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BackgroundRestraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan.MethodsUsing Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture.ResultsWe developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo.ConclusionsOur method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors.

With the advent of gene editing technologies like CRISPR/Cas9, it has become possible to edit genomic regions of interest for research and therapeutic purposes. These technologies have also been adapted to alter gene expression without changing their DNA sequence, allowing epigenetic edits. While genetic editors make edits by cutting the genome at specified regions, epigenetic editors leverage the same targeting mechanism but act based on the epigenetic modifier fused to them, such as a methyltransferase. Here, we discuss two recently employed epigenetic editors (epi‐editors) that silenced target genes involved in disease to mitigate their effects. Neumann et al. reported the construction of an epigenetic editor called CHARM that could methylate and silence the prion gene in mouse brains and subsequently switch itself off. Additionally, Capelluti et al. developed an epi‐editor called EvoETR that knocked down Pcsk9 in the murine liver to reduce LDL levels. We aim to highlight the design principles underlying the design of these epi‐editors to inform future editor designs. [ABSTRACT FROM AUTHOR]

Abstract Background DNA methylation may have a regulatory role in monogenic sensorineural hearing loss and complex, polygenic phenotypic forms of hearing loss, including age-related hearing impairment or Meniere disease. The purpose of this systematic review is to critically assess the evidence supporting a functional role of DNA methylation in phenotypes associated with hearing loss. Results The search strategy yielded a total of 661 articles. After quality assessment, 25 records were selected (12 human DNA methylation studies, 5 experimental animal studies and 8 studies reporting mutations in the DNMT1 gene). Although some methylation studies reported significant differences in CpG methylation in diverse gene promoters associated with complex hearing loss phenotypes (ARHI, otosclerosis, MD), only one study included a replication cohort that supported a regulatory role for CpG methylation in the genes TCF25 and POLE in ARHI. Conversely, several studies have independently confirmed pathogenic mutations within exon 21 of the DNMT1 gene, which encodes the DNA (cytosine-5)-methyltransferase 1 enzyme. This methylation enzyme is strongly associated with a rare disease defined by autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN). Of note, rare variants in DNMT1 and DNMT3A genes have also been reported in noise-induced hearing loss. Conclusions Evidence supporting a functional role for DNA methylation in hearing loss is limited to few genes in complex disorders such as ARHI. Mutations in the DNMT1 gene are associated with ADCA-DN, suggesting the CpG methylation in hearing loss genes deserves further attention in hearing research.

Abstract Background Restraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan. Methods Using Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture. Results We developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo. Conclusions Our method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors.

Abstract Identifying novel epigenetic biomarkers is a promising way to improve the clinical management of patients with breast cancer. Our study aimed to determine the methylation pattern of 25 tumor suppressor genes (TSG) and select the best methylation biomarker associated with clinicopathological features in the cohort of Slovak patients diagnosed with invasive ductal carcinoma (IDC). Overall, 166 formalin-fixed, paraffin-embedded (FFPE) tissues obtained from patients with IDC were included in the study. The methylation status of the promoter regions of 25 TSG was analyzed using semiquantitative methylation-specific MLPA (MS-MLPA). We identified CDH13 as the most frequently methylated gene in our cohort of patients. Further analysis by ddPCR confirmed an increased level of methylation in the promoter region of CDH13. A significant difference in CDH13 methylation levels was observed between IDC molecular subtypes LUM A versus HER2 (P = 0.0116) and HER2 versus TNBC (P = 0.0234). In addition, significantly higher methylation was detected in HER2+ versus HER2- tumors (P = 0.0004) and PR− versus PR+ tumors (P = 0.0421). Our results provide evidence that alteration in CDH13 methylation is associated with clinicopathological features in the cohort of Slovak patients with IDC. In addition, using ddPCR as a methylation-sensitive method represents a promising approach characterized by higher precision and technical simplicity to measure the methylation of target CpGs in CDH13 compared to other conventional methods such as MS-MLPA.

Stem cell therapies hold promise in addressing the burden of neurodegenerative diseases with human embryonic neural stem cells (hNSC-H9s) and bone marrow-derived human mesenchymal stem cells (hMSCs) as viable candidates. The induction of hMSC neurospheres (hMSC-IN) generate a more lineage-restricted common neural progenitor-like cell population, potentially tunable by heparan sulfate proteoglycans (HSPGs). We examined CpG (5 mC) site methylation patterns using Illumina Infinium 850 K EPIC arrays in hNSC-H9, hMSCs and hMSC-IN cultures with HSPG agonist heparin at early and late phases of growth. We identified key regulatory CpG sites in syndecans (SDC2; SDC4) that potentially regulate gene expression in monolayers. Unique hMSC-IN hypomethylation in glypicans (GPC3 ; GPC4) underscore their significance in neural lineages with Sulfatase 1 and 2 (SULF1 & 2) CpG methylation changes potentially driving the neurogenic shift. hMSC-INs methylation levels at SULF1 CpG sites and SULF2 :cg25401628 were more closely aligned with hNSC-H9 cells than with hMSCs. We further suggest SOX2 regulation governed by lncSOX2-Overall Transcript (lncSOX2-OT) methylation changes with preferential activation of ENO2 over other neuronal markers within hMSC-INs. Our findings illuminate epigenetic dynamics governing neural lineage commitment of hMSC-INs offering insights for targeted mechanisms for regenerative medicine and therapeutic strategies. • Key CpG sites in the promoter regions of Syndecans (SDC2, SDC4) are potentially regulate gene expression patterns in monolayer cultures of human Mesenchymal Stem Cell (hMSC) and human Neural Stem Cell (hNSC-H9). • Hypomethylation of glypican (GPC3) and Sulfatase 1 and 2 (SULF1-2) in hMSC induced neurospheres (INs) suggest their novel roles in cultures and potentially contributing to increased neurogenic potential. • Evidence for CpG site hypomethylation in the 5′UTR of long non-coding SRY-box 2 Overall Transcript (SOX2-OT) regulating SOX2 gene expression. • Preferential methylation changes at the transcription starting site of Enolase 2 (ENO2) over other neural markers within hMSC-INs drive neurogenic shift. [ABSTRACT FROM AUTHOR]

Background: JUB1, a NAC domain containing hydrogen peroxide-induced transcription factor, plays a critical role in plant immunity. Little is known about how JUB1 responds to leaf rust disease in wheat. Recent discoveries in genomics have also unveiled a multitude of sORFs often assumed to be non-functional, to argue for the necessity of including them as potential regulatory players of translation. However, whether methylation on sORFs spanning the 3'UTR of regulatory genes like JUB1 modulate gene expression, remains unclear. Methods and results: In this study, we identified the methylation states of two sORFs in 3'UTR of a homologous gene of JUB1 in wheat, TaJUB1-L, at cytosine residues in CpG, CHH and CHG sites at different time points of disease progression in two near-isogenic lines of wheat (HD2329), with and without Lr24 gene during leaf rust pathogenesis. Here, we report a significant demethylation of the CpG dinucleotides occurring in the sORFs of the 3'UTR in the resistant isolines after 24 h post-infection. Also, the up-regulated gene expression observed through RT-qPCR was directly proportional to the demethylation of the CpG sites in the sORFs. Conclusions: Our findings indicate that TaJUB1-L might be a positive regulator in providing tolerance during leaf rust pathogenesis and cytosine methylation at 3'UTR might act as a switch for its expression control. These results enrich the potential benefit of conventional methylation assay techniques for unraveling the unexplored enigma in epigenetics during plant-pathogen interaction in a cost-effective and confidentially conclusive manner. [ABSTRACT FROM AUTHOR]

