Genome-wide lone strand adenine methylation in Deinococcus radiodurans R1: Regulation of gene expression through DR0643-dependent adenine methylation.

DNA methylation is a covalent modification of adenine or cytosine in the genome of an organism and is found in diverse microbes including the radiation resistant bacterium Deinococcus radiodurans R1. Although earlier findings have confirmed repression or de-repression of certain genes in adenine methyltransferase (DR_0643/Dam1DR) deficient D. radiodurans mutant however, the overall regulatory aspects of Dam1DR-mediated adenine methylation remain mostly unexplored. In the present study, we compared the genome-wide methylome and the corresponding transcriptome of D. radiodurans WT and Δdam1 mutant to explore the correlation between methylation and gene expression. In D. radiodurans, deletion of DR_0643 ORF (Δdam1) led to hypomethylation of 512 genes resulting in differential expression of 168 genes (99 genes are upregulated and 69 genes are downregulated). The modification patterns deduced for Dam1DR (DR_0643) and Dam2DR (DR_2267) were non-palindromic and atypical. Moreover, we observed methylation at opportunistic sites that show adenine methylation only in D. radiodurans Δdam1 and not in D. radiodurans WT. Correlation between the methylome and transcriptome suggests that hypomethylation at Dam1DR specific sites had both negative as well as a positive effects on gene expression. Pathways such as amino acid metabolism, transport, oxidative phosphorylation, quorum sensing, signal transduction, two-component system, glycolysis/gluconeogenesis, TCA cycle, glyoxylate and dicarboxylate metabolism were modulated by Dam1DR-mediated adenine methylation in D. radiodurans. Processes such as DNA repair, recombination, ATPase and transmembrane transporter activity were enriched when Dam1DR mutant was subjected to radiation stress. We further evaluated the molecular interactions and mode of binding between Dam1DR protein and S-adenosyl methionine using molecular docking followed by MD simulation. To get a better insight into the methylation mechanism, the Dam1DR-SAM complex was also docked with a DNA molecule to elucidate DNA-Dam1DR structural interaction during methyl-group transfer reaction. In summary, our work presents comprehensive and integrative approaches to investigate both functional and structural aspects of DNA adenine methyltransferase (Dam1DR) in D. radiodurans biology.

Functional and regulatory aspects of oxidative stress response in X monosomy.

The partial/complete loss of one X chromosome in a human female leads to Turner syndrome (TS). TS individuals display a range of phenotypes including short stature, osteoporosis, ovarian malfunction, diabetes, and thyroid dysfunction. Epigenetic factors and regulatory networks are distinctly different in X monosomy (45, X). In a lifetime, an individual is exposed to a variety of stress conditions. To study whether X monosomy cells display a differential response upon exposure to mild stress as compared to normal 46, XX cells and whether this may contribute to various co-morbidities in aneuploid individuals, we have carried out a transcriptomic analysis of human fibroblasts 45, X and 46, XX after exposure to mild oxidative stress. Under these conditions, over 350 transcripts were seen to be differentially expressed in 45, X and 46, XX cells. Pathways associated with oxidative stress were differentially expressed highlighting the differential regulation of genes and associated phenotypes. It could be seen that X monosomy cells are more susceptible to oxidative stress as compared to normal cells and have altered molecular pathways both in normal conditions and also upon exposure to mild oxidative stress. To explore this aspect in detail, we have mapped the expressions of transcription factors (TFs) in 45, X and 46, XX cells. The network of transcription activating factors is differentially regulated in 45, X and 46, XX cells under stress exposure. It is tempting to speculate that the altered ability of 45, X (Turner) cells to respond to stress may play a significant role in the physiological function and altered phenotypes in Turner syndrome.

Analysis of drug resistance marker genes of Plasmodium falciparum after implementation of artemisinin-based combination therapy in Pune district, India.

