated and unmethylated CpG content in genomic DNA. CXXC-OLuc excites the BOBO-1 DNA-intercalating dye depending on the unmethylated CpG content. Thus, the emission intensities of BOBO-1 and BOBO-3 excited by CXXC-OLuc and MBD-FLuc, respectively, can be simultaneously measured, thereby enabling the determination of global DNA methylation level in a single step. Here, we describe the detailed protocols for the expression of MBD-FLuc, CXXC-FLuc, and CXXC-OLuc in Escherichia coli and determine the global DNA methylation levels using these BRET assays.
Topics: CpG Islands; DNA; DNA Methylation; DNA-Binding Proteins; Energy Transfer; Luciferases; Luciferases, Firefly
PubMed: 35836075
DOI: 10.1007/978-1-0716-2473-9_20 -
Clinical Laboratory Apr 2023During viral infections such as SARS-CoV-2, epigenetic changes within the promoter region of the immune system genes would possibly occur and have an effect on the...
BACKGROUND
During viral infections such as SARS-CoV-2, epigenetic changes within the promoter region of the immune system genes would possibly occur and have an effect on the immune system response as well as disease outcome. We aimed to evaluate and compare the methylation level of the IFITM1 gene promoter in different stages of COVID-19 disease with a healthy control group.
METHODS
In this cross-sectional study, 75 COVID-19 patients (25 mild, 25 severe, and 25 critical in addition to 25 age- and gender-matched healthy volunteers) have been included. DNA was extracted from the peripheral white blood cells using a commercial DNA extraction kit. PCR was performed using two types of primers designed for the methylated and unmethylated forms of the IFITM1 gene promoter.
RESULTS
The mean age of the patient and healthy volunteer groups was 52.733 ± 13.780 and 49.120 ± 12.490, respectively. Out of a hundred participants, 52 were male. The results demonstrated that severe (p = 0.03, OR 6.729) and critical (p = 0.001, OR 11.156) patients were much more likely to show methylation of the IFITM1 gene in contrast with mild patients. Moreover, IFITM1 methylation was significantly higher in COVID-19 patients in comparison with the healthy volunteer group (p = 0.004, OR 3.17). Furthermore, IFITM1 methylation in male patients with critical status, (p = 0.01) was significantly higher than in male patients with mild status. In addition, IFITM1 methylation of male (p = 0.03) and female (p = 0.01) critical patients was considerably higher compared to males and females of volunteer group.
CONCLUSIONS
Increased methylation of the IFITM1 gene in the severe and critical stage of COVID-19 diseases may indicate the role of SARS-CoV-2 infection in increasing methylation of this antiviral gene. This might be involved in suppressing the immune system, promoting SARS-CoV-2 replication and disease outcome.
Topics: Humans; Male; Female; COVID-19; SARS-CoV-2; Methylation; Cross-Sectional Studies; Promoter Regions, Genetic; DNA Methylation
PubMed: 37057950
DOI: 10.7754/Clin.Lab.2022.220622 -
Analytical Biochemistry Jul 2023DNA methylation as a ubiquitously regulation is closely associated with cell proliferation and differentiation. Growing data shows that aberrant methylation contributes...
DNA methylation as a ubiquitously regulation is closely associated with cell proliferation and differentiation. Growing data shows that aberrant methylation contributes to disease incidence, especially in tumorigenesis. The approach for identifying DNA methylation usually depends on treatment of sodium bisulfite, which is time-consuming and conversion-insufficient. Here, with a special biosensor, we establish an alternative approach for detecting DNA methylation. The biosensor is consisted of two parts, which are gold electrode and nanocomposite (AuNPs/rGO/g-CN). Nanocomposite was fabricated by three components, which are gold nanoparticles (AuNPs), reduced graphene oxide (rGO) and graphite carbon nitride (g-CN). For methylated DNA detection, the target DNA was captured by probe DNA immobilized on the gold electrode surface through thiolating process and subjected to hybrid with anti-methylated cytosine conjugated to nanocomposite. When the methylated cytosines in target DNA were recognized by anti-methylated cytosine, a change of electrochemical signals will be observed. With different size of target DNAs, the concentration and methylation level were tested. It is shown that in short size methylated DNA fragment, the linear range and LOD of concentration is 10M-10M and 0.74 fM respectively; in longer size methylated DNA, the linear range of methylation proportion and LOD of copy number is 3%-84% and 10 respectively. Also, this approach has a high sensitivity and specificity as well as anti-disturbing ability.