Background: DNA methylation may have a regulatory role in monogenic sensorineural hearing loss and complex, polygenic phenotypic forms of hearing loss, including age-related hearing impairment or Meniere disease. The purpose of this systematic review is to critically assess the evidence supporting a functional role of DNA methylation in phenotypes associated with hearing loss. Results: The search strategy yielded a total of 661 articles. After quality assessment, 25 records were selected (12 human DNA methylation studies, 5 experimental animal studies and 8 studies reporting mutations in the DNMT1 gene). Although some methylation studies reported significant differences in CpG methylation in diverse gene promoters associated with complex hearing loss phenotypes (ARHI, otosclerosis, MD), only one study included a replication cohort that supported a regulatory role for CpG methylation in the genes TCF25 and POLE in ARHI. Conversely, several studies have independently confirmed pathogenic mutations within exon 21 of the DNMT1 gene, which encodes the DNA (cytosine-5)-methyltransferase 1 enzyme. This methylation enzyme is strongly associated with a rare disease defined by autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN). Of note, rare variants in DNMT1 and DNMT3A genes have also been reported in noise-induced hearing loss. Conclusions: Evidence supporting a functional role for DNA methylation in hearing loss is limited to few genes in complex disorders such as ARHI. Mutations in the DNMT1 gene are associated with ADCA-DN, suggesting the CpG methylation in hearing loss genes deserves further attention in hearing research. [ABSTRACT FROM AUTHOR]

Identifying novel epigenetic biomarkers is a promising way to improve the clinical management of patients with breast cancer. Our study aimed to determine the methylation pattern of 25 tumor suppressor genes (TSG) and select the best methylation biomarker associated with clinicopathological features in the cohort of Slovak patients diagnosed with invasive ductal carcinoma (IDC). Overall, 166 formalin-fixed, paraffin-embedded (FFPE) tissues obtained from patients with IDC were included in the study. The methylation status of the promoter regions of 25 TSG was analyzed using semiquantitative methylation-specific MLPA (MS-MLPA). We identified CDH13 as the most frequently methylated gene in our cohort of patients. Further analysis by ddPCR confirmed an increased level of methylation in the promoter region of CDH13. A significant difference in CDH13 methylation levels was observed between IDC molecular subtypes LUM A versus HER2 (P = 0.0116) and HER2 versus TNBC (P = 0.0234). In addition, significantly higher methylation was detected in HER2+ versus HER2- tumors (P = 0.0004) and PR− versus PR+ tumors (P = 0.0421). Our results provide evidence that alteration in CDH13 methylation is associated with clinicopathological features in the cohort of Slovak patients with IDC. In addition, using ddPCR as a methylation-sensitive method represents a promising approach characterized by higher precision and technical simplicity to measure the methylation of target CpGs in CDH13 compared to other conventional methods such as MS-MLPA. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion. Methods: Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging. Results: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation. Conclusions/interpretation: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. Data availability: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465. [ABSTRACT FROM AUTHOR]

Recombination-activating gene 1 (RAG1) is a vital player in V(D)J recombination, a fundamental process in primary B cell and T cell receptor diversification of the adaptive immune system. Current vertebrate RAG evolved from RAG transposon; however, it has been modified to play a crucial role in the adaptive system instead of being irreversibly silenced by CpG methylation. By interrogating a range of publicly available datasets, the current study investigated whether RAG1 has retained a disproportionate level of its original CpG dinucleotides compared to other genes, thereby rendering it more exposed to methylation-mediated mutation. Here, we show that 57.57% of RAG1 pathogenic mutations and 51.6% of RAG1 disease-causing mutations were associated with CpG methylation, a percentage that was significantly higher than that of its RAG2 cofactor alongside the whole genome. The CpG scores and densities for all RAG ancestors suggested that RAG transposon was CpG denser. The percentage of the ancestral CpG of RAG1 and RAG2 were 6% and 4.2%, respectively, with no preference towards CG containing codons. Furthermore, CpG loci of RAG1 in sperms were significantly higher methylated than that of RAG2. In conclusion, RAG1 has been exposed to CpG mediated methylation mutagenesis more than RAG2 and the whole genome, presumably due to its late entry to the genome later with an initially higher CpG content. [ABSTRACT FROM AUTHOR]

Allele‐specific epigenetic events regulate the expression of specific genes such as tumor suppressor genes. Methods to biochemically identify epigenetic regulators remain limited. Here, we used insertional chromatin immunoprecipitation (iChIP) to address this issue. iChIP combined with quantitative mass spectrometry identified DNA methyltransferase 1 (DNMT1) and epigenetic regulators as proteins that potentially interact with a region of the p16INK4A gene that is CpG‐methylated in one allele in HCT116 cells. Some of the identified proteins are involved in the CpG methylation of this region, and of these, DEAD‐box helicase 24 (DDX24) contributes to CpG methylation by regulating the protein levels of DNMT1. Thus, iChIP is a useful method to identify proteins which bind to a target locus of interest. [ABSTRACT FROM AUTHOR]

Abstract Background E-cadherin, a major actor of cell adhesion in the intestinal barrier, is encoded by the CDH1 gene associated with susceptibility to Crohn Disease (CD) and colorectal cancer. Since epigenetic mechanisms are suspected to contribute to the multifactorial pathogenesis of CD, we studied CpG methylation at the CDH1 locus. The methylation of the CpG island (CGI) and of the 1st enhancer, two critical regulatory positions, was quantified in surgical specimens of inflamed ileal mucosa and in peripheral blood mononuclear cells (PBMC) of 21 CD patients. Sixteen patients operated on for a non-inflammatory bowel disease, although not normal controls, provided a macroscopically normal ileal mucosa and PBMC for comparison. Results In ileal mucosa, 19/21 (90%) CD patients vs 8/16 control patients (50%) (p

Abstract Background Surrogate production by germline stem cell transplantation is a powerful method to produce donor-derived gametes via a host, a practice known as surrogacy. The gametes produced by surrogates are often analysed on the basis of their morphology and species-specific genotyping, which enables conclusion to be drawn about the donor’s characteristics. However, in-depth information, such as data on epigenetic changes, is rarely acquired. Germ cells develop in close contact with supporting somatic cells during gametogenesis in vertebrates, and we hypothesize that the recipient’s gonadal environment may cause epigenetic changes in produced gametes and progeny. Here, we extensively characterize the DNA methylome of donor-derived sperm and their intergenerational effects in both inter- and intraspecific surrogates. Results We found more than 3000 differentially methylated regions in both the sperm and progeny derived from inter- and intraspecific surrogates. Hypermethylation in the promoter regions of the protocadherin gamma gene in the intraspecific surrogates was found to be associated with germline transmission. On the contrary, gene expression level and the embryonic development of the offspring remained unaffected. We also discovered MAPK/p53 pathway disruption in interspecific surrogates due to promoter hypermethylation and identified that the inefficient removal of meiotic-arrested endogenous germ cells in hybrid gonads led to the production of infertile spermatozoa. Conclusions Donor-derived sperm and progeny from inter- and intraspecific surrogates were more globally hypermethylated than those of the donors. The observed changes in DNA methylation marks in the surrogates had no significant phenotypic effects in the offspring.