The global emergence and spread of malaria parasites resistant to antimalarial drugs is a major problem in malaria control and elimination. In this study, samples from Pune district were characterized to determine prevalence of molecular markers of resistance to chloroquine (pfcrt codons C72S, M74I, N75E, K76T and pfmdr-1 N86Y, Y184F), pyrimethamine (pfdhfr C50R, N51I, C59R, S108N), sulfadoxine (pfdhps, S436A, A437G, K540E, A581G), and artemisinin (pfkelch13, C580Y, R539T). The pfcrt K76T mutation was found in 78% samples as CVMNT, SVMNT and CVIET haplotype. The pfmdr-1 N86Y and Y184F mutations were found in 54% of samples. The pfdhfr double mutation C59R + S108N was present in 67% of samples, while the pfdhfr triple mutation (N51I + C59R + S108N) was not detected. The pfdhps mutations A437G and K540E were found in 67% of samples. Single mutants of pfdhps were rare, with K540E detected in only 6 patient samples. Similarly, pfdhps A581G was found in 13 of the isolates. The molecular markers associated with artemisinin resistance (mutations in pfkelch13 C580Y, R539T) were not detected in any of the isolates. These results suggest an emerging problem with multidrug-resistant P. falciparum. Though the genotype conventionally associated with artemisinin resistance was not observed, chloroquine-resistant genotype has reached complete fixation in the population. Moreover, the prevalence of mutations in both pfdhfr and pfdhps, with the presence of the quadruple mutant, indicates that continued monitoring is required to assess whether sulfadoxine-pyrimethamine can be used efficiently as a partner drug for artemisinin for the treatment of P. falciparum.

N6-methyladenine and epigenetic immunity of Deinococcus radiodurans.

DNA methylation is ubiquitously found in all three domains of life. This epigenetic modification on adenine or cytosine residues serves to regulate gene expression or to defend against invading DNA in bacteria. Here, we report the significance of N6-methyladenine (6mA) to epigenetic immunity in Deinococcus radiodurans. Putative protein encoded by DR_2267 ORF (Dam2DR) contributed 35% of genomic 6mA in D. radiodurans but did not influence gene expression or radiation resistance. Dam2DR was characterized to be a functional S-adenosyl methionine (SAM)-dependent N6-adenine DNA methyltransferase (MTase) but with no endonuclease activity. Adenine methylation from Dam2DR or Dam1DR (N6-adenine MTase encoded by DR_0643) improved DNA uptake during natural transformation. To the contrary, methylation from Escherichia coli N6-adenine MTase (DamEC that methylates adenine in GATC sequence) on donor plasmid drastically reduced DNA uptake in D. radiodurans, even in presence of Dam2DR or Dam1DR methylated adenines. With these results, we conclude that self-type N6-adenine methylation on donor DNA had a protective effect in absence of additional foreign methylation, a separate methylation-dependent Restriction Modification (R-M) system effectively identifies and limits uptake of G6mATC sequence containing donor DNA. This is the first report demonstrating presence of epigenetic immunity in D. radiodurans.

Construction, analysis and validation of co-expression network to understand stress adaptation in Deinococcus radiodurans R1.

Systems biology based approaches have been effectively utilized to mine high throughput data. In the current study, we have performed system-level analysis for Deinococcus radiodurans R1 by constructing a gene co-expression network based on several microarray datasets available in the public domain. This condition-independent network was constructed by Weighted Gene Co-expression Network Analysis (WGCNA) with 61 microarray samples from 9 different experimental conditions. We identified 13 co-expressed modules, of which, 11 showed functional enrichments of one or more pathway/s or biological process. Comparative analysis of differentially expressed genes and proteins from radiation and desiccation stress studies with our co-expressed modules revealed the association of cyan with radiation response. Interestingly, two modules viz darkgreen and tan was associated with radiation as well as desiccation stress responses. The functional analysis of these modules showed enrichment of pathways important for adaptation of radiation or desiccation stress. To decipher the regulatory roles of these stress responsive modules, we identified transcription factors (TFs) and then calculated a Biweight mid correlation between modules hub gene and the identified TFs. We obtained 7 TFs for radiation and desiccation responsive modules. The expressions of 3 TFs were validated in response to gamma radiation using qRT-PCR. Along with the TFs, selected close neighbor genes of two important TFs, viz., DR_0997 (CRP) and DR_2287 (AsnC family transcriptional regulator) in the darkgreen module were also validated. In our network, among 13 hub genes associated with 13 modules, the functionality of 5 hub genes which are annotated as hypothetical proteins (hypothetical hub genes) in D. radiodurans genome has been revealed. Overall the study provided a better insight of pathways and regulators associated with relevant DNA damaging stress response in D. radiodurans.