Topics: Graphite; Gold; DNA Methylation; Electrochemical Techniques; Metal Nanoparticles; Biosensing Techniques; DNA; Nanocomposites; Electrodes
PubMed: 37146956
DOI: 10.1016/j.ab.2023.115180 -
Molecules (Basel, Switzerland) Jun 2021The almiramide -methylated lipopeptides exhibit promising activity against trypanosomatid parasites. A structure-activity relationship study has been performed to...
The almiramide -methylated lipopeptides exhibit promising activity against trypanosomatid parasites. A structure-activity relationship study has been performed to examine the influences of -methylation and conformation on activity against various strains of leishmaniasis protozoan and on cytotoxicity. The synthesis and biological analysis of twenty-five analogs demonstrated that derivatives with a single methyl group on either the first or fifth residue amide nitrogen exhibited greater activity than the permethylated peptides and relatively high potency against resistant strains. Replacement of amino amide residues in the peptide, by turn inducing α amino γ lactam (Agl) and -aminoimidazalone (Nai) counterparts, reduced typically anti-parasitic activity; however, peptide amides possessing Agl residues at the second residue retained significant potency in the unmethylated and permethylated series. Systematic study of the effects of methylation and turn geometry on anti-parasitic activity indicated the relevance of an extended conformer about the central residues, and conformational mobility by tertiary amide isomerization and turn geometry at the extremities of the active peptides.
Topics: Amides; Isomerism; Leishmania; Lipopeptides; Methylation; Protein Conformation; Structure-Activity Relationship
PubMed: 34204673
DOI: 10.3390/molecules26123606 -
BMC Bioinformatics Jan 2022DNA methylation is commonly measured using bisulfite sequencing (BS-seq). The quality of a BS-seq library is measured by its bisulfite conversion efficiency. Libraries...
BACKGROUND
DNA methylation is commonly measured using bisulfite sequencing (BS-seq). The quality of a BS-seq library is measured by its bisulfite conversion efficiency. Libraries with low conversion rates are typically excluded from analysis resulting in reduced coverage and increased costs.
RESULTS
We have developed a probabilistic method and software, LuxRep, that implements a general linear model and simultaneously accounts for technical replicates (libraries from the same biological sample) from different bisulfite-converted DNA libraries. Using simulations and actual DNA methylation data, we show that including technical replicates with low bisulfite conversion rates generates more accurate estimates of methylation levels and differentially methylated sites. Moreover, using variational inference speeds up computation time necessary for whole genome analysis.
CONCLUSIONS
In this work we show that taking into account technical replicates (i.e. libraries) of BS-seq data of varying bisulfite conversion rates, with their corresponding experimental parameters, improves methylation level estimation and differential methylation detection.
Topics: DNA Methylation; Data Analysis; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA; Sulfites
PubMed: 35030989
DOI: 10.1186/s12859-021-04546-1 -
The Journal of Experimental Biology Jul 2020The epigenome determines heritable patterns of gene expression in the absence of changes in DNA sequence. The result is programming of different cellular-, tissue- and... (Review)
Review
The epigenome determines heritable patterns of gene expression in the absence of changes in DNA sequence. The result is programming of different cellular-, tissue- and organ-specific phenotypes from a single organismic genome. Epigenetic marks that comprise the epigenome (e.g. methylation) are placed upon or removed from chromatin (histones and DNA) to direct the activity of effectors that regulate gene expression and chromatin structure. Recently, the cytoskeleton has been identified as a second target for the cell's epigenetic machinery. Several epigenetic 'readers, writers and erasers' that remodel chromatin have been discovered to also remodel the cytoskeleton, regulating structure and function of microtubules and actin filaments. This points to an emerging paradigm for dual-function remodelers with 'chromatocytoskeletal' activity that can integrate cytoplasmic and nuclear functions. For example, the SET domain-containing 2 methyltransferase (SETD2) has chromatocytoskeletal activity, methylating both histones and microtubules. The SETD2 methyl mark on chromatin is required for efficient DNA repair, and its microtubule methyl mark is required for proper chromosome segregation during mitosis. This unexpected convergence of SETD2 activity on histones and microtubules to maintain genomic stability suggests the intriguing possibility of an expanded role in the cell for chromatocytoskeletal proteins that read, write and erase methyl marks on the cytoskeleton as well as chromatin. Coordinated use of methyl marks to remodel both the epigenome and the (epi)cytoskeleton opens the possibility for integrated regulation (which we refer to as 'epiregulation') of other higher-level functions, such as muscle contraction or learning and memory, and could even have evolutionary implications.