Background: E-cadherin, a major actor of cell adhesion in the intestinal barrier, is encoded by the CDH1 gene associated with susceptibility to Crohn Disease (CD) and colorectal cancer. Since epigenetic mechanisms are suspected to contribute to the multifactorial pathogenesis of CD, we studied CpG methylation at the CDH1 locus. The methylation of the CpG island (CGI) and of the 1st enhancer, two critical regulatory positions, was quantified in surgical specimens of inflamed ileal mucosa and in peripheral blood mononuclear cells (PBMC) of 21 CD patients. Sixteen patients operated on for a non-inflammatory bowel disease, although not normal controls, provided a macroscopically normal ileal mucosa and PBMC for comparison. Results: In ileal mucosa, 19/21 (90%) CD patients vs 8/16 control patients (50%) (p < 0.01) had a methylated CDH1 promoter CGI. In PBMC, CD patients with methylated CGI were 11/21 (52%) vs 7/16 controls (44%), respectively. Methylation in the 1st enhancer of CDH1 was also higher in the CD group for each of the studied CpGs and for their average value (45 ± 17% in CD patients vs 36 ± 17% in controls; p < 0.001). Again, methylation was comparable in PBMC. Methylation of CGI and 1st enhancer were not correlated in mucosa or PBMC. Conclusions: Methylation of several CpGs at the CDH1 locus was increased in the inflamed ileal mucosa, not in the PBMC, of CD patients, suggesting the association of CDH1 methylation with ileal inflammation. Longitudinal studies will explore if this increased methylation is a risk marker for colorectal cancer. [ABSTRACT FROM AUTHOR]

Background: Surrogate production by germline stem cell transplantation is a powerful method to produce donor-derived gametes via a host, a practice known as surrogacy. The gametes produced by surrogates are often analysed on the basis of their morphology and species-specific genotyping, which enables conclusion to be drawn about the donor’s characteristics. However, in-depth information, such as data on epigenetic changes, is rarely acquired. Germ cells develop in close contact with supporting somatic cells during gametogenesis in vertebrates, and we hypothesize that the recipient’s gonadal environment may cause epigenetic changes in produced gametes and progeny. Here, we extensively characterize the DNA methylome of donor-derived sperm and their intergenerational effects in both inter- and intraspecific surrogates. Results: We found more than 3000 differentially methylated regions in both the sperm and progeny derived from inter- and intraspecific surrogates. Hypermethylation in the promoter regions of the protocadherin gamma gene in the intraspecific surrogates was found to be associated with germline transmission. On the contrary, gene expression level and the embryonic development of the offspring remained unaffected. We also discovered MAPK/p53 pathway disruption in interspecific surrogates due to promoter hypermethylation and identified that the inefficient removal of meiotic-arrested endogenous germ cells in hybrid gonads led to the production of infertile spermatozoa. Conclusions: Donor-derived sperm and progeny from inter- and intraspecific surrogates were more globally hypermethylated than those of the donors. The observed changes in DNA methylation marks in the surrogates had no significant phenotypic effects in the offspring. [ABSTRACT FROM AUTHOR]

Until recently, studying the murine methylome was restricted to sequencing-based methods. In this study we compared the global DNA methylation levels of hairless mouse epidermis using the recently released Infinium Mouse Methylation BeadChip from Illumina and whole genome bisulphite sequencing (WGBS). We also studied the effect of sample storage conditions by using fresh and fresh-frozen epidermis. The DNA methylation levels of 123,851 CpG sites covered by both the BeadChip and WGBS were compared. DNA methylation levels obtained with WGBS and the BeadChip were strongly correlated (Pearson correlation r = 0.984). We applied a threshold of 15 reads for the WGBS methylation analysis. Even at a threshold of 10 reads, we observed no substantial difference in DNA methylation levels compared with that obtained with the BeadChip. The DNA methylation levels from the fresh and the fresh-frozen samples were strongly correlated when analysed with both the BeadChip (r = 0.999) and WGBS (r = 0.994). We conclude that the two methods of analysis generally work equally well for studies of DNA methylation of mouse epidermis and find that fresh and fresh-frozen epidermis can generally be used equally well. The choice of method will depend on the specific study’s aims and the available resources in the laboratory.

African American (AA) men have the highest incidence and mortality rate from Prostate cancer (PCa) than any other racial/ethnic group. To date, PCa genomic studies have largely under-represented tumour samples from AA men. We measured genome-wide DNA methylation in benign and tumor prostate tissues from AA men using the Illumina Infunium 850 K EPIC array. mRNA expression database from a subset of the AA biospecimen were used to assess correlation of transcriptome and methylation datasets. Genome-wide methylation analysis identified 11,460 probes that were significant (p

Abstract Epigenetic modifications of histones are associated with development and pathogenesis of disease. Existing approaches cannot provide insights into long-range interactions and represent the average chromatin state. Here we describe BIND&MODIFY, a method using long-read sequencing for profiling histone modifications and transcription factors on individual DNA fibers. We use recombinant fused protein A-M.EcoGII to tether methyltransferase M.EcoGII to protein binding sites to label neighboring regions by methylation. Aggregated BIND&MODIFY signal matches bulk ChIP-seq and CUT&TAG. BIND&MODIFY can simultaneously measure histone modification status, transcription factor binding, and CpG 5mC methylation at single-molecule resolution and also quantifies correlation between local and distal elements.