Genome Sequence of Bordetella pertussis Vaccine Strain BP 165.

Whole-cell and acellular pertussis vaccines are used globally against Bordetella pertussis Various vaccine reference strains are used globally for the production of such vaccines. We report here a draft genome sequence for Bordetella pertussis strain BP 165, which is used by the Serum Institute of India in the production of acellular pertussis vaccine.

Characterization and DNA methylation modulatory activity of gold nanoparticles synthesized by Pseudoalteromonas strain.

Marine extremophiles are shown to tolerate extreme environmental conditions and have high metal reducing properties. Here, we report intracellular synthesis of gold nanoparticles (AuNP) by marine extremophilic bacteria Pseudoalteromonas sp. Bac178 which was isolated from the OMZ of Arabian Sea. Preliminary observations suggest that these bacteria use different pathways which may involves the membrane as well as intracellular proteins for the gold salt reduction. Characterization of the biosynthesised nanoparticles by various techniques such as Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), X-ray diffraction (XRD) and Energy-dispersive X-ray spectroscopy (EDS) confirmed the presence of crystalline gold. These biologically synthesized AuNP were investigated for cytotoxicity and oxidative stress generation in human normal fibroblast and melanoma cells (A375). As AuNP are envisaged to find many applications in the medical field, it was of interest to study the effect of AuNP at the epigenetic level. They were found to be non-cytotoxic, non-genotoxic and non-oxidative stress generating over a range of concentrations. Exposure to these AuNP is observed to cause alterations in global DNA methylation as well as in the expression of DNA methyltransferase (DNMT) genes. Since biosynthesized AuNP are being used in various applications and therapies, their epigenetic modulatory activity needs careful consideration.

An exploration of microbial and associated functional diversity in the OMZ and non-OMZ areas in the Bay of Bengal.

Depletion of oxygen in certain marine areas creates oxygen minimum zones (OMZs), which can alter the species composition and abundance. We have carried out high-throughput 16S rRNA gene amplicon profiling from the Bay of Bengal (BOB) OMZ and non-OMZ areas. Typically, a total of 35 families of micro-organisms were identified as biomarkers for OMZ and non-OMZ regions in the BOB. Our analysis has identified families Pseudoalteromonadaceae, OM60 and Synechococcaceae to be abundant in oxygenated water, whereas organisms belonging to families Pelagibacteraceae and Caulobacteraceae, which are involved in sulphur and nitrogen metabolism, were prominent in the OMZ areas. Predictive functional analysis for these identified bacteria clearly that suggested an abundance of microbes with assimilatory sulphurreducing genes (cysl and csH) in the non-OMZ, while bacteria involved in dissimilatory sulphate reduction (known to carry aprA and aprB genes) were enriched in the OMZ areas. Comparative analysis with OMZ areas from Peru and Chile revealed that OMZ areas in the BOB are characterized by specific and distinctive bacterial diversity. Overall, the current analysis provides valuable documentation about the bacterial populations and their characteristics, which can generate pointers for their functional significance in the BOB.

Drosophila DNA/RNA methyltransferase contributes to robust host defense in aging animals by regulating sphingolipid metabolism.

