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Abstract—Age-associated transformation of methylation patterns is considered to be an important predictor of human biological age. Changes in the level of CpG-dinucleotide methylation contribute to a shift in the function of a number of genes, including those associated with the functioning of the immune system. One such gene is CSF1. The protein product of this gene is associated with inflammatory aging, making it an important biomarker of age-related diseases. We studied the methylation profile of the promoter-associated CpG island of the CSF1 gene by MALDI-TOF mass spectrometry. Dependences between the character of CpG-site methylation within the investigated regions and the relative level of the gene mRNA and its protein product in people of different age groups were sought. For two CpG sites, a high level of correlation with the studied parameters is shown. A search for the landing sites of transcription factor binding sites associated with gene transcription showed that these CpG dinucleotides are part of motifs for the NFI family transcription factors and the EGR1 factor. We hypothesize that these CpG sites play an important role in the regulation of CSF1 gene expression. [ABSTRACT FROM AUTHOR]

Ankylosing spondylitis (AS) is a chronic inflammatory autoimmune disease, mainly characterized by perifibrocartilage osteitis of the sacroiliac joints and spinal enthesitis. To date, the exact pathogenesis of AS remains elusive. It is generally believed that AS is a multifactorial disease involving genetics, infection, environment, and immunity. Among them, genetic factors are the primary determinants of disease risk and severity. In recent years, epigenetic mechanisms such as DNA methylation have been extensively surveyed with respect to the pathogenesis of AS. This review summarizes the latest research progress of methylation in AS, from whole-genome sequencing to individual differentially methylated gene. And finally, the role of methylase in AS inflammation, autophagy, and osteogenic differentiation was explored. In summary, the results of this review attempt to explain the role of methylation in the occurrence and development of AS and point out the shortcomings of current methylation research, providing directions for subsequent methylation research in AS. [ABSTRACT FROM AUTHOR]

Long-standing, continuous blurring and controversies in the field of phylogenetic interspecies relations, associated with insufficient explanations for dynamics and variability of speeds of evolution in mammals, hint at a crucial missing link. It has been suggested that transgenerational epigenetic inheritance and the concealed mechanisms behind play a distinct role in mammalian evolution. Here, a comprehensive sequence alignment approach in hominid species, i.e., Homo sapiens, Homo neanderthalensis, Denisovan human, Pan troglodytes, Pan paniscus, Gorilla gorilla, and Pongo pygmaeus, comprising conserved CpG islands of housekeeping genes, uncover evidence for a distinct variability of CpG dinucleotides. Applying solely these evolutionary consistent and inconsistent CpG sites in a classic phylogenetic analysis, calibrated by the divergence time point of the common chimpanzee (P. troglodytes) and the bonobo or pygmy chimpanzee (P. paniscus), a "phylo-epigenetic" tree has been generated, which precisely recapitulates branch points and branch lengths, i.e., divergence events and relations, as they have been broadly suggested in the current literature, based on comprehensive molecular phylogenomics and fossil records of many decades. It is suggested here that CpG dinucleotide changes at CpG islands are of superior importance for evolutionary developments. These changes are successfully inherited through the germ line, determining emerging methylation profiles, and they are a central component of transgenerational epigenetic inheritance. It is hidden in the DNA, what will happen on it later. [ABSTRACT FROM AUTHOR]

Strong epigenetic pan-cancer biomarkers are required to meet several current, urgent clinical needs and to further improve the present chemotherapeutic standard. We have concentrated on the investigation of epigenetic alteration of the hTERT gene, which is frequently epigenetically dysregulated in a number of cancers in specific developmental stages. Distinct DNA methylation profiles were identified in our data on early urothelial cancer. An efficient EpihTERT assay could be developed utilizing suitable combinations with sequence-dependent thermodynamic parameters to distinguish between differentially methylated states. We infer from this data set, the epigenetic context, and the related literature that a CpG-rich, 2800 bp region, a prominent CpG island, surrounding the transcription start of the hTERT gene is the crucial epigenetic zone for the development of a potent biomarker. In order to accurately describe this region, we have named it "Acheron" (Ἀχέρων). In Greek mythology, this is the river of woe and misery and the path to the underworld. Exploitation of the DNA methylation profiles focused on this region, e.g., idiolocal normalized Methylation Specific PCR (IDLN-MSP), opens up a wide range of new possibilities for diagnosis, determination of prognosis, follow-up, and detection of residual disease. It may also have broad implications for the choice of chemotherapy. [ABSTRACT FROM AUTHOR]

1. This study focused on the relationship between MITF mRNA expression and plumage colour in quail and the effect of promoter methylation on the expression of MITF mRNA. 2. The CDS region of MITF mRNA was cloned by RT-PCR, followed by DNA sequencing. The RT-qPCR method was used to analyse the expression levels of MITF mRNA in dorsal skin tissue in Korean quail and Beijing white quail. The promoter region of the MITF gene was cloned, and the CpG island was predicted by the CpGplot program. The methylation levels of the CpG island were analysed using BS-PCR technology. 3. Quail MITF mRNA contains a 1,476 bp complete ORF, which encodes a 492 amino acid residue protein. The MITF protein has no signal peptide or transmembrane region. The expression of MITF mRNA in dorsal tissue of Korean quail was significantly higher than that in Beijing white quail (p < 0.01). Abundant cis-elements and a 346 bp CpG island were found in the promoter region of the MITF gene. The average methylation level of the CpG island was 22 (22%) in Korean quail, and 46 (30%) in Beijing white quail (p < 0.05). 4. The hypermethylation of the MITF gene promoter region in Beijing white quail resulted in a decrease in expression level, which was related to white feather colour. [ABSTRACT FROM AUTHOR]

Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene are linked to Charcot–Marie–Tooth (CMT) disease, a hereditary neurodegenerative condition. The protein encoded by this gene is involved in mitochondrial fission and calcium homeostasis. Recently, GDAP1 has also been implicated in the survival of patients with certain cancers. Despite its significant role in specific cellular processes and associated diseases, the mechanisms regulating GDAP1 expression are largely unknown. Here, we show for the first time that methylation of the CpG island in the proximal promoter of the GDAP1 gene inhibits its activity. Treating cells with low GDAP1 expression using methyltransferase and HDAC inhibitors induced the expression of this gene and its encoded protein. This induction was associated with promoter demethylation and increased association of acetylated histones with the GDAP1 promoter. Thus, we identified a mechanism that could be used to manipulate GDAP1 expression.

Gliomas, the most common type of malignant primary brain tumor, were conventionally classified through WHO Grades I-IV (now 1-4), with low-grade gliomas being entities belonging to Grades 1 or 2. While the focus of the WHO Classification for Central Nervous System (CNS) tumors had historically been on histopathological attributes, the recently released fifth edition of the classification (WHO CNS5) characterizes brain tumors, including gliomas, using an integration of histological and molecular features, including their epigenetic changes such as histone methylation, DNA methylation, and histone acetylation, which are increasingly being used for the classification of low-grade gliomas. This review describes the current understanding of the role of DNA methylation, demethylation, and histone modification in pathogenesis, clinical behavior, and outcomes of brain tumors, in particular of low-grade gliomas. The review also highlights potential diagnostic and/or therapeutic targets in associated cellular biomolecules, structures, and processes. Targeting of MGMT promoter methylation, TET-hTDG-BER pathway, association of G-CIMP with key gene mutations, PARP inhibition, IDH and 2-HG-associated processes, TERT mutation and ARL9-associated pathways, DNA Methyltransferase (DNMT) inhibition, Histone Deacetylase (HDAC) inhibition, BET inhibition, CpG site DNA methylation signatures, along with others, present exciting avenues for translational research. This review also summarizes the current clinical trial landscape associated with the therapeutic utility of epigenetics in low-grade gliomas. Much of the evidence currently remains restricted to preclinical studies, warranting further investigation to demonstrate true clinical utility.