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive histological type of cancer in this location. Distant metastases are present in approximately 30% of patients at the time of first examination. Therefore, the ability to predict the occurrence of metastases in patients at early stages of the disease is an urgent task aimed at personalized treatment. Samples of tumor and paired histologically normal kidney tissue from patients with metastatic and non-metastatic ccRCC were studied. Gene expression was analyzed using real-time PCR. The level of gene methylation was evaluated using bisulfite conversion followed by quantitative methylation-specific PCR. Two groups of genes were analyzed in this study. The first group includes genes whose expression is significantly reduced during metastasis: CA9, NDUFA4L2, EGLN3, and BHLHE41 (p < 0.001, ROC analysis). The second group includes microRNA genes: MIR125B-1, MIR137, MIR375, MIR193A, and MIR34B/C, whose increased methylation levels are associated with the development of distant metastases (p = 0.002 to <0.001, ROC analysis). Based on the data obtained, a combined panel of genes was formed to identify patients whose tumors have a high metastatic potential. The panel can estimate the probability of metastasis with an accuracy of up to 92%. [ABSTRACT FROM AUTHOR]

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is aberrantly expressed in about one-third of GH-secreting pituitary adenomas (GH-PAs) and has been associated with a paradoxical increase of GH after a glucose load. The reason for such an overexpression has not yet been clarified. In this work, we aimed to evaluate whether locus-specific changes in DNA methylation patterns could contribute to this phenomenon. By cloning bisulfite-sequencing PCR, we compared the methylation pattern of the GIPR locus in GIPR-positive (GIPR+) and GIPR-negative (GIPR−) GH-PAs. Then, to assess the correlation between Gipr expression and locus methylation, we induced global DNA methylation changes by treating the lactosomatotroph GH3 cells with 5-aza-2′-deoxycytidine. Differences in methylation levels were observed between GIPR+ and GIPR− GH-PAs, both within the promoter (31.9% vs. 68.2%, p < 0.05) and at two gene body regions (GB_1 20.7% vs. 9.1%; GB_2 51.2% vs. 65.8%, p < 0.05). GH3 cells treated with 5-aza-2′-deoxycytidine showed a ~75% reduction in Gipr steady-state level, possibly associated with the observed decrease in CpGs methylation. These results indicate that epigenetic regulation affects GIPR expression in GH-PAs, even though this possibly represents only a part of a much more complex regulatory mechanism. [ABSTRACT FROM AUTHOR]

IntroductionModulation of epigenetic mechanisms that contribute to retinal development may render the eye susceptible to age-related macular degeneration (AMD). Progression of AMD involves alterations of epigenome such as CpG methylation and histone modifications, and study of the epigenetic regulation of molecular/ cellular pathways associated with AMD might identify target epigenetic markers for treatment of AMD.Areas coveredIn this review, we provide an overview of the influence of epigenetic factors on signaling pathways/ related genes associated with AMD, mainly hypoxia, angiogenesis, inflammation, complement, and oxidative stress; and discuss the critical role of microRNAs in AMD.Expert opinionBetter understanding of epigenetic-mediated and microRNA-mediated regulation of the AMD disease-related pathways would help to assess the risk of developing AMD besides providing valuable insight on potential target candidates for AMD therapy.

Highlights PLCD1 is methylated and downregulated in RCC and functions as a tumor suppressor gene Inactivation of PLCD1 contributes to RCC tumorigenesis. PLCD1 inhibits WNT/β-catenin and EGFR-FAK-ERK signaling in RCC.

Abstract Background Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder characterized by asymmetric muscle wasting and weakness. FSHD can be subdivided into two types: FSHD1, caused by contraction of the D4Z4 repeat on chromosome 4q35, and FSHD2, caused by mild contraction of the D4Z4 repeat plus aberrant hypomethylation mediated by genetic variants in SMCHD1, DNMT3B, or LRIF1. Genetic diagnosis of FSHD is challenging because of the complex procedures required. Methods We applied Nanopore CRISPR/Cas9-targeted resequencing for the diagnosis of FSHD by simultaneous detection of D4Z4 repeat length and methylation status at nucleotide level in genetically-confirmed and suspected patients. Results We found significant hypomethylation of contracted 4q-D4Z4 repeats in FSHD1, and both 4q- and 10q-D4Z4 repeats in FSHD2. We also found that the hypomethylation in the contracted D4Z4 in FSHD1 is moderately correlated with patient phenotypes. Conclusions Our method contributes to the development for the diagnosis of FSHD using Nanopore long-read sequencing. This finding might give insight into the mechanisms by which repeat contraction causes disease pathogenesis.

nc886 is a medium-sized non-coding RNA that is transcribed by RNA polymerase III (Pol III) and plays diverse roles in tumorigenesis, innate immunity, and other cellular processes. Although Pol III-transcribed ncRNAs were previously thought to be expressed constitutively, this concept is evolving, and nc886 is the most notable example. The transcription of nc886 in a cell, as well as in human individuals, is controlled by multiple mechanisms, including its promoter CpG DNA methylation and transcription factor activity. Additionally, the RNA instability of nc886 contributes to its highly variable steady-state expression levels in a given situation. This comprehensive review discusses nc886's variable expression in physiological and pathological conditions and critically examines the regulatory factors that determine its expression levels. [ABSTRACT FROM AUTHOR]

Children born preterm are at heightened risk of neurodevelopmental impairments, including Autism Spectrum Disorder (ASD). The placenta is a key regulator of neurodevelopmental processes, though the precise underlying molecular mechanisms remain unclear. Here, we employed a multi‐omic approach to identify placental transcriptomic and epigenetic modifications related to ASD diagnosis at age 10, among children born preterm. Working with the extremely low gestational age (ELGAN) cohort, we hypothesized that a pro‐inflammatory placental environment would be predictive of ASD diagnosis at age 10. Placental messenger RNA (mRNA) expression, CpG methylation, and microRNA (miRNA) expression were compared among 368 ELGANs (28 children diagnosed with ASD and 340 children without ASD). A total of 111 genes displayed expression levels in the placenta that were associated with ASD. Within these ASD‐associated genes is an ASD regulatory complex comprising key genes that predicted ASD case status. Genes with expression that predicted ASD case status included Ewing Sarcoma Breakpoint Region 1 (EWSR1) (OR: 6.57 (95% CI: 2.34, 23.58)) and Bromodomain Adjacent To Zinc Finger Domain 2A (BAZ2A) (OR: 0.12 (95% CI: 0.03, 0.35)). Moreover, of the 111 ASD‐associated genes, nine (8.1%) displayed associations with CpG methylation levels, while 14 (12.6%) displayed associations with miRNA expression levels. Among these, LRR Binding FLII Interacting Protein 1 (LRRFIP1) was identified as being under the control of both CpG methylation and miRNAs, displaying an OR of 0.42 (95% CI: 0.17, 0.95). This gene, as well as others identified as having functional epimutations, plays a critical role in immune system regulation and inflammatory response. In summary, a multi‐omic approach was used to identify functional epimutations in the placenta that are associated with the development of ASD in children born preterm, highlighting future avenues for intervention. Lay Summary: Children who are born preterm have an increased risk for neurodevelopmental impairments, including autism spectrum disorder (ASD). The placenta is known to play a pivotal role in child development, and is posited to regulate neurodevelopment as well. Our research reveals a significant number of placental genes expressed in relation to ASD, and that many of these genes are under the control of epigenetic processes. This research is important for understanding how the placenta may contribute to the development of ASD later in life in children born preterm. [ABSTRACT FROM AUTHOR]