Drosophila methyltransferase (Mt2) has been implicated in the methylation of both DNA and tRNA. In this study, we demonstrate that loss of Mt2 activity leads to an age-dependent decline of immune function in the adult fly. A newly eclosed adult has mild immune defects that are exacerbated in a 15 day old Mt2-/- fly. The age-dependent effects appear to be systemic, including disturbances in lipid metabolism, changes in cell shape of hemocytes and significant fold-changes in levels of transcripts related to host defense. Lipid imbalance, as measured by quantitative lipidomics, correlates with immune dysfunction, with high levels of immunomodulatory lipids, sphingosine-1-phosphate (S1P) and ceramides, along with low levels of storage lipids. Activity assays on fly lysates confirm the age-dependent increase in S1P and concomitant reduction of S1P lyase activity. We hypothesize that Mt2 functions to regulate genetic loci such as S1P lyase and this regulation is essential for robust host defense as the animal ages. Our study uncovers novel links between age--dependent Mt2 function, innate immune response and lipid homeostasis.

Levels of DNA cytosine methylation in the Drosophila genome.

Insects provide an accessible system to study the contribution of DNA methylation to complex epigenetic phenotypes created to regulate gene expression, chromatin states, imprinting and dosage compensation. The members of genus Drosophila have been used as a model system to study aspects of biology like development, behaviour and genetics. Despite the popularity of Drosophila melanogaster as a genetic and epigenetic model organism, DNA methylation studies are limited due to low levels of genomic 5-methylcytosine. Our study employs a sensitive liquid chromatography-mass spectrometry (LCMS) based method to quantify the levels of 5-methylcytosine from the genomic DNA in different members of the genus Drosophila. Our results reveal that, despite being phylogenetically related, there is a marked variation in the levels of 5-methylcytosine between the genomes of the members of genus Drosophila. Also, there is a change in the genomic levels of 5-methylcytosine through each life cycle stage of holometabolous development in D. melanogaster.

Epigenetics with special reference to the human X chromosome inactivation and the enigma of Drosophila DNA methylation.

Epigenetics confers adaptability and survival advantage to an organism. Most epigenetic processes demonstrate memory and heritability. DNA methylation is an epigenetic process that adds imprints which can be inherited during cell division and across generations. DNA methylation adds an additional level of information to the basic DNA sequence and can influence chromatin organization and the function of the DNA sequence. In bacteria, it works as a defence strategy and preserves genome integrity. DNA methylation in eukaryotes has been implicated in a large number of cellular regulatory processes and is implied in development, differentiation, life style diseases and cancer. Mammals have an intricate DNA methylation machinery with dNMT1, 3A and 3B enzymes. The human X chromosome inactivation, an example of differential regulation of homologous chromosomes, is known to involve many epigenetic processes with intricate interactions of lncRNAs, miRNAs and DNA methylation. Drosophila possesses very low levels of DNA methylation with only dNMT2 gene. Since Drosophila is an important model organism for study of development and differentiation, the implications of this sparse DNA methylation and the lack of DNA methylation machinery in Drosophila is discussed.

Metals in mangrove ecosystems and associated biota: A global perspective.

Mangrove forests prevalent along the intertidal regions of tropical and sub-tropical coastlines are inimitable and dynamic ecosystems. They protect and stabilize coastal areas from deleterious consequences of natural disasters such as hurricanes and tsunamis. Although there are reviews on ecological aspects, industrial uses of mangrove-associated microorganisms and occurrence of pollutants in a region-specific manner, there is no exclusive review detailing the incidence of metals in mangrove sediments and associated biota in these ecosystems on a global level. In this review, mangrove forests have been classified in a continent-wise manner. Most of the investigations detail the distribution of metals such as zinc, chromium, arsenic, copper, cobalt, manganese, nickel, lead and mercury although in some cases levels of vanadium, strontium, zirconium and uranium have also been studied. Seasonal, tidal, marine, riverine, and terrestrial components are seen to influence occurrence, speciation, bioavailability and fate of metals in these ecosystems. In most of the cases, associated plants and animals also accumulate metals to different extents and are of ecotoxicological relevance. Levels of metals vary in a region specific manner and there is disparity in the pollution status of different mangrove areas. Protecting these vulnerable ecosystems from metal pollutants is important from environmental safety point of view.