Glioblastoma (GBM) is the most aggressive and frequent type of primary brain cancer in adult patients. One of the key molecular features associated with GBM pathogenesis is the dysfunction of PTEN oncosuppressor. In addition to PTEN gene, humans and several primates possess processed PTEN pseudogene (PTENP1) that gives rise to long non-coding RNA lncPTENP1-S. Regulation and functions of PTEN and PTENP1 are highly interconnected, however, the exact molecular mechanism of how these two genes affect each other remains unclear. Here, we analyzed the methylation level of the CpG islands (CpGIs) in the promoter regions of PTEN and PTENP1 in patient-derived GBM neurospheres. We found that increased PTEN methylation corelates with decreased PTEN mRNA level. Unexpectedly, we showed the opposite trend for PTENP1. Using targeted methylation and demethylation of PTENP1 CpGI, we demonstrated that DNA methylation increases lncPTENP1-S expression in the presence of wild type PTEN protein but decreases lncPTENP1-S expression if PTEN protein is absent. Further experiments revealed that PTEN protein binds to PTENP1 promoter region and inhibits lncPTENP1-S expression if its CpGI is demethylated. Interestingly, we did not detect any effect of lncPTENP1-S on the level of PTEN mRNA, indicating that in GBM cells PTENP1 is a downstream target of PTEN rather than its upstream regulator. Finally, we studied the functions of lncPTENP1-S and demonstrated that it plays a pro-oncogenic role in GBM cells by upregulating the expression of cancer stem cell markers and decreasing cell adhesion. • CpG methylation has different effect on PTEN and PTENP1 transcription level. • CpG methylation upregulates PTENP1 transcription if PTEN protein is present. • PTEN protein inhibits PTENP1 transcription if PTENP1 CpGI is demethylated. • lncPTENP1-S promotes malignancy of glioblastoma cells. [ABSTRACT FROM AUTHOR]

Abstract Background Chrysanthemum is a popular ornamental plant worldwide. MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factors play an important role in everything from stress resistance to plant growth and development. However, the MYB family of chrysanthemums has not been the subject of a detailed bioinformatics and expression investigation. Results In this study, we examined 324 CnMYB transcription factors from Chrysanthemum nankingense genome data, which contained 122 Cn1R-MYB, 183 CnR2R3-MYB, 12 Cn3R-MYB, 2 Cn4R-MYB, and 5 atypical CnMYB. The protein motifs and classification of CnMYB transcription factors were analyzed. Among them, motifs 1, 2, 3, and 4 were found to encode the MYB DNA-binding domain in R2R3-MYB proteins, while in other-MYB proteins, the motifs 1, 2, 3, 4, 5, 6, 7, and 8 encode the MYB DNA-binding domain. Among all CnMYBs, 44 genes were selected due to the presence of CpG islands, while methylation is detected in three genes, including CnMYB9, CnMYB152, and CnMYB219. We analyzed the expression levels of each CnMYB gene in ray floret, disc floret, flower bud, leaf, stem, and root tissues. Based on phylogenetic analysis and gene expression analysis, three genes appeared likely to control cellulose and lignin synthesis in stem tissue, and 16 genes appeared likely to regulate flowering time, anther, pollen development, and flower color. Fifty-one candidate genes that may be involved in stress response were identified through phylogenetic, stress-responseve motif of promoter, and qRT-PCR analyses. According to genes expression levels under stress conditions, six CnMYB genes (CnMYB9, CnMYB172, CnMYB186, CnMYB199, CnMYB219, and CnMYB152) were identified as key stress-responsive genes. Conclusions This research provides useful information for further functional analysis of the CnMYB gene family in chrysanthemums, as well as offers candidate genes for further study of cellulose and lignin synthesis, flowering traits, salt and drought stress mechanism.

Retinoid X receptor (RXR) heterodimerizes with the PPAR nuclear hormone receptor and regulates its downstream events. We investigated the effects of RXR agonists (LG100754, bexarotene, AGN194204, and LG101506) on lenalidomide's anti-myeloma activity, T cell functions, and the level of glucose and lipids in vivo. Genetic overexpression and CRISPR/Cas9 knockout experiments were conducted in multiple myeloma (MM) cell lines and Jurkat T cell lines to determine the roles of CRBN in RXR-agonist mediated effects. A xenograft mouse model of MM was established to determine the combination effect of LG100754 and lenalidomide. The combination of RXR agonists and lenalidomide demonstrated synergistic activity in increasing CRBN expression and killing myeloma cells. Mechanistically, the RXR agonists reduced the binding of PPARs to the CRBN promoter, thereby relieving the repressor effect of PPARs on CRBN transcription. RXR agonists downregulated the exhaustion markers and increased the activation markers of Jurkat T cells and primary human T cells. Co-administration of LG100754 and lenalidomide showed enhanced anti-tumor activity in vivo. LG100754 retained its glucose- and lipid-lowering effects. RXR agonists demonstrate potential utility in enhancing drug sensitivity and T-cell function in the treatment of myeloma. [ABSTRACT FROM AUTHOR]

Abstract The inheritance of acquired traits in mammals is a highly controversial topic in biology. Recently, Takahashi et al. (Cell 186:715–731, 2023) have reported that insertion of CpG-free DNA into a CpG island (CGI) can induce DNA methylation of the CGI and that this aberrant methylation pattern can be transmitted across generations, even after removal of the foreign DNA. These results were interpreted as evidence for transgenerational inheritance of acquired DNA methylation patterns. Here, we discuss several interpretational issues raised by this study and consider alternative explanations.

Objective: Alzheimer's disease (AD) is a neurodegenerative disease characterized by neuropathology and cognitive decline and associated with age. The comprehensive deoxyribonucleic acid methylation (DNAm)-transcriptome profile association analysis conducted in this study aimed to establish whole-genome DNAm profiles and explore DNAm-related genes and their potential functions. More appropriate biomarkers were expected to be identified in terms of AD. Materials and methods: Illumina 450KGSE59685 dataset AD (n = 54) and HC (n = 21) and ribonucleic-acid-sequencing data GSE118553 dataset AD patients (n = 21) and HCs (n = 13) were obtained from the gene expression omnibus database before a comprehensive DNAm-transcriptome profile association analysis, and we performed functional enrichment analysis by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses (KEGG). Three transgenic mice and three wild-type mice were used to validate the hub genes. Results: A total of 18,104 DNAm sites in healthy controls (n = 21) and AD patients (n = 54) were surveyed across three brain regions (superior temporal gyrus, entorhinal cortex, and dorsolateral prefrontal cortex). With the addition of the transcriptome analysis, eight hypomethylated-related highly expressed genes and 61 hypermethylated-related lowly expressed genes were identified. Based on 69 shared differentially methylated genes (DMGs), the function enrichment analysis indicated Guanosine triphosphate enzymes (GTPase) regulator activity, a synaptic vesicle cycle, and tight junction functioning. Following this, mice-based models of AD were constructed, and five hub DMGs were verified, which represented a powerful, disease-specific DNAm signature for AD. Conclusion: The results revealed that the cross-brain region DNAm was altered in those with AD. The alterations in DNAm affected the target gene expression and participated in the key biological processes of AD. The study provides a valuable epigenetic resource for identifying DNAm-based diagnostic biomarkers, developing effective drugs, and studying AD pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Chrysanthemum is a popular ornamental plant worldwide. MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factors play an important role in everything from stress resistance to plant growth and development. However, the MYB family of chrysanthemums has not been the subject of a detailed bioinformatics and expression investigation. Results: In this study, we examined 324 CnMYB transcription factors from Chrysanthemum nankingense genome data, which contained 122 Cn1R-MYB, 183 CnR2R3-MYB, 12 Cn3R-MYB, 2 Cn4R-MYB, and 5 atypical CnMYB. The protein motifs and classification of CnMYB transcription factors were analyzed. Among them, motifs 1, 2, 3, and 4 were found to encode the MYB DNA-binding domain in R2R3-MYB proteins, while in other-MYB proteins, the motifs 1, 2, 3, 4, 5, 6, 7, and 8 encode the MYB DNA-binding domain. Among all CnMYBs, 44 genes were selected due to the presence of CpG islands, while methylation is detected in three genes, including CnMYB9, CnMYB152, and CnMYB219. We analyzed the expression levels of each CnMYB gene in ray floret, disc floret, flower bud, leaf, stem, and root tissues. Based on phylogenetic analysis and gene expression analysis, three genes appeared likely to control cellulose and lignin synthesis in stem tissue, and 16 genes appeared likely to regulate flowering time, anther, pollen development, and flower color. Fifty-one candidate genes that may be involved in stress response were identified through phylogenetic, stress-responseve motif of promoter, and qRT-PCR analyses. According to genes expression levels under stress conditions, six CnMYB genes (CnMYB9, CnMYB172, CnMYB186, CnMYB199, CnMYB219, and CnMYB152) were identified as key stress-responsive genes. Conclusions: This research provides useful information for further functional analysis of the CnMYB gene family in chrysanthemums, as well as offers candidate genes for further study of cellulose and lignin synthesis, flowering traits, salt and drought stress mechanism. [ABSTRACT FROM AUTHOR]