The cyp19a1 gene codes aromatase that converts androgen to estrogen, which plays a central role in early female differentiation and ovarian development in teleosts. For the blotched snakehead (Channa maculata), an important aquaculture fish that is susceptible to hormone-induced sex reversal, two aromatase genes were characterized in the present study, cyp19a1a and cyp19a1b. We analyzed gene expression and the epigenetic state of cyp19a1a and cyp19a1b in different adult tissues: the gonad and brain from normal XX females (XX-F), normal XY males (XY-M), sex-reversal females (XY-F) induced by estrogen, and YY super-males (YY-M), and gonads at different development stages. Cyp19a1a exhibited strong female-biased expression patterns in the ovary, and cyp19a1b dominantly expressed in the brain with no sex bias. Cyp19a1a's expression pattern in the XY-F ovary was similar to that in the XX-F ovary, with a relatively high expression level, which was far higher than that in XY-M and YY-M testis. Meanwhile, CpG methylation levels of cyp19a1a promoter were lower in XX-F and XY-F ovaries compared with XY-M and YY-M testis. A significantly negative correlation between the CpG methylation levels and cyp19a1a expression was elucidated in XX-F, XY-M, XY-F, and YY-M gonads. Furthermore, the strong female-biased cyp19a1a expression was closely related to ovarian differentiation and maturation, and the overall methylation levels of cyp19a1a promoter were inversely correlated with cyp19a1a expression. There were no detectable sexually dimorphic differences in cyp19a1b expression and CpG methylation levels of cyp19a1b promoter in the brain and gonad between sexes in C. maculata, thus the function of cyp19a1b in C. maculata needs further research. Our research illustrates that cyp19a1a is closely related to estrogen production, ovary differentiation/maintenance, and sex reversal, and epigenetic modification plays a crucial part in maintaining the sexual dimorphic expression of cyp19a1a, ovarian differentiation and oogenesis in C. maculata. [ABSTRACT FROM AUTHOR]

Recent studies suggest that Hypocretin (HCRT, Orexin) are involved in stress regulation of depression through the hypothalamic-pituitary-adrenal (HPA) axis. However, the molecular mechanism by which Hypocretin regulate neurobiological responses is unknown. Herein, the effects of chronic stress on the epigenetic modification of HCRT and its association with depression were explored with regard to a potential role in cancer progression. In the study, Sprague Dawley (SD) rats were used to establish an animal model of cancer with depression by administrating n-nitrosodiethylamine (DEN) and chronic unpredictable mild stress (CUMS). RNA-sequencing was used to detect differentially expressed genes in the hippocampus of rats and quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the results of RNA-sequencing. The status of HCRT promoter methylation was assessed by methylation specific polymerase chain reaction. Behavioral tests showed that rats exposed to CUMS had significant depressive-like behaviors. The number of liver tumors and tumor load in depressed rats exposed to CUMS was higher than in SD rats without CUMS. RNA-sequencing revealed that HCRT was one of the most siginificantly downregulated gene in the hippocampus of SD rats with CUMS compared to non-stressed group, which was validated by qRT-PCR. HCRT mRNA expression was downregulated and the promoter for HCRT was hyper-methylated in those with depression. These results identified a critical role for chronic psychological stressors in tumorigenesis and cancer progression, via epigenetic HCRT downregulation. Such epigenetic downregulation may be the molecular basis for the association of cancer with depression.

Background: PLCD1, located at 3p22, encodes an enzyme that mediates cellular metabolism and homeostasis, intracellular signal transduction and movement. PLCD1 plays a pivotal role in tumor suppression of several types of cancers; however, its expression and underlying molecular mechanisms in renal cell carcinoma (RCC) pathogenesis remain elusive. Methods: RT-PCR and Western blot were used to detect PLCD1 expression in RCC cell lines and normal tissues. Bisulfite treatment, MSP and BGS were utilized to explore the CpG methylation status of PLCD1 promoter. Online databases were analyzed for the association between PLCD1 expression/methylation and patient survival. In vitro experiments including CCK8, colony formation, wound-healing, transwell migration and invasion, immunofluorescence and flow cytometry assays were performed to evaluate tumor cell behavior. Luciferase assay and Western blot were used to examine effect of PLCD1 on WNT/β‐catenin and EGFR‐FAK-ERK signaling. Results: We found that PLCD1 was widely expressed in multiple adult normal tissues including kidney, but frequently downregulated or silenced in RCC due to its promoter CpG methylation. Restoration of PLCD1 expression inhibited the viability, migration and induced G2/M cell cycle arrest and apoptosis in RCC cells. PLCD1 restoration led to the inhibition of signaling activation of WNT/β-catenin and EGFR-FAK-ERK pathways, and the EMT program of RCC cells. Conclusions: Our results demonstrate that PLCD1 is a potent tumor suppressor frequently inactivated by promoter methylation in RCC and exerts its tumor suppressive functions via suppressing WNT/β‐catenin and EGFR‐FAK-ERK signaling. These findings establish PLCD1 as a promising prognostic biomarker and treatment target for RCC. Highlights: PLCD1 is methylated and downregulated in RCC and functions as a tumor suppressor gene Inactivation of PLCD1 contributes to RCC tumorigenesis. PLCD1 inhibits WNT/β-catenin and EGFR-FAK-ERK signaling in RCC. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) latency patterns are well defined in EBV-associated epithelial, NK/T-cell, and B-cell malignancies, with links between latency stage and tumorigenesis deciphered in various studies. In vitro studies suggest that the oncogenic activity of EBV in T-cells might be somewhat different from that in EBV-tropic B lymphoid cells, prompting us to study this much less investigated viral gene expression pattern and its regulation in nine EBV+ peripheral T-cell lymphoma (PTCL) biopsies. Using frozen specimens, RT-PCR showed 6/7 cases with a latency II pattern of EBV gene expression. Analyses of EBNA1 promoter usage and CpG methylation status in these six cases showed that only Qp was used, while Cp, Wp, and Fp were all silent. However, the remaining case showed an exceptionally unique latency III type with lytic activation, as evidenced by EBV lytic clonality and confirmed by the full usage of Cp and Qp as well as weakly lytic Fp and Wp, fully unmethylated Cp and marginally unmethylated Wp. Further immunostaining of the eight cases revealed a few focally clustered LMP1+ cells in 7/8 cases, with rare isolated LMP1+ cells detected in another case. Double immunostaining confirmed that the LMP1+ cells were of the T-cell phenotype (CD3+). In 6/8 cases, sporadically scattered Zta+ cells were detected. Double staining of EBER-ISH with T-cell (CD45RO/UCHL1) or B-cell (CD20) markers confirmed that the vast majority of EBER+ cells were of the T-cell phenotype. Predominant type-A EBV variant and LMP1 30-bp deletion variant were present, with both F and f variants detected. In summary, the EBV gene expression pattern in PTCL was found to be mainly of latency II (BART+EBNA1(Qp)+LMP1+LMP2A+BZLF1+), similar to that previously reported in EBV-infected nasopharyngeal epithelial, NK/T-cell, and Hodgkin malignancies; however, fully lytic infection could also be detected in occasional cases. Rare cells with sporadic immediate-early gene expression were commonly detected in PTCL. These findings have implications for the future development of EBV-targeting therapeutics for this cancer. [ABSTRACT FROM AUTHOR]