Characterization of structural and functional role of selenocysteine in selenoprotein H and its impact on DNA binding.

Selenoproteins are a group of proteins which contain selenocysteine (Sec or U) in their primary structure. Selenoproteins play a critical role in antioxidant defense, hormone metabolism, immune responses and muscle development. The selenoprotein H (SELENOH) is essential in the regulation of gene expression in response to redox status and antioxidant defense. It has Sec residue located in conserved CXXU motif similar to other selenoproteins. However, exact biological function of Sec residue in SELENOH is not known in detail. Therefore, it is essential to understand the structural and functional role of Sec in SELENOH. In the present study, homology modelling and MD simulation were performed to understand the role of Sec residue in SELENOH. The modelled 3D structure of wild-SELENOH along with two mutants (Mut-U44C and Mut-41CS-SC44) was subjected to MD simulation. Based on simulation results, we demonstrate that wild-SELENOH structure is dynamically stabilized by network of intramolecular hydrogen bonding and internal residue contacts facilitated by Sec residue. In contrast, notable differences have been observed in residue contacts and stability in other two mutant structures. Additionally, docking studies revealed that 3PRGRKRK9 motif of wild-SELENOH interacts with HSE and STRE of DNA molecule as observed experimentally. Similar to earlier reports, our sequence analysis study pinpoints conserved 3PRGRKRK9 motif present in SELENOH perform dual role as AT-hook motif and NLS. Overall, the obtained results clearly illustrate Sec residue plays an important role to restore functionally active conformation of SELENOH. The present study broadened our current understanding regarding the role of selenocysteine in protein structure and function.

Aneuploidy: an important model system to understand salient aspects of functional genomics.

Maintaining a balance in gene dosage and protein activity is essential to sustain normal cellular functions. Males and females have a wide range of genetic as well as epigenetic differences, where X-linked gene dosage is an essential regulatory factor. Basic understanding of gene dosage maintenance has emerged from the studies carried out using mouse models with FCG (four core genotype) and chromosomal aneuploidy as well as from mono-chromosomal hybrid cells. In mammals, aneuploidy often leads to embryonic lethality particularly in early development with major developmental and structural abnormalities. Thus, in-depth analysis of the causes and consequences of gene dosage alterations is needed to unravel its effects on basic cellular and developmental functions as well as in understanding its medical implications. Cells isolated from individuals with naturally occurring chromosomal aneuploidy can be considered as true representatives, as these cells have stable chromosomal alterations/gene dosage imbalance, which have occurred by modulation of the basic molecular machinery. Therefore, innovative use of these natural aneuploidy cells/organisms with recent molecular and high-throughput techniques will provide an understanding of the basic mechanisms involved in gene dosage balance and the related consequences for functional genomics.

Manganese- and 1-methyl-4-phenylpyridinium-induced neurotoxicity display differences in morphological, electrophysiological and genome-wide alterations: implications for idiopathic Parkinson's disease.

Idiopathic Parkinson's disease and manganese-induced atypical parkinsonism are characterized by movement disorder and nigrostriatal pathology. Although clinical features, brain region involved and responsiveness to levodopa distinguish both, differences at the neuronal level are largely unknown. We studied the morphological, neurophysiological and molecular differences in dopaminergic neurons exposed to the Parkinson's disease toxin 1-methyl-4-phenylpyridinium ion (MPP+ ) and manganese (Mn), followed by validation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and Mn mouse models. Morphological analysis highlighted loss of neuronal processes in the MPP+ and not the Mn model. Cellular network dynamics of dopaminergic neurons characterized by spike frequency and inter-spike intervals indicated major neuronal population (~ 93%) with slow discharge rates (0-5 Hz). While MPP+ exposure suppressed the firing of these neurons, Mn neither suppressed nor elevated the neuronal activity. High-throughput transcriptomic analysis revealed up-regulation of 694 and 603 genes and down-regulation of 428 and 255 genes in the MPP+ and Mn models respectively. Many differentially expressed genes were unique to either models and contributed to neuroinflammation, metabolic/mitochondrial function, apoptosis and nuclear function, synaptic plasticity, neurotransmission and cytoskeleton. Analysis of the Janus kinase-signal transducer and activator of transcription pathway with implications for neuritogenesis and neuronal proliferation revealed contrasting profile in both models. Genome-wide DNA methylomics revealed differences between both models and substantiated the epigenetic basis of the difference in the Janus kinase-signal transducer and activator of transcription pathway. We conclude that idiopathic Parkinson's disease and atypical parkinsonism have divergent neurotoxicological manifestation at the dopaminergic neuronal level with implications for pathobiology and evolution of novel therapeutics. Cover Image for this issue: doi. 10.1111/jnc.13821.