Background: The role of alcohol in carcinogenesis has received increasing attention in recent years. Evidence shows its impacts on various aspects, including epigenetics alteration. The DNA methylation patterns underlying alcohol-associated cancers are not fully understood. Methods: We investigated the aberrant DNA methylation patterns in four alcoholassociated cancers based on the Illumina HumanMethylation450 BeadChip. Pearson coefficient correlations were identified between differential methylated CpG probes and annotated genes. Transcriptional factor motifs were enriched and clustered using MEME Suite, and a regulatory network was constructed. Results: In each cancer, differential methylated probes (DMPs) were identified, and 172 hypermethylated and 21 hypomethylated pan-cancer DMPs (PDMPs) were examined further. Annotated genes significantly regulated by PDMPs were investigated and enriched in transcriptional misregulation in cancers. The CpG island chr19:58220189--58220517 was hypermethylated in all four cancers and silenced in the transcription factor ZNF154. Various biological effects were exerted by 33 hypermethylated and seven hypomethylated transcriptional factor motifs grouped into five clusters. Eleven pan-cancer DMPs were identified to be associated with clinical outcomes in the four alcohol-associated cancers, which might provide a potential point of view for clinical outcome prediction. Conclusion: This study provides an integrated insight into DNA methylation patterns in alcohol-associated cancers and reveals the corresponding features, influences, and potential mechanisms. [ABSTRACT FROM AUTHOR]

Background: Saffron (Crocus sativus) is high valued spice crop, but due to its sterile nature, the crop is propagated exclusively through corms. Thus, the genetic base of this crop is very narrow, however, frequency of phenotypic variability is observed; and suggested the potential role of epigenetics in saffron crop growth and development. Methods and results: To facilitate epigenetic studies in saffron, we developed 1525 methylation-specific PCR (MSP) markers using MethPrimer. For this purpose, we used 6767 EST sequences of saffron available on the NCBI database. We also mine CpG islands (2555) and found that 32.7% of EST sequences had CpG islands. Out of 1525 MSP markers developed during the present study, 725 covered the CpG islands and 800 were without CpG islands. PCR amplification was found successful for 82% of MSP markers. A preliminary analysis suggested that 53.7% of genomic sites were methylated and more prominent (60% methylations) in non-CpG island regions, although, more comprehensive studies are required to validate it further. Conclusions: The epigenetic resource developed during the present study will strengthen the epigenetic studies like epiQTL mapping, epiGWAS to explore the molecular mechanisms and genomic/epigenomic regions associated with phenotype; and further may be utilized for saffron improvement programs through epibreeding. [ABSTRACT FROM AUTHOR]

Abstract Background Abnormal DNA methylation of tumor suppressor gene promoter has been found in breast cancer. Therefore, the current study set out to explore how DNA methyltransferase 1 (DNMT1) affects breast cancer through mediating miR-497/GPRC5A axis. Methods After loss and gain-of-function approaches were conducted in MCF-7/ADR and MCF-7 cells, cell viability, IC50 value, invasion, migration and apoptosis were measured, respectively. In addition, drug resistance, metastasis and apoptosis-related protein expression were examined using immunoblotting. ChIP and dual-luciferase reporter gene assays were carried out to validate relationship among DNMT1, miR-497, and GPRC5RA. Subcutaneous xenograft tumor model in nude mice was established to detect effects of DNMT1 on growth and metastasis of breast cancer in vivo. Results It was found that DNMT1 was notably increased, while miR-497 was poorly-expressed in breast cancer. Highly-expressed DNMT1 could promote chemotherapy resistance and metastasis of breast cancer. Meanwhile, DNMT1 modified methylation of CpG island in miR-497 promoter region, thereby repressing miR-497 level. In addition, miR-497 targeted GPRC5A expression to curb chemotherapy resistance and metastasis of breast cancer cells. Lastly, in vivo experiments showed that knockdown of DNMT1 could suppress breast cancer growth and metastasis. Conclusions Collectively, our findings indicated that DNMT1 may inhibit miR-497 and boost the expression of GPRC5A through methylation, thus augmenting breast cancer chemotherapy resistance and metastasis, which provides novel mechanistic insight into the unrecognized roles of DNMT1 in breast cancer.

Abstract Background Cytosine DNA methylation is a heritable epigenetic mark present in most eukaryotic groups. While the patterns and functions of DNA methylation have been extensively studied in mouse and human, their conservation in other vertebrates remains poorly explored. In this study, we interrogated the distribution and function of DNA methylation in primary fibroblasts of seven vertebrate species including bio-medical models and livestock species (human, mouse, rabbit, dog, cow, pig, and chicken). Results Our data highlight both divergence and conservation of DNA methylation patterns and functions. We show that the chicken genome is hypomethylated compared to other vertebrates. Furthermore, compared to mouse, other species show a higher frequency of methylation of CpG-rich DNA. We reveal the conservation of large unmethylated valleys and patterns of DNA methylation associated with X-chromosome inactivation through vertebrate evolution and make predictions of conserved sets of imprinted genes across mammals. Finally, using chemical inhibition of DNA methylation, we show that the silencing of germline genes and endogenous retroviruses (ERVs) are conserved functions of DNA methylation in vertebrates. Conclusions Our data highlight conserved properties of DNA methylation in vertebrate genomes but at the same time point to differences between mouse and other vertebrate species.

Spontaneous mutations are evolutionary engines as they generate variants for the evolutionary downstream processes that give rise to speciation and adaptation. Single nucleotide mutations (SNM) are the most abundant type of mutations among them. Here, we perform a meta-analysis to quantify the influence of selected global genomic parameters (genome size, genomic GC content, genomic repeat fraction, number of coding genes, gene count, and strand bias in prokaryotes) and local genomic features (local GC content, repeat content, CpG content and the number of SNM at CpG islands) on spontaneous SNM rates across the tree of life (prokaryotes, unicellular eukaryotes, multicellular eukaryotes) using wild-type sequence data in two different taxon classification systems. We find that the spontaneous SNM rates in our data are correlated with many genomic features in prokaryotes and unicellular eukaryotes irrespective of their sample sizes. On the other hand, only the number of coding genes was correlated with the spontaneous SNM rates in multicellular eukaryotes primarily contributed by vertebrates data. Considering local features, we notice that local GC content and CpG content significantly were correlated with the spontaneous SNM rates in the unicellular eukaryotes, while local repeat fraction is an important feature in prokaryotes and certain specific uni- and multi-cellular eukaryotes. Such predictive features of the spontaneous SNM rates often support non-linear models as the best fit compared to the linear model. We also observe that the strand asymmetry in prokaryotes plays an important role in determining the spontaneous SNM rates but the SNM spectrum does not. [Display omitted] • Global and local genomic features are correlated with SNM rates, but the patterns vary across taxa • Prokaryotes and unicellular eukaryotes show stronger correlations with genomic features as compared with multicellular eukaryotes. • Strand bias in the propensity of SNMs is found to be correlated with SNM rate and spectrum in prokaryotes. [ABSTRACT FROM AUTHOR]

Makorin RING finger protein 3 (MKRN3) is an important factor located on chromosome 15 in the imprinting region associated with Prader-Willi syndrome. Imprinted MKRN3 is expressed in hypothalamic regions essential for the onset of puberty and mutations in the gene have been found in patients with central precocious puberty. The pubertal process is largely controlled by epigenetic mechanisms that include, among other things, DNA methylation at CpG dinucleotides of puberty-related genes. In the present study, we investigated the methylation status of the Mkrn3 promoter in the hypothalamus of the female mouse before, during and after puberty. Initially, we mapped the 32 CpG dinucleotides in the promoter, the 5’UTR and the first 50 nucleotides of the coding region of the Mkrn3 gene. Moreover, we identified a short CpG island region (CpG islet) located within the promoter. Methylation analysis using bisulfite sequencing revealed that CpG dinucleotides were methylated regardless of developmental stage, with the lowest levels of methylation being found within the CpG islet region. In addition, the CpG islet region showed significantly lower methylation levels at the pre-pubertal stage when compared with the pubertal or post-pubertal stage. Finally, in silico analysis of transcription factor binding sites on the Mkrn3 CpG islet identified the recruitment of 29 transcriptional regulators of which 14 were transcriptional repressors. Our findings demonstrate the characterization and differential methylation of the CpG dinucleotides located in the Mkrn3 promoter that could influence the transcriptional activity in pre-pubertal compared to pubertal or post-pubertal period. Further studies are needed to clarify the possible mechanisms and effects of differential methylation of the Mkrn3 promoter.