A limited number of studies are devoted to regulating TRIP6 expression in cancer. Hence, we aimed to unveil the regulation of TRIP6 expression in MCF-7 breast cancer cells (with high TRIP6 expression) and taxane-resistant MCF-7 sublines (manifesting even higher TRIP6 expression). We found that TRIP6 transcription is regulated primarily by the cyclic AMP response element (CRE) in hypomethylated proximal promoters in both taxane-sensitive and taxane-resistant MCF-7 cells. Furthermore, in taxane-resistant MCF-7 sublines, TRIP6 co-amplification with the neighboring ABCB1 gene, as witnessed by fluorescence in situ hybridization (FISH), led to TRIP6 overexpression. Ultimately, we found high TRIP6 mRNA levels in progesterone receptor-positive breast cancer and samples resected from premenopausal women. [ABSTRACT FROM AUTHOR]

The mammalian DNA methylation landscape is established and maintained by the combined activities of the two key epigenetic modifiers, DNA methyltransferases (DNMT) and Ten-eleven-translocation (TET) enzymes. Once DNMTs produce 5-methylcytosine (5mC), TET proteins fine-tune the DNA methylation status by consecutively oxidizing 5mC to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives. The 5mC and oxidized methylcytosines are essential for the maintenance of cellular identity and function during differentiation. Cytosine modifications with DNMT and TET enzymes exert pleiotropic effects on various aspects of hematopoiesis, including self-renewal of hematopoietic stem/progenitor cells (HSPCs), lineage determination, differentiation, and function. Under pathological conditions, these enzymes are frequently dysregulated, leading to loss of function. In particular, the loss of DNMT3A and TET2 function is conspicuous in diverse hematological disorders, including myeloid and lymphoid malignancies, and causally related to clonal hematopoiesis and malignant transformation. Here, we update recent advances in understanding how the maintenance of DNA methylation homeostasis by DNMT and TET proteins influences normal hematopoiesis and malignant transformation, highlighting the potential impact of DNMT3A and TET2 dysregulation on clonal dominance and evolution of pre-leukemic stem cells to full-blown malignancies. Clarification of the normal and pathological functions of DNA-modifying epigenetic regulators will be crucial to future innovations in epigenetic therapies for treating hematological disorders. [ABSTRACT FROM AUTHOR]

Synucleinopathies are a group of neurodegenerative diseases with common pathological lesions associated with the excessive accumulation and abnormal intracellular deposition of toxic species of α-synuclein. The shared clinical features are chronic progressive decline of motor, cognitive, and behavioral functions. These disorders include Parkinson's disease, dementia with Lewy body, and multiple system atrophy. Vigorous research in the mechanisms of pathology of these illnesses is currently under way to find disease-modifying treatment and molecular markers for early diagnosis. α-Synuclein is a prone-to-aggregate, small amyloidogenic protein with multiple roles in synaptic vesicle trafficking, neurotransmitter release, and intracellular signaling events. Its expression is controlled by several mechanisms, one of which is epigenetic regulation. When transmitted to the nucleus, α-synuclein binds to DNA and histones and participates in epigenetic regulatory functions controlling specific gene transcription. Here, we discuss the various aspects of α-synuclein involvement in epigenetic regulation in health and diseases. [ABSTRACT FROM AUTHOR]

African American (AA) men have the highest incidence and mortality rate from Prostate cancer (PCa) than any other racial/ethnic group. To date, PCa genomic studies have largely underrepresented tumour samples from AA men. We measured genome-wide DNA methylation in benign and tumor prostate tissues from AA men using the Illumina Infunium 850 K EPIC array. mRNA expression database from a subset of the AA biospecimen were used to assess correlation of transcriptome and methylation datasets. Genome-wide methylation analysis identified 11,460 probes that were significant (p < 0.01) and differentially methylated in AA PCa compared to normal prostate tissues and showed significant (p < 0.01) inverse-correlation with mRNA expression. Ingenuity pathway analysis and Gene Ontology analysis in our AA dataset compared with TCGA dataset showed similarities in methylation patterns: top candidate genes with significant hypermethylation and corresponding down-regulated gene expression were associated with biological pathways in hemidesmosome assembly, mammary gland development, epidermis development, hormone biosynthesis, and cell communication. In addition, top candidate genes with significant hypomethylation and corresponding up-regulated gene expression were associated with biological pathways in macrophage differentiation, cAMP-dependent protein kinase activity, protein destabilization, transcription co-repression, and fatty acid biosynthesis. In contrast, differences in genome-wide methylation in our AA dataset compared with TCGA dataset were enriched for genes in steroid signalling, immune signalling, chromatin structure remodelling and RNA processing. Overall, differential methylation of AMIGO3, IER3, UPB1, GRM7, TFAP2C, TOX2, PLSCR2, ZNF292, ESR2, MIXL1, BOLL, and FGF6 were significant and uniquely associated with PCa progression in our AA cohort. [ABSTRACT FROM AUTHOR]

Until recently, studying the murine methylome was restricted to sequencing-based methods. In this study we compared the global DNA methylation levels of hairless mouse epidermis using the recently released Infinium Mouse Methylation BeadChip from Illumina and whole genome bisulphite sequencing (WGBS). We also studied the effect of sample storage conditions by using fresh and fresh-frozen epidermis. The DNA methylation levels of 123,851 CpG sites covered by both the BeadChip and WGBS were compared. DNA methylation levels obtained with WGBS and the BeadChip were strongly correlated (Pearson correlation r = 0.984). We applied a threshold of 15 reads for the WGBS methylation analysis. Even at a threshold of 10 reads, we observed no substantial difference in DNA methylation levels compared with that obtained with the BeadChip. The DNA methylation levels from the fresh and the fresh-frozen samples were strongly correlated when analysed with both the BeadChip (r = 0.999) and WGBS (r = 0.994). We conclude that the two methods of analysis generally work equally well for studies of DNA methylation of mouse epidermis and find that fresh and fresh-frozen epidermis can generally be used equally well. The choice of method will depend on the specific study's aims and the available resources in the laboratory. [ABSTRACT FROM AUTHOR]

Objectives: The study aimed to find differential gene mutations, DNA methylation, and expression profiles among different categories of cervical cancer samples. Methods: The study was based on freely available gene mutations, promoter methylation, and gene expression status of The Cancer Genome Atlas (TCGA) cervical cancer samples and adjacent normal tissues in the Genomic Data Commons (GDC) portal. The association of CpG island methylation with gene expression was determined through negative correlation analysis. Results: We identified that the ErbB signaling pathway and proteoglycans pathway was significantly associated with adenocarcinoma cervical cancers patients. In these pathways, missense mutation especially S310F in the ERBB2 gene as well as G12D and A146T in the KRAS gene were significantly associated with adenocarcinoma cases. Furthermore, a comparison of SCC cases with adjacent control tissues revealed differential hypermethylation of two CpG positions of the KAAG1 gene and differential downregulation of NPY1R and NPY5R genes in cervical squamous cell carcinoma compared to cervical adenocarcinoma cases and adjacent normal tissues. Specifically, the hypermethylation of the promoter region of the KAAG1 gene might be responsible for the carcinogenesis of cervical squamous cells exclusively and methylation marks can be reversible by the widely used drug, azacytidine. In contrast, adenocarcinoma cervical cancer cases may be treated with floxuridine which is successfully utilized for other tissue‐specific adenocarcinoma cases. Conclusions: These results provide valuable insight into the differential molecular markers among the categories of cervical cancer, which helps our ability to classify these cancers and for targeted therapy. [ABSTRACT FROM AUTHOR]