Transcriptome Analysis Reveals Altered Expression of Memory and Neurotransmission Associated Genes in the REM Sleep Deprived Rat Brain.

Sleep disorders are associated with cognitive impairment. Selective rapid eye movement sleep (REMS) deprivation (REMSD) alters several physiological processes and behaviors. By employing NGS platform we carried out transcriptomic analysis in brain samples of control rats and those exposed to REMSD. The expression of genes involved in chromatin assembly, methylation, learning, memory, regulation of synaptic transmission, neuronal plasticity and neurohypophysial hormone synthesis were altered. Increased transcription of BMP4, DBH and ATP1B2 genes after REMSD supports our earlier findings and hypothesis. Alteration in the transcripts encoding histone subtypes and important players in chromatin remodeling was observed. The mRNAs which transcribe neurotransmitters such as OXT, AVP, PMCH and LNPEP and two small non-coding RNAs, namely RMRP and BC1 were down regulated. At least some of these changes are likely to regulate REMS and may participate in the consequences of REMS loss. Thus, the findings of this study have identified key epigenetic regulators and neuronal plasticity genes associated to REMS and its loss. This analysis provides a background and opens up avenues for unraveling their specific roles in the complex behavioral network particularly in relation to sustained REMS-loss associated changes.

Micro RNAs and DNA methylation are regulatory players in human cells with altered X chromosome to autosome balance.

The gene balance hypothesis predicts that an imbalance in the dosage sensitive genes affects the cascade of gene networks that may influence the fitness of individuals. The phenotypes associated with chromosomal aneuploidies demonstrate the importance of gene dosage balance. We have employed untransformed human fibroblast cells with different number of X chromosomes to assess the expression of miRNAs and autosomal genes in addition to the DNA methylation status. High throughput NGS analysis using illumina Next seq500 has detected several autosomal as well as X linked miRNAs as differentially expressed in X monosomy and trisomy cells. Two of these miRNAs (hsa-miR-125a-5p and 335-5p) are likely to be involved in regulation of the autosomal gene expression. Additionally, our data demonstrates altered expression and DNA methylation signatures of autosomal genes in X monosomy and trisomy cells. In addition to miRNAs, expression of DNMT1 which is an important epigenetic player involved in many processes including cancer, is seen to be altered. Overall, present study provides a proof for regulatory roles of micro RNAs and DNA methylation in human X aneuploidy cells opening up possible new ways for designing therapeutic strategies.

Putative DNA modification methylase DR_C0020 of Deinococcus radiodurans is an atypical SAM dependent C-5 cytosine DNA methylase.