Simple Summary: Cereblon (CRBN) is a direct binding target of immunomodulatory drugs (IMiDs) that are commonly used to treat multiple myeloma (MM). Many patients with MM have comorbidities, including diabetes and/or dyslipidemia, and are treated with peroxisome proliferator-activated receptor (PPAR) agonists. This study aimed to further analyze the effects and mechanisms underlying the drug-to-drug interactions between IMiDs and PPAR agonists in MM. We found that PPAR agonists reduced CRBN expression by inducing DNA methylation and increasing protein degradation. PPAR agonists and IMiDs showed opposing metabolic effects in MM cells. Our retrospective study suggested an inferior response and outcome when PPARs and IMiDs were concurrently administered. Our study has important implications for the care of patients with MM and provides a foundation for exploring novel compounds or PPAR partial agonists/antagonists that can increase CRBN expression while retaining their lipid-lowering or insulin-sensitizing functions. Our previous study demonstrated that peroxisome proliferator-activated receptor (PPAR) agonists downregulated cereblon (CRBN) expression and reduced the anti-myeloma activity of lenalidomide in vitro and in vivo. We aimed to determine whether DNA methylation and protein degradation contribute to the effects of PPAR agonists. CRBN promoter methylation status was detected using methylation-specific polymerase chain reaction. The CRBN protein degradation rate was measured using a cycloheximide chase assay. Metabolomic analysis was performed in multiple myeloma (MM) cells treated with PPAR agonists and/or lenalidomide. Our retrospective study determined the effect of co-administration of PPAR agonists with immunomodulatory drugs on the outcomes of patients with MM. CpG islands of the CRBN promoter region became highly methylated upon treatment with PPAR agonists, whereas treatment with PPAR antagonists resulted in unmethylation. The CRBN protein was rapidly degraded after treatment with PPAR agonists. Lenalidomide and fenofibrate showed opposite effects on acylcarnitines and amino acids. Co-administration of immunomodulatory drugs and PPAR agonists was associated with inferior treatment responses and poor survival. Our study provides the first evidence that PPAR agonists reduce CRBN expression through various mechanisms including inducing methylation of CRBN promoter CpG island, enhancing CRBN protein degradation, and affecting metabolomics of MM cells. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal disease. Skeletal muscles and motor neurons (MNs) degenerate. ALS is a complex disease involving many genes in multiple tissues, the environment, cellular metabolism, and lifestyles. We hypothesized that epigenetic anomalies in DNA and RNA occur in ALS and examined this idea in: (1) mouse models of ALS, (2) human ALS, and (3) mouse ALS with therapeutic targeting of DNA methylation. Human superoxide dismutase-1 (hSOD1) transgenic (tg) mice were used. They expressed nonconditionally wildtype (WT) and the G93A and G37R mutant variants or skeletal muscle-restricted WT and G93A and G37R mutated forms. Age-matched non-tg mice were controls. hSOD1 mutant mice had increased DNA methyltransferase enzyme activity in spinal cord and skeletal muscle and increased 5-methylcytosine (5mC) levels. Genome-wide promoter CpG DNA methylation profiling in skeletal muscle of ALS mice identified hypermethylation notably in cytoskeletal genes. 5mC accumulated in spinal cord MNs and skeletal muscle satellite cells in mice. Significant increases in DNA methyltransferase-1 (DNMT1) and DNA methyltransferase-3A (DNMT3A) levels occurred in spinal cord nuclear and chromatin bound extracts of the different hSOD1 mouse lines. Mutant hSOD1 interacted with DNMT3A in skeletal muscle. 6-methyladenosine (6mA) RNA methylation was markedly increased or decreased in mouse spinal cord depending on hSOD1-G93A model, while fat mass and obesity associated protein was depleted and methyltransferase-like protein 3 was increased in spinal cord and skeletal muscle. Human ALS spinal cord had increased numbers of MNs and interneurons with nuclear 5mC, motor cortex had increased 5mC-positive neurons, while 6mA was severely depleted. Treatment of hSOD1-G93A mice with DNMT inhibitor improved motor function and extended lifespan by 25%. We conclude that DNA and RNA epigenetic anomalies are prominent in mouse and human ALS and are potentially targetable for disease-modifying therapeutics. [ABSTRACT FROM AUTHOR]

Hypermethylation of CpG islands in the promoter regions of four genes (p53, ATM, SOD3, ESR1) was studied in blood leukocytes of irradiated humans (100 subjects: Chernobyl Nuclear Power Plant clean-up workers, nuclear specialists, residents of territories with radionuclide contamination) and 140 unirradiated subjects (control group) using methylation-sensitive polymerase chain reaction (PCR) assay. The obtained data significantly supplemented and allowed us to summarize results from several years of the study of radiation-induced hypermethylation in the gene promoters in various contingents of individuals exposed to chronic or fractionated radiation in a wide range of doses. The differential significance of age and radiation exposure in methylation of CpG islands in promoter regions of different genes was revealed, which is demonstrated by unidirectional effects observed in independent samples of irradiated individuals. The ROC curve analysis showed a high prognostic potential of consideration of detected epigenetic disorders as biomarkers of radiation exposure (AUC = 0.846 ± 0.015, p = 1.5E-48). Hypermethylation of the CpG islands in the RASSF1A and p14/ARF genes depends on age, and epigenetic modification of the p16/INK4A and GSTP1 loci is highly significantly associated with radiation exposure, which was verified on two test samples of the examined individuals. The revealed dose-dependent hypermethylation of the studied genes is indirectly confirmed by a positive associative relationship between the level of chromosomal aberrations and the frequency of methylated loci (r = 0.604, p = 2.3E-11). [ABSTRACT FROM AUTHOR]

Background: Cytosine DNA methylation is a heritable epigenetic mark present in most eukaryotic groups. While the patterns and functions of DNA methylation have been extensively studied in mouse and human, their conservation in other vertebrates remains poorly explored. In this study, we interrogated the distribution and function of DNA methylation in primary fibroblasts of seven vertebrate species including bio-medical models and livestock species (human, mouse, rabbit, dog, cow, pig, and chicken). Results: Our data highlight both divergence and conservation of DNA methylation patterns and functions. We show that the chicken genome is hypomethylated compared to other vertebrates. Furthermore, compared to mouse, other species show a higher frequency of methylation of CpG-rich DNA. We reveal the conservation of large unmethylated valleys and patterns of DNA methylation associated with X-chromosome inactivation through vertebrate evolution and make predictions of conserved sets of imprinted genes across mammals. Finally, using chemical inhibition of DNA methylation, we show that the silencing of germline genes and endogenous retroviruses (ERVs) are conserved functions of DNA methylation in vertebrates. Conclusions: Our data highlight conserved properties of DNA methylation in vertebrate genomes but at the same time point to differences between mouse and other vertebrate species. [ABSTRACT FROM AUTHOR]