Background: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder characterized by asymmetric muscle wasting and weakness. FSHD can be subdivided into two types: FSHD1, caused by contraction of the D4Z4 repeat on chromosome 4q35, and FSHD2, caused by mild contraction of the D4Z4 repeat plus aberrant hypomethylation mediated by genetic variants in SMCHD1, DNMT3B, or LRIF1. Genetic diagnosis of FSHD is challenging because of the complex procedures required.Methods: We applied Nanopore CRISPR/Cas9-targeted resequencing for the diagnosis of FSHD by simultaneous detection of D4Z4 repeat length and methylation status at nucleotide level in genetically-confirmed and suspected patients.Results: We found significant hypomethylation of contracted 4q-D4Z4 repeats in FSHD1, and both 4q- and 10q-D4Z4 repeats in FSHD2. We also found that the hypomethylation in the contracted D4Z4 in FSHD1 is moderately correlated with patient phenotypes.Conclusions: Our method contributes to the development for the diagnosis of FSHD using Nanopore long-read sequencing. This finding might give insight into the mechanisms by which repeat contraction causes disease pathogenesis. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is the major causative pathogen associated with hepatocellular carcinoma and liver cirrhosis. The main virion component, the Core (C) protein, is involved in multiple aspects of HCV pathology including oncogenesis and immune evasion. In this study, we established a next-generation bisulfite sequencing (NGS-BS) protocol to analyze the CpG methylation profile at the tumor suppressor gene SHP-1 P2 promoter as a model system. Our data show that HCV C protein expression in the immortalized T cells correlated with a specific CpG methylation profile at the SHP-1 P2. The NGS-BS on HCV-positive (HCV+) patient-derived PBMCs revealed a considerably different CpG methylation profile compared to the HCV C protein immortalized T cells. Notably, the CpG methylation profile was very similar in healthy and HCV+ PBMCs, suggesting that the SHP-1 P2 CpG methylation profile is not altered in the HCV+ individuals. Collectively, the NGS-BS is a highly sensitive method that can be used to quantitatively characterize the CpG methylation status at the level of individual CpG position and also allows the characterization of cis-acting effects on epigenetic regulation. [ABSTRACT FROM AUTHOR]

The clinical manifestations of psoriatic arthritis (PsA) are highly heterogeneous and no reliable diagnostic biomarkers exist. We explored the role of DNA methylation CpG markers in the diagnosis of PsA. DNA methylation array was used to screen for differentially methylated sites (DMSs) in the discovery phase (PsA, n = 25; healthy controls [HCs], n = 19; psoriasis vulgaris [PsV], n = 20). In the validation phase, pyrosequencing was used to identify the DMSs in an expanded cohort (PsA, n = 60; HCs, n = 91; PsV, n = 48; rheumatoid arthritis [RA], n = 60). Logistic regression prediction models were established based on the identified DMSs for the diagnosis of PsA. A total of 17 DMSs differentiating PsA and HCs as well as 11 DMSs differentiating PsA and PsV were screened in the discovery phase. A total of six DMSs (chr14: cg07940072, chr14: 38061320, chr9: cg15734589, chr6: cg12800266, chr3: cg12992827, chr6: cg24500972) differentiating PsA and HCs and two DMSs (chr12: cg16459382, chr2: cg16348668) differentiating PsA and PsV were identified using pyrosequencing. Three logistic regression prediction models were established based on the identified DMSs, which distinguished PsA, RA, PsV, and HCs (P < 0.001). The models performed well in differentiating PsA from HCs, RA, and PsV (AUC: 0.858, 0.851, and 0.976, respectively). The models based on methylated CpG sites are useful for distinguishing patients with PsA from HCs and those with RA or PsV and are a highly sensitive and specific diagnostic biomarker for PsA. • DNA methylation markers distinguish psoriatic arthritis from psoriasis vulgaris. • DNA methylation plays a crucial role in psoriasis and psoriatic arthritis. • DNA methylation serves as a good biomarker for the diagnosis of psoriatic arthritis. [ABSTRACT FROM AUTHOR]

In the vast majority of instances, the causes of male infertility are unknown, although scientists have lately attempted to correlate epigenetics to infertility mechanisms. DNA methylation is an important biological modification that gives phenotypic diversity and adaptability. The aim of this study was whether the methylation status of the methylenetetrahydrofolate reductase (MTHFR) gene promoter in Iraqi men's spermatozoa is correlated with male infertility. Semen samples were collected from 100 infertile and healthy fertile men. Divided into 4 groups included 25 subjects of healthy fertile men as a control group(C) and infertile groups, which divided into (3) subgroups: Asthenozoospermia (A) (n=25), Oligoasthenozoospermia (OA) (n = 25) and severe Oligoasthenoteratozoospermia (SOA) (n =25). Sperm DNAs and RNAs from all samples were isolated; followed by sperm-specific methylation detection using the EpiJET DNA Methylation Analysis Kit, which is simple digestion was performed with two restriction enzymes (MspI/HpaII). The methylation percentage (mean ± SD) of the MTHFR gene promoter was quantified in each studied group as follow:[ (C) (1.76±0.46) (A) (3.59±0.89), (OA) (26.10±4.14) and (SOA)(29.13±4.88)]. The percentage of MTHFR promoter methylation infertile men was significantly higher than that in the healthy control group (p = 0.00). Methylation level was determined via receiver operator characteristic (ROC) analysis as the cut-off values of each infertile subgroup were as follows: [(A) (2.43%), (OA) (7.59%) and (SOA) (10.96%)]. According to WHO guidelines, sperm concentration, progressive motility and morphologically normal sperm were determining (WHO, 2010). The qRT-PCR was used to evaluate MTHFR gene expression in all sperm samples. The results revealed a substantial reduction in fold expression (2-ΔΔCt) in all infertile groups when compared to the control g roup, with a P-value (0.002). In this research findings suggest that epigenetic impairment (hypermethylation) of the MTHFR promoter region in sperm DNA from OA and SOA patients may increase male infertility risk. [ABSTRACT FROM AUTHOR]