Topics: Chromatin; Cytoskeleton; DNA Methylation; Epigenesis, Genetic; Epigenome; Histones; Methylation; Microtubules
PubMed: 32620673
DOI: 10.1242/jeb.220632 -
Plant & Cell Physiology Jun 2020Soil salinity is a major source of abiotic plant stress, adversely affecting plant growth, development and productivity. Although the physiological and molecular...
Soil salinity is a major source of abiotic plant stress, adversely affecting plant growth, development and productivity. Although the physiological and molecular mechanisms that underlie plant responses to salt stress are becoming increasingly understood, epigenetic modifications, such as histone methylations and their potential regulation of the transcription of masked genes at the genome level in response to salt stress, remain largely unclear. Castor bean, an important nonedible oil crop, has evolved the capacity to grow under salt stress. Here, based on high-throughput RNA-seq and ChIP-seq data, we systematically investigated changes in genomic transcription and histone methylation using typical histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 tri-methylated lysine 27 (H3K27me3) markers in castor bean leaves subjected to salt stress. The results showed that gain or loss of histone methylation was closely associated with activated or repressed gene expression, though variations in both transcriptome and histone methylation modifications were relatively narrow in response to salt stress. Diverse salt responsive genes and switched histone methylation sites were identified in this study. In particular, we found for the first time that the transcription of the key salt-response regulator RADIALIS-LIKE SANT (RSM1), a MYB-related transcription factor involved in ABA(abscisic acid)-mediated salt stress signaling, was potentially regulated by bivalent H3K4me3-H3K27me3 modifications. Combining phenotypic variations with transcriptional and epigenetic changes, we provide a comprehensive profile for understanding histone modification, genomic transcription and their associations in response to salt stress in plants.
Topics: Ricinus communis; Gene Expression Regulation, Plant; Histone Code; Histones; Methylation; Plant Leaves; Salt Stress; Transcription, Genetic
PubMed: 32186723
DOI: 10.1093/pcp/pcaa037 -
ACS Chemical Biology Jul 2020The methylation of amide nitrogen atoms can improve the stability, oral availability, and cell permeability of peptide therapeutics. Chemical -methylation of peptides is...
The methylation of amide nitrogen atoms can improve the stability, oral availability, and cell permeability of peptide therapeutics. Chemical -methylation of peptides is challenging. Omphalotin A is a ribosomally synthesized, macrocylic dodecapeptide with nine backbone -methylations. The fungal natural product is derived from the precursor protein, OphMA, harboring both the core peptide and a SAM-dependent peptide α--methyltransferase domain. OphMA forms a homodimer and its α--methyltransferase domain installs the methyl groups on the hydrophobic core dodecapeptide and some additional C-terminal residues of the protomers. These post-translational backbone -methylations occur in a processive manner from the N- to the C-terminus of the peptide substrate. We demonstrate that OphMA can methylate polar, aromatic, and charged residues when these are introduced into the core peptide. Some of these amino acids alter the efficiency and pattern of methylation. Proline, depending on its sequence context, can act as a tunable stop signal. Crystal structures of OphMA variants have allowed rationalization of these observations. Our results hint at the potential to control this fungal α--methyltransferase for biotechnological applications.
Topics: Agaricales; Amino Acid Sequence; Fungal Proteins; Methylation; Methyltransferases; Mutation; Peptides, Cyclic; Protein Domains; Protein Precursors; Protein Processing, Post-Translational; Substrate Specificity
PubMed: 32491837
DOI: 10.1021/acschembio.0c00237