Simple Summary: Jinghai yellow Chickens have a small size, high quality, early maturity, and stress resistance characteristics. There are particular individuals in its population with high and low egg numbers at 300 days of age. As a quantitative trait, egg production is not only influenced by the environment but is also controlled by micro-effect polygenes. In this paper, we focus on the ZP2 gene, which may impact the egg production of Jinghai yellow chickens. We also analyze the role of essential methylation sites in regulating the mRNA expression of the gene and further explore the molecular regulation mechanism of the ZP2 gene. Egg production in chickens is a quantitative trait. The aim of this study was to investigate the effect of promoter methylation of the Zona pellucida 2 (ZP2) gene on egg production. Real-time fluorescence quantification showed that the expression of the ZP2 gene in the ovaries of 300-day-old Jinghai yellow chickens in the high-laying group was significantly higher than that in the low-laying group (p < 0.01). A series of deletion fragments of the ZP2 gene promoter in Jinghai yellow chickens had different promoter activities in DF-1 cells, and the core region of the ZP2 gene promoter was found to be between −1552 and −1348. Four CpG islands in the promoter region of the ZP2 gene were detected by software prediction. The overall degree of methylation of the ZP2-1 amplified fragment was negatively correlated with mRNA expression to some extent (R = −0.197); the overall degree of methylation of the ZP2-2 amplified fragment was also negatively correlated with mRNA expression to some extent (R = −0.264), in which the methylation of methylcytosine (mC)-9, mC-20, and mC-21 sites was significantly negatively correlated with mRNA expression (p < 0.05). In addition, the mC-20 and mC-21 sites are located on the Sp1 transcription factor binding site, and it is speculated that these two sites may be the main sites for regulating transcription. In summary, the methylation sites mC-20 and mC-21 of the ZP2 gene may inhibit the binding of Sp1 and DNA, affect the transcription of the ZP2 gene, and then affect the number of eggs produced by the Jinghai yellow chickens. [ABSTRACT FROM AUTHOR]

Background: Epigenetic changes occur in response to environmental factors during the pathogenesis of necrotizing enterocolitis (NEC) in animal models, but the DNA methylation signature in human patients with NEC has not been examined. Aim: To illustrate the signature and function of DNA methylation in the intestine of human NEC. Methods: DNA methyltransferases (DNMTs) were compared between intestinal tissue with NEC and control. Genome-wide DNA methylation was analyzed by reduced representation bisulfite sequencing (RRBS). The biological functions of the potential methylation regulated genes were analyzed by Gene Ontology. Gene methylation and expression were confirmed by bisulfite genomic sequencing (BGS) and RT-qPCR. Results: By screening the expression of DNMTs, we identified a marked reduction in DNMT3A at both the mRNA and protein levels in NEC. Genome-wide variation of DNA methylation was detected in NEC lesions. The CG methylation level in almost all unique regions except CpG islands (CGIs) was lower in NEC compared with control. A total of 287 differentially methylated regions (DMRs) were identified across the whole genome in NEC, 123 of them are located on the CGI in the promoter. The DMR-associated genes were linked to intestinal epithelial permeability, platelet aggregation, and lymphocyte proliferation. Four genes (ZNF335, MPL, RASAL3, and KDM6A) with roles in the regulation of lymphocytes that may predispose the intestine to imbalanced immune processes were further confirmed to be hypermethylated and transcriptionally downregulated. Conclusions: These findings underscore the novel relationship between epigenetic changes and lymphocyte regulation in human NEC, which may have potential diagnostic and therapeutic relevance for NEC. [ABSTRACT FROM AUTHOR]

In the mammalian genome, most CpGs are methylated. However, CpGs within the CpG islands (CGIs) are largely unmethylated, which are important for gene expression regulation. The mechanism underlying the low methylation levels at CGIs remains largely elusive. KDM2 proteins (KDM2A and KDM2B) are H3K36me2 demethylases known to bind specifically at CGIs. Here, we report that depletion of each or both KDM2 proteins, or mutation of all their JmjC domains that harbor the H3K36me2 demethylation activity, leads to an increase in DNA methylation at selective CGIs. The Kdm2a/2b double knockout shows a stronger increase in DNA methylation compared to the single mutant of Kdm2a or Kdm2b, indicating that KDM2A and KDM2B redundantly regulate DNA methylation at CGIs. In addition, the increase of CGI DNA methylation upon mutations of KDM2 proteins is associated with the chromatin environment. Our findings reveal that KDM2A and KDM2B function redundantly in regulating DNA methylation at a subset of CGIs in an H3K36me2 demethylation-dependent manner., (Copyright © 2024. Published by Elsevier Ltd.)

Mutations in the ganglioside-induced differentiation-associated protein 1 ( GDAP1 ) gene are linked to Charcot-Marie-Tooth (CMT) disease, a hereditary neurodegenerative condition. The protein encoded by this gene is involved in mitochondrial fission and calcium homeostasis. Recently, GDAP1 has also been implicated in the survival of patients with certain cancers. Despite its significant role in specific cellular processes and associated diseases, the mechanisms regulating GDAP1 expression are largely unknown. Here, we show for the first time that methylation of the CpG island in the proximal promoter of the GDAP1 gene inhibits its activity. Treating cells with low GDAP1 expression using methyltransferase and HDAC inhibitors induced the expression of this gene and its encoded protein. This induction was associated with promoter demethylation and increased association of acetylated histones with the GDAP1 promoter. Thus, we identified a mechanism that could be used to manipulate GDAP1 expression., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Abstract Background In silico analysis of transcription start sites, promoter regions, transcription factors and their binding sites, and CpG islands for the Trametes hirsuta strain 072 genome were performed to understand the regulation mechanisms of gene expression and its genetic variations in the genomes. Therefore, a computational survey was carried out for the Trametes hirsuta strain 072 genome with the open reading frames from the National Center for Biotechnology Information database. Seventeen functional sequences were used to analyze promoter regions and their regulatory elements. Result The present study revealed that 94% of Trametes hirsuta strain 072 genes contained more than two TSSs. Among these identified TSSs, a TSS with the highest predictive score was considered to determine a promoter region of the genes. Moreover, a total of five common candidate motifs such as MotI, MotII, MotIII, MotIV, and MotV were identified. Among these motifs, motif IV was investigated as the common promoter motif for 41.17% of genes that serve as binding sites for transcription factors (TFs) involved in the expression regulation of Trametes hirsuta strain 072 genes. Motif IV was also compared to registered motifs in publically available databases to see if they are similar to known regulatory motifs for TF using TOMTOM web server. Hence, it was revealed that MotIV might serve as the binding site mainly for the leucine zipper TF gene family to regulate a gene expression of Trametes hirsuta strain 072. Regarding CpG island determination, it was concluded that there is no CpG island in both promoter and gene body regions of the Trametes hirsuta strain 072 genome. Conclusions This study provides a better insight into further molecular characterization which aimed to efficiently exploit a white rot fungus, Trametes hirsuta strain 072, for several biotechnological applications aimed to revitalize a severely contaminated environment.

Abstract Background Potato (Solanum tuberosum L.) is one of the most important food crops in the world. Pathogens remain as one of the major constraints limiting potato productivity. Thus, understanding of gene regulation mechanism of pathogenesis-related genes such as glucan endo-1,3-beta-glucosidase is a foundation for genetic engineering of potato for disease resistance and reduces the use of fungicides. In the present study, 19 genes were selected and attempts were made through in silico methods to identify and characterize the promoter regions, regulatory elements, and CpG islands of glucan endo-1,3-beta-glucosidase gene in Solanum tuberosum cultivar DM 1-3 516 R44. Results The current analysis revealed that single transcription start sites (TSSs) were present in 12/19 (63.2%) of promoter regions analyzed. The predictive score at a cutoff value of 0.8 for the majority (84.2%) of the promoter regions ranged from 0.90 to 1.00. The locations for 42% of the TSSs were below −500 bp relative to the start codon (ATG). MβGII was identified as the common promoter motif for 94.4% of the genes with an E value of 3.5e−001. The CpG analysis showed low CpG density in the promoter regions of most of the genes except for gene ID102593331 and ID: 102595860. The number of SSRs per gene ranged from 2 to 9 with repeat lengths of 2 to 6 bp. Evolutionary distances ranged from 0.685 to 0.770 (mean = 0.73), demonstrating narrower genetic diversity range. Phylogeny was inferred using the UPGMA method, and gene sequences from different species were found to be clustered together. Conclusion In silico identified regulatory elements in promoter regions will contribute to our understanding of the regulatory mechanism of glucan endo-1,3-beta-glucosidase genes and provide a promising target for genetic engineering to improve disease resistance in potatoes.