Background: Immune evasion in glioblastoma (GBM) shields cancer cells from cytotoxic immune response. Methods: We investigated CpG methylation in promoters, genes, and pathways in 22 pairs of formalin-fixed paraffin-embedded sequential (FFPE) GBM using restricted resolution bisulfite sequencing (RRBS) and bioinformatic analyses. Results: Gene ontology revealed hypermethylation in elements of the innate and adaptive immune system when recurrent GBM samples (GBMrec) were compared to control (CG) and primary GBM samples (GBMprim). Higher methylation levels of the IL-7 signaling pathway and response to IL-7 were found in GBMrec suggesting a progressive blockade of the IL-7 driven T cell response in sequential GBM. Analyses of the Cancer Genome Atlas array-based data confirmed hypermethylation of the IL-7 pathway in recurrent compared with primary GBM. We also quantified DNA CpG methylation in promoter and gene regions of the IL-7 ligand and IL-7 α-receptor subunit in individual samples of a large RRBS-based sequential cohort of GBM in a Viennese database and found significantly higher methylation levels in the IL-7 receptor α-subunit in GBMrec compared with GBMprim. Conclusions: This study revealed the progressive suppression of the IL-7 receptor-mediated pathway as a means of immune evasion by GBM and thereby highlighted it as a new treatment target. [ABSTRACT FROM AUTHOR]

nc886 is a regulatory noncoding RNA that is transcribed by RNA polymerase III (Pol III), is variably expressed in different biological contexts, and plays roles in inflammation and cancer. Epigenetic mechanisms play an intriguing role in regulating nc886 expression. As a maternally imprinted gene and metastable epiallele, nc866 exhibits polymorphic imprinting, with a methylation status that is influenced by environmental and biological factors. Consequently, the promoter DNA methylation status and the different resulting RNA expression levels of nc886 are associated with physiological and pathological conditions. In this review, we summarize the literature and explore the significance in relation to diverse roles of nc886.

The regulation of genes can be mathematically described by input-output functions that are typically assumed to be time invariant. This fundamental assumption underpins the design of synthetic gene circuits and the quantitative understanding of natural gene regulatory networks. Here, we found that this assumption is challenged in mammalian cells. We observed that a synthetic reporter gene can exhibit unexpected transcriptional memory, leading to a shift in the dose-response curve upon a second induction. Mechanistically, we investigated the cis-dependency of transcriptional memory, revealing the necessity of promoter DNA methylation in establishing memory. Furthermore, we showed that the synthetic transcription factor's effective DNA binding affinity underlies trans-dependency, which is associated with its capacity to undergo biomolecular condensation. These principles enabled modulating memory by perturbing either cis- or trans-regulation of genes. Together, our findings suggest the potential pervasiveness of transcriptional memory and implicate the need to model mammalian gene regulation with time-varying input-output functions. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

DNA methylation is one of the major epigenetic mechanisms crucial for gene regulation and genome stability. De novo DNA methyltransferase DNMT3C is required for silencing evolutionarily young transposons during mice spermatogenesis. Mutation of DNMT3C led to a sterility phenotype that cannot be rescued by its homologs DNMT3A and DNMT3B. However, the structural basis of DNMT3C-mediated DNA methylation remains unknown. Here, we report the structure and mechanism of DNMT3C-mediated DNA methylation. The DNMT3C methyltransferase domain recognizes CpG-containing DNA in a manner similar to that of DNMT3A and DNMT3B, in line with their high sequence similarity. However, two evolutionary covariation sites, C543 and E590, diversify the substrate interaction among DNMT3C, DNMT3A, and DNMT3B, resulting in distinct DNA methylation activity and specificity between DNMT3C, DNMT3A, and DNMT3B in vitro. In addition, our combined structural and biochemical analysis reveals that the disease-causing rahu mutation of DNMT3C compromises its oligomerization and DNA-binding activities, explaining the loss of DNA methylation activity caused by this mutation. This study provides a mechanistic insight into DNMT3C-mediated DNA methylation that complements DNMT3A- and DNMT3B-mediated DNA methylation in mice, unraveling a regulatory mechanism by which evolutionary conservation and diversification fine-tune the activity of de novo DNA methyltransferases., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Renal cell carcinoma (RCC) is one of the most common types of cancers, representing about 2.3% of all malignancies throughout the world. In RCC, Cathepsin B (CtsB) plays a major role in signalling pathways, processing, and intracellular protein degradation. CtsB is involved in tumour cell invasion, matrix remodelling and metastasis and this mechanism was controlled by natural inhibitors. In this study targeted methylation changes were analyzed in primary renal cancer and metastatic tissues and the expression of CtsB was analyzed in RCC and compared with normal cells. The levels of CpG methylation of Runt-trelated transcription factor 3 (RUNX3) were analyzed. The decreased level of RUNX3 was observed in metastatic tissues due to CpG methylation. The increased methylation in CpG islands increased metastasis and was associated with RUNX3. The amount of CtsB mRNA was high in the RCC tissues than in surrounding normal tissues. Increased expression of CtsB was observed in RCC. Overexpression of CtsB was confirmed by Western blotting analysis and expression of CtsB increased the proliferation of RCC. The present finding revealed that CtsB has a major role in the development and proliferation of RCC.

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive histological type of cancer in this location. Distant metastases are present in approximately 30% of patients at the time of first examination. Therefore, the ability to predict the occurrence of metastases in patients at early stages of the disease is an urgent task aimed at personalized treatment. Samples of tumor and paired histologically normal kidney tissue from patients with metastatic and non-metastatic ccRCC were studied. Gene expression was analyzed using real-time PCR. The level of gene methylation was evaluated using bisulfite conversion followed by quantitative methylation-specific PCR. Two groups of genes were analyzed in this study. The first group includes genes whose expression is significantly reduced during metastasis: CA9, NDUFA4L2, EGLN3, and BHLHE41 (p < 0.001, ROC analysis). The second group includes microRNA genes: MIR125B-1, MIR137, MIR375, MIR193A, and MIR34B/C, whose increased methylation levels are associated with the development of distant metastases (p = 0.002 to

Background: Dysregulation of DNA damage response and altered DNA methylation in acute myeloid leukemia (AML) have been reported, but the impact of methylation of DNA repair genes has not yet been researched. We aimed to predict the prognosis of non-APL AML patients based on the known CpG site methylation levels of DNA repair genes through The Cancer Genome Atlas AML project (TCGA-LAML). Methods: We utilized TCGA-LAML cohort (174 non-APL AML) for the methylation data of 22 DNA repair genes. Results: In univariate analysis among 174 non-APL AML patients of the TCGA-LAML cohort, the hypermethylation of MLH1, RAD51, and ATM showed superior overall survival (OS) than non-hypermethylated groups, while hypermethylation of RAD23A, RAD23B, MLH1, MSH2, BRCA1, BRCA2, RAD50, and PARP1 was associated with poor OS. We demonstrated that CpG hypermethylation levels of DNA repair genes differed according to the AML cytogenetic risk groups. In multivariate analysis, hypermethylation of MLH1 and RAD51 showed better OS than non-hypermethylated patients, but hypermethylation of MSH2 and RAD50 showed worse OS than non-hypermethylated patients. Conclusion: Methylation of 4 DNA repair genes, such as MLH1, RAD51, MSH2, and RAD50, have the potential to be independent risk factors in non-APL AML patients. [ABSTRACT FROM AUTHOR]