BACKGROUND: Control of cellular processes by epigenetic modification of cytosine in DNA is widespread among living organisms, but, is hitherto unknown in the extremely radioresistant microbe D. radiodurans. METHODS: C-5 methyl cytosines (m5C) were detected by immuno-blotting with m5C-specific antibody. Site of cytosine methylation by DR_C0020 encoded protein was investigated by bisulfite sequencing. The DR_C0020 knockout mutant (Δdcm), constructed by site directed mutagenesis, was assessed for effect on growth, radiation resistance and proteome. Proteins were identified by mass spectrometry. RESULTS: Methylated cytosines were detected in the D. radiodurans genome. The DR_C0020 encoded protein (Dcm, NCBI accession: WP_034351354.1), whose amino acid sequence resembles m4C methylases, was shown to be the lone SAM-dependent C-5 cytosine methyltransferase. Purified Dcm protein was found to methylate CpN sequence with a preference for methylation of two consecutive cytosines. The Δdcm strain completely lost m5C modification from its genome, had no effect on growth but became radiation sensitive. The Δdcm cells exhibited minor alterations in the abundance of several proteins involved primarily in protein homeostasis, oxidative stress defense, metabolism, etc. CONCLUSION: DR_C0020 encoded SAM-dependent methyltransferase Dcm is solely responsible for C-5cytosine methylation at CpN sites in the genome of D. radiodurans and regulates protein homeostasis under normal growth conditions. The protein is an unusual case of an amino methyltransferase that has evolved to producing m5C. GENERAL SIGNIFICANCE: Although, dispensable under optimal growth conditions, the presence of m5C may be important for recognition of parent strand and, thus, could contribute to the extraordinary DNA repair in D. radiodurans.

Epigenetic modulation upon exposure of lung fibroblasts to TiO2 and ZnO nanoparticles: alterations in DNA methylation.

Titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) are promising candidates for numerous applications in consumer products. This will lead to increased human exposure, thus posing a threat to human health. Both these types of NPs have been studied for their cell toxicity, immunotoxicity, and genotoxicity. However, effects of these NPs on epigenetic modulations have not been studied. Epigenetics is an important link in the genotype and phenotype modulation and misregulation can often lead to lifestyle diseases. In this study, we have evaluated the DNA methylation-based epigenetic changes upon exposure to various concentrations of NPs. The investigation was designed to evaluate global DNA methylation, estimating the corresponding methyltransferase activity and expression of Dnmt gene using lung fibroblast (MRC5) cell line as lungs are the primary route of entry and target of occupational exposure to TiO2 and ZnO NPs. Enzyme-linked immunosorbent assay-based immunochemical assay revealed dose-related decrease in global DNA methylation and DNA methyltransferase activity. We also found direct correlation between the concentration of NPs, global methylation levels, and expression levels of Dnmt1, 3A, and 3B genes upon exposure. This is the first study to investigate effect of exposure to TiO2 and ZnO on DNA methylation levels in MRC5 cells. Epigenetic processes are known to play an important role in reprogramming and adaptation ability of an organism and can have long-term consequences. We suggest that changes in DNA methylation can serve as good biomarkers for early exposure to NPs since they occur at concentrations well below the sublethal levels. Our results demonstrate a clear epigenetic alteration in response to metal oxide NPs and that this effect was dose-dependent.

Ensemble-Based Virtual Screening and Experimental Validation of Inhibitors Targeting a Novel Site of Human DNMT1.

Human DNA methyltransferase1 (hDNMT1) is responsible for preserving DNA methylation patterns that play important regulatory roles in differentiation and development. Misregulation of DNA methylation has thus been linked to many syndromes, life style diseases, and cancers. Developing specific inhibitors of hDNMT1 is an important challenge in the area since the currently targeted cofactor and substrate binding site share structural features with various proteins. In this work, we generated a structural model of the active form of hDNMT1 and identified that the 5-methylcytosine (5-mC) binding site of the hDNMT1 is structurally unique to the protein. This site has been previously demonstrated to be critical for methylation activity. We further performed multiple nanosecond time scale atomistic molecular dynamics simulations of the structural model followed by virtual screening of the Asinex database to identify inhibitors targeting the 5-mC site. Two compounds were discovered that inhibited hDNMT1 in vitro, one of which also showed inhibition in vivo corroborating the screening procedure. This study thus identifies and attempts to validate for the first time a unique site of hDNMT1 that could be harnessed for rationally designing highly selective and potent hypomethylating agents.