Retinoid X receptor (RXR) heterodimerizes with the PPAR nuclear hormone receptor and regulates its downstream events. We investigated the effects of RXR agonists (LG100754, bexarotene, AGN194204, and LG101506) on lenalidomide’s anti-myeloma activity, T cell functions, and the level of glucose and lipids in vivo. Genetic overexpression and CRISPR/Cas9 knockout experiments were conducted in multiple myeloma (MM) cell lines and Jurkat T cell lines to determine the roles of CRBN in RXR-agonist mediated effects. A xenograft mouse model of MM was established to determine the combination effect of LG100754 and lenalidomide. The combination of RXR agonists and lenalidomide demonstrated synergistic activity in increasing CRBN expression and killing myeloma cells. Mechanistically, the RXR agonists reduced the binding of PPARs to the CRBN promoter, thereby relieving the repressor effect of PPARs on CRBN transcription. RXR agonists downregulated the exhaustion markers and increased the activation markers of Jurkat T cells and primary human T cells. Co-administration of LG100754 and lenalidomide showed enhanced anti-tumor activity in vivo. LG100754 retained its glucose- and lipid-lowering effects. RXR agonists demonstrate potential utility in enhancing drug sensitivity and T-cell function in the treatment of myeloma.

Nanopore sequencing has brought the technology to the next generation in the science of sequencing. This is achieved through research advancing on: pore efficiency, creating mechanisms to control DNA translocation, enhancing signal-to-noise ratio, and expanding to long-read ranges. Heterogeneity regarding epigenetics would be broad as mutations in the epigenome are sensitive to cause new challenges in cancer research. Epigenetic enzymes which catalyze DNA methylation and histone modification are dysregulated in cancer cells and cause numerous heterogeneous clones to evolve. Detection of this heterogeneity in these clones plays an indispensable role in the treatment of various cancer types. With single-cell profiling, the nanopore sequencing technology could provide a simple sequence at long reads and is expected to be used soon at the bedside or doctor's office. Here, we review the advancements of nanopore sequencing and its use in the detection of epigenetic heterogeneity in cancer. [ABSTRACT FROM AUTHOR]

Aim: To explore the methylation pattern, its role in transcriptional regulation and potential modifiers of methylation of the TLR9 gene in chronic periapical inflammation. Methodology: In this cross‐sectional study, apical lesions of endodontic origin (ALEO, n = 61) and healthy periodontal ligaments (HPL, n = 15) were included. Products from bisulfited and PCR‐amplified DNA were analysed for their methylation profiles in the promoter region and at each CpG island. Additionally, TLR9 mRNA levels were quantified by qPCR and bivariate and multiple modelling were performed to better understand the influence of methylation on gene transcription. Results: TLR9 mRNA levels were upregulated in ALEO compared to HPL (p <.001). TLR9 promoter CpG sites and CpG +2086 in the intragenic island 1 were demethylated in ALEO compared to HPL (p <.05). Multivariate analysis, adjusted by smoking and gender, revealed that demethylation of TLR9 promoter sites enhanced transcriptional activity, specifically demethylated CpGs at positions −736 and −683, (p =.02), which are close to CRE binding. Although ALEO reduced the global methylation of the gene promoter and intragenic‐island 2 (p <.05) by −42.5 and −9.5 percentage points, respectively, age reduced the global methylation of intragenic‐island 3 within the exon 2. Conclusions: Demethylations of TLR9 promoter CpG sites, along with the intragenic DNA methylation status, were involved in higher transcription in ALEO. Hence, chronic periapical inflammation and ageing modify the methylation status both in the gene promoter and in intragenic CpG islands. [ABSTRACT FROM AUTHOR]

Suppressor of cytokine signaling 1 (SOCS-1) is implicated in both virus infection and carcinogenesis. This study investigated the role of HCV infection on SOCS-1 in normal and HCV-infected tissues and revealed a possible mechanism underlying HCV-induced hepatocellular carcinoma (HCC) genesis. In total, 10 HCV-HCC tissues, seven adjacent tissues, seven distal tissues, and 16 normal liver tissues were collected. SOCS-1 expression in tissue sections was detected by immunohistochemistry. After viral load was quantified, the correlation between SOCS-1 expression and viral load was analyzed in different tissues. Then, HCV replicon model was used to detect a relationship between HCV and SOCS-1. Subsequently, methylation-specific PCR (MSP) was applied to show the methylation status of SOCS-1 genes in normal tissues and HCV-replicating cell lines. A correlation between gene methylation, SOCS-1 expression, and HCV was analyzed. The lowest expression of SOCS-1 was observed in HCV-HCC tissues. Tissues with a higher HCV viral load showed lower SOCS-1 expression (p = 0.0282). Consistently, SOCS-1 mRNA and protein were lower in HCV-replicating cell lines than in uninfected ones. Furthermore, gene methylation was found in all examined tissues but higher in HCC tissues, and it is positively correlated with HCV viral load (r 2 = 0.7309, p < 0.0001). HCV infection would upregulate methylation of the SOCS-1 gene in HCV-replicating cell lines. The downregulation of SOCS-1 in normal and HCV-replicating cell lines may result from HCV infection through epigenetic regulation, in which gene methylation in the CpG island of SOCS-1 promoters upon HCV infection suppresses its expression. [ABSTRACT FROM AUTHOR]

Suppressor of cytokine signaling 1 (SOCS-1) is implicated in both virus infection and carcinogenesis. This study investigated the role of HCV infection on SOCS-1 in normal and HCV-infected tissues and revealed a possible mechanism underlying HCV-induced hepatocellular carcinoma (HCC) genesis. In total, 10 HCV-HCC tissues, seven adjacent tissues, seven distal tissues, and 16 normal liver tissues were collected. SOCS-1 expression in tissue sections was detected by immunohistochemistry. After viral load was quantified, the correlation between SOCS-1 expression and viral load was analyzed in different tissues. Then, HCV replicon model was used to detect a relationship between HCV and SOCS-1. Subsequently, methylation-specific PCR (MSP) was applied to show the methylation status of SOCS-1 genes in normal tissues and HCV-replicating cell lines. A correlation between gene methylation, SOCS-1 expression, and HCV was analyzed. The lowest expression of SOCS-1 was observed in HCV-HCC tissues. Tissues with a higher HCV viral load showed lower SOCS-1 expression (p = 0.0282). Consistently, SOCS-1 mRNA and protein were lower in HCV-replicating cell lines than in uninfected ones. Furthermore, gene methylation was found in all examined tissues but higher in HCC tissues, and it is positively correlated with HCV viral load (r2 = 0.7309, p < 0.0001). HCV infection would upregulate methylation of the SOCS-1 gene in HCV-replicating cell lines. The downregulation of SOCS-1 in normal and HCV-replicating cell lines may result from HCV infection through epigenetic regulation, in which gene methylation in the CpG island of SOCS-1 promoters upon HCV infection suppresses its expression.

Background: Abnormal DNA methylation of tumor suppressor gene promoter has been found in breast cancer. Therefore, the current study set out to explore how DNA methyltransferase 1 (DNMT1) affects breast cancer through mediating miR-497/GPRC5A axis. Methods: After loss and gain-of-function approaches were conducted in MCF-7/ADR and MCF-7 cells, cell viability, IC50 value, invasion, migration and apoptosis were measured, respectively. In addition, drug resistance, metastasis and apoptosis-related protein expression were examined using immunoblotting. ChIP and dual-luciferase reporter gene assays were carried out to validate relationship among DNMT1, miR-497, and GPRC5RA. Subcutaneous xenograft tumor model in nude mice was established to detect effects of DNMT1 on growth and metastasis of breast cancer in vivo. Results: It was found that DNMT1 was notably increased, while miR-497 was poorly-expressed in breast cancer. Highly-expressed DNMT1 could promote chemotherapy resistance and metastasis of breast cancer. Meanwhile, DNMT1 modified methylation of CpG island in miR-497 promoter region, thereby repressing miR-497 level. In addition, miR-497 targeted GPRC5A expression to curb chemotherapy resistance and metastasis of breast cancer cells. Lastly, in vivo experiments showed that knockdown of DNMT1 could suppress breast cancer growth and metastasis. Conclusions: Collectively, our findings indicated that DNMT1 may inhibit miR-497 and boost the expression of GPRC5A through methylation, thus augmenting breast cancer chemotherapy resistance and metastasis, which provides novel mechanistic insight into the unrecognized roles of DNMT1 in breast cancer. [ABSTRACT FROM AUTHOR]

Background: Medullary thyroid cancer (MTC) is a rare type of neuroendocrine tumor. This study aimed to investigate the gene and protein expression of RAP1GAP and DNA methylation patterns of its CpG74a, CpG74b, and CpG24 in an Iranian population with MTC. Methods: In this case-control study, we selected 55 individuals who underwent thyroidectomy in Erfan hospital, Tehran, between 2018 and 2020. Samples were divided into normal thyroid tissues (control; n = 20), benign nodule (n = 20), and MTC (n = 15). DNA methylation patterns were investigated using MSP (methylation-specific PCR). The protein level and mRNA expression of RAP1GAP were also evaluated using western blotting and real-time PCR, respectively. Results: The hyper-methylation rates of CpG24 and CpG74a in the MTC samples were considerably higher than the controls (83% versus 15% and 74% versus 17%, respectively; P < 0.001). The methylation/unmethylation ratio of CpG74a, and CpG24 was considerably higher than the controls (P < 0.001). The methylation/unmethylation ratio of CpG24 in the benign nodules was also considerably greater than the controls (P < 0.001). The mRNA expression and the protein level of RAP1GAP in the MTC group were considerably lower than the controls (P = 0.005 and P = 0.035, respectively). In the MTC group, aberrant methylation of CpG74a and CpG24 was significantly correlated with decreasing expression of the RaplGap gene (R2: 0.23; P = 0.032 and R²: 0.56; P = 0a.001, respectively). Conclusion: Hyper-methylation in CpG24 and CpG74a and decreasing expression of RAP1GAP can be considered as diagnostic biomarkers for MTC. [ABSTRACT FROM AUTHOR]

Introduction: Apical periodontitis (AP) is a common oral disease caused by the inflammatory destruction of the periapical tissues due to the infection of the root canal system of the tooth. It also contributes to systemic bacterial translocation, where peripheric mononuclear blood cells (PBMCs) can act as carriers. Toll-like receptor (TLR) 2 mediates the response to infection and activates inflammatory responses. DNA methylation can be induced by bacteria and contributes to the modulation of this response. Despite the evidence that supports the participation of PBMCs in immune-inflammatory disorders, the inflammatory profile and epigenetic regulatory mechanisms of PBMCs in AP individuals are unknown. Aim: To determine TLR2 gene methylation and inflammatory profiles of PBMCs in AP. Methods: Cross-sectional exploratory study. Otherwise, healthy individuals with AP (n=27) and controls (n=30) were included. PMBCs were isolated by a Ficoll gradient, cultured for 24 hours, and both RNA and DNA were extracted. DNA was bisulfite-treated, and specific sites at the promoter region of the TLR2 gene were amplified by qPCR using validated primers. To verify its amplification, agarose gels were performed. Then, the PCR product was sequenced. mRNA expression of TLR2 was determined by qPCR. The soluble levels of 105 inflammatory mediators were first explored with Proteome Profiler Human Cytokine Array Kit. Consequently, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, IL-6Rα, IL-1β, and IL-12p70 levels were measured by Multiplex assay. Results: PBMCs from individuals with AP demonstrated a proinflammatory profile showing higher soluble levels of TNF-α, IL-6, and IL-1β compared to controls (p<0.05). Higher TLR2 expression and higher global methylation pattern of the promoter region of the gene were found in AP compared to controls (p<0.05). The CpGs single-sites at positions -166 and -146 were completely methylated, while the site -102 was totally unmethylated, independently of the presence of AP. DNA methylation of CpG single-sites in positions -77 and +24 was positively associated with TLR2 expression. Conclusions: PBMCs from AP subjects show a hyperinflammatory phenotype and TLR2 upregulation in association with single CpG-sites' methylation from the TLR2 gene promoter, thereby contributing to a sustained systemic inflammatory load in individuals with periapical endodontic diseases. [ABSTRACT FROM AUTHOR]

The inhibition of 5-α reductase type 2 (SRD5A2) by finasteride is commonly used for the management of urinary obstruction resulting from benign prostatic enlargement (BPE). Certain BPE patients showing no SRD5A2 protein expression are resistant to finasteride therapy. Our previous work showed that methylated cytosine-phosphate-guanine (CpG) islands in the SRD5A2 gene might account for the absence or reduction of SRD5A2 protein expression. Here, we found that the expression of the SRD5A2 protein was variable and that weak expression of the SRD5A2 protein (scored 0–100) occurred in 10.0% (4/40) of benign adult prostates. We showed that the expression of SRD5A2 was negatively correlated with DNA methyltransferase 1 (DNMT1) expression. In vitro SRD5A2-negative BPH-1 cells were resistant to finasteride treatment, and SRD5A2 was re-expressed in BPH-1 cells when SRD5A2 was demethylated by 5-Aza-2'-deoxycytidine (5-Aza-CdR) or N-phthalyl-L-tryptophan (RG108). Furthermore, we determined the exact methylation ratios of CpG dinucleotides in a CpG island of SRD5A2 through MassArray quantitative methylation analysis. Ten methylated CpG dinucleotides, including four CpG dinucleotides in the promoter and six CpG dinucleotides in the first exon, were found in a CpG island located from −400 bp to +600 bp in SRD5A2, which might lead to the silencing of SRD5A2 and the absence or reduction of SRD5A2 protein expression. Finasteride cannot exert a therapeutic effect on patients lacking SRD5A2, which may partially account for the resistance to finasteride observed in certain BPE patients.

The enzymes that are independently encoded by GA2ox, GA3ox and GA20ox genes control gibberellins (GA) biosynthesis and catabolism. This study analysed the promoter regions, transcription start site (TSS), motifs and CpG islands (CGIs) in 15 GA oxidase genes of Arabidopsis thaliana using 1-kb sequences. Excluding ATGA2ox5 and ATGA3ox4, promoter regions and TSS analysis showed that 60.00% of the genes have more than one TSS in 1000 bp. Relative to the ATG, 80.00% of TSSs with highest score were in the range of −100 bp. Sixty percent of investigated GA genes have TSS in their 5′ untranslated regions (UTRs) with highest prediction score at 0.8 cut-off values. A total of 5 motifs were discovered in 15 GA oxidase genes. More than half (53.33%) of the motifs were distributed throughout promoter regions on both strands. Positional clustering of more than half (55.31%) of motifs was found between +1 and −500 bp. MfIV was detected (100%) in all promoter regions of GA genes. Using the Plant CARE database, various similar motifs were identified in all promoter regions of GA genes. Including two gibberellin-responsive elements (GARE-motif, P-box), numerous motifs such as CAAT-box, TATA-box, ATC-motif, Gap-box and others were distinguished in the promoter regions of GA genes. CGIs were not detected in the promoter sequences of all GA oxidase genes. However, 86.66% of CGIs were discovered in the gene bodies of GA oxidase genes. Analysing the regulatory elements of GA genes is the first step to control crop height, increase yield and resistance to lodging.

Potassium transporter genes are essential for plant salt stress tolerance. Identification of gene regulatory elements is vital for the recognition of gene expression patterns. Thus, understanding gene regulatory systems of potassium transporter genes is useful to improve the salt tolerance of crop plants. The present study was aimed at in silico analysis of promoter and regulatory elements of potassium transporter genes in coffee, peanut, soybean, maize, sorghum and potato crops. A total of 19 potassium transporter genes were identified, and the transcription start site (TSS), conserved motif, CpG islands were analysed using various computational tools. The highest promoter prediction score (1) was obtained in one gene sequence (LOC113728271) for Coffea arabica transcription start site, whereas the lowest score (0.8) was recorded in one gene sequence (LOC8065633) for Sorghum bicolor transcription start site. The analysis also showed that 66% of the genes contained more than one transcription start site whereas 63.16% had only one transcription start site. Five motifs were identified. Motif 1 was found as the common promoter motif residing on 78.95% of potassium transporter promoter sequences with an E-value of 3.4e − 002.C2H2 zinc finger transcription factors are predicted to bind to these conserved motifs with high statistical probability (2.28e − 03). Very few CpG islands were observed both in the promoter and body region of the gene sequences using two algorithms. The present study could contribute for better understanding of gene expression and the improvement of crops’ tolerance to environmental stresses through molecular assisted breeding or genetic engineering techniques.

Abstract Background The development and application of CRISPR technologies for the modification of the genome are rapidly expanding. Advances in the field describe new CRISPR components that are strategically engineered to improve the precision and reliability of CRISPR editing within the genome sequence. Genome modification using induced genome breaks that are targeted and mediated by CRISPR components leverage cellular mechanisms for repair like homology directed repair (HDR) to incorporate genomic edits with increased precision. Results In this report, we describe the gain of methylation at typically hypomethylated CpG island (CGI) locations affected by the CRISPR-mediated incorporation of donor DNA using HDR mechanisms. With characterization of CpG methylation patterns using whole genome bisulfite sequencing, these CGI methylation disruptions trace the insertion of the donor DNA during the genomic edit. These insertions mediated by homology-directed recombination disrupt the generational methylation pattern stability of the edited CGI within the cells and their cellular lineage within the animal strain, persisting across generations. Our approach describes a statistically based workflow for indicating locations of modified CGIs and provides a mechanism for evaluating the directed modification of the methylome of the affected CGI at the CpG-level. Conclusions With advances in genome modification technology comes the need to detect the level and persistence of methylation change that modifications to the genomic sequence impose upon the collaterally edited methylome. Any modification of the methylome of somatic or germline cells could have implications for gene regulation mechanisms governed by the methylation patterns of CGI regions in the application of therapeutic edits of more sensitively regulated genomic regions. The method described here locates the directed modification of the mouse epigenome that persists over generations. While this observance would require supporting molecular observations such as direct sequence changes or gene expression changes, the observation of epigenetic modification provides an indicator that intentionally directed genomic edits can lead to collateral, unintentional epigenomic changes post modification with generational persistence.

IntroductionApical periodontitis (AP) is a common oral disease caused by the inflammatory destruction of the periapical tissues due to the infection of the root canal system of the tooth. It also contributes to systemic bacterial translocation, where peripheric mononuclear blood cells (PBMCs) can act as carriers. Toll-like receptor (TLR) 2 mediates the response to infection and activates inflammatory responses. DNA methylation can be induced by bacteria and contributes to the modulation of this response. Despite the evidence that supports the participation of PBMCs in immune-inflammatory disorders, the inflammatory profile and epigenetic regulatory mechanisms of PBMCs in AP individuals are unknown.AimTo determine TLR2 gene methylation and inflammatory profiles of PBMCs in AP.MethodsCross-sectional exploratory study. Otherwise, healthy individuals with AP (n=27) and controls (n=30) were included. PMBCs were isolated by a Ficoll gradient, cultured for 24 hours, and both RNA and DNA were extracted. DNA was bisulfite-treated, and specific sites at the promoter region of the TLR2 gene were amplified by qPCR using validated primers. To verify its amplification, agarose gels were performed. Then, the PCR product was sequenced. mRNA expression of TLR2 was determined by qPCR. The soluble levels of 105 inflammatory mediators were first explored with Proteome Profiler Human Cytokine Array Kit. Consequently, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, IL-6Rα, IL-1β, and IL-12p70 levels were measured by Multiplex assay.ResultsPBMCs from individuals with AP demonstrated a proinflammatory profile showing higher soluble levels of TNF-α, IL-6, and IL-1β compared to controls (p

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common types of cancer in the world and a reason behind different oncogenes activation and tumor suppressor genes inactivation. Hyper-methylation of tumor suppressor genes including RASSF1a, GSTP1, p16, and APC cause gene silencing as well as tumor cell invasion. STAT 1 gene is a part of signaling cascade of JAK/STAT and any dysregulation in signaling has been implicated in tumor formation. OBJECTIVE: The current investigation focus on the methylation role of STAT1 gene as a non-invasive biomarker in the progression and diagnosis of hepatocellular carcinoma. METHODS: STAT1 gene methylation status in 46 HCV induced hepatocellular carcinoma patients and 40 non-HCC controls were examined by methylation specific PCR. STAT1 gene expression was examined by real time PCR and further validated by various bioinformatics tools. RESULTS: STAT1 methylation in HCV-induced HCC (67.4%) was significantly higher compared to the non-HCC controls (p < 0.01). However, mRNA expression of STAT1 gene in methylated groups was significantly lower compared to unmethylated groups (p < 0.05). Furthermore, insilco analysis of STAT1 validated our results and shown expression of STAT1 mRNA was lower in liver cancer with the median 24.3 (p = 0.085). CONCLUSION: After using peripheral blood samples we observed that STAT1 silencing caused by aberrant methylation could be used as potential non-invasive biomarker for the diagnosis of HCV induced hepatocellular carcinoma. We conclude that blood as a sample source could be used instead of biopsy for early detection of HCC. [ABSTRACT FROM AUTHOR]

BACKGROUND: Genetic and epigenetic dysregulation of Wnt signaling pathway is widely linked up with abnormal proliferation and/or epithelial-to-mesenchymal transition, in different cancer cell types. OBJECTIVE: In the present research, we have tested whether promoter DNA methylation of a set of Wnt/non-Wnt genes such as [cadherin-2 (CDH2)], "present in circulation", could serve as "bone-marrow biopsy surrogate" and help in diagnosing the status, sub-type or treatment outcome in pediatric acute lymphoblastic leukemia (ALL) patients. METHODS: Promoter DNA methylation was quantified in the bisulfite modified blood from the pediatric ALL patients (n = 86) in comparison with age-matched cancer-free subjects (n = 28), using real-time methylation specific PCR followed by rigorous statistical validations. RESULTS: The observed methylation index, sensitivity and specificity of selected molecular markers (viz., SALL1, WNT5 α , LRP1b, CDH2) in patients' liquid-biopsies was clinically significant showing high positive correlation in the pre-B ALL cases (p -value < 0.001). A substantial drop in promoter methylation signal of the follow-up/post-treatment patients was also noted (p -value < 0.001), which suggested an impending role of minimally invasive liquid-biopsy approach in the diagnosis and/or therapeutic monitoring of pediatric leukemia. CONCLUSIONS: Whilst the reported metadata provides useful insight into the plausible involvement of epigenetic glitches in leukemogensis, our findings strengthen the remarkable functional consequences of dysregulated Wnt signaling genes in the hematological malignancies besides offering a novel panel of epigenetic marks. [ABSTRACT FROM AUTHOR]

The estimation of hypermethylation of the cell cycle (RASSF1A, p16/INK4A, p14/ARF) and detoxification (GSTP1) gene promoters was carried out in blood leukocytes of humans exposed to radiation as a result of the Chernobyl accident (98 individuals: accident liquidators, 76 individuals; adult residents of the territories with radionuclide contamination, 135–688 kBq/m2, 22 individuals) depending on their cytogenetic status. The results of multiple regression analysis ("Frequency of aberrations ~ age + number of hypermethylated genes") indicate a correlation of the total level of chromosomal type aberrations with the considered epigenetic disturbances (β = 0.256; р = 0.011), but not with age (β = –0.138; p = 0.165). The frequency of these cytogenetic disorders increases with an increase in the number of methylated loci. Thus, a positive association between the damages to the genome induced by radiation exposure in the range of small and medium doses (long time ago) and hypermethylation of promoters of the genes of the main protective systems of the cells was detected. [ABSTRACT FROM AUTHOR]