DNA Immunoprecipitation Research Articles

Gene body methylation safeguards ribosomal DNA transcription by preventing PHF6-mediated enrichment of repressive histone mark H4K20me3

DNA methylation shows complex correlations with gene expression, and the role of promoter hypermethylation in repressing gene transcription has been well addressed. Emerging evidence indicates that gene body methylation promotes transcription; however, the underlying mechanisms remain to be further investigated. Here, using methylated DNA immunoprecipitation sequencing (MeDIP-seq), bisulfite genomic sequencing, and immunofluorescent labeling, we show that gene body methylation is indeed positively correlated with rRNA gene (rDNA) transcription. Mechanistically, gene body methylation is largely maintained by DNA methyltransferase 1 (DNMT1), deficiency or downregulation of which during myoblast differentiation or nutrient deprivation results in decreased gene body methylation levels, leading to increased gene body occupancy of plant homeodomain (PHD) finger protein 6 (PHF6). PHF6 binds to hypomethylated rDNA gene bodies where it recruits histone methyltransferase SUV4-20H2 to establish the repressive histone modification, H4K20me3, ultimately inhibiting rDNA transcription. These findings demonstrate that DNMT1-mediated gene body methylation safeguards rDNA transcription by preventing enrichment of repressive histone modifications, suggesting that gene body methylation serves to maintain gene expression in response to developmental and/or environmental stresses.

Young Breast Cancer: Novel Gene Methylation in WBC.

Introduction:Breast cancer is a highly diverse disease, and epigenomic alterations, as principle changes in the pathogenesis of breast cancer, have recently been noticed in epimarker research on peripheral blood. Methods:In this study, DNA samples isolated from the white blood cells of 30 breast cancer patients were compared to 30 healthy controls using methylated DNA immunoprecipitation microarray (MeDIP-chip) to determine differentially methylated region as a potential epimarker in cancer and control cases. Results:A total of 1799 differentially methylated regions were identified, including ZNF154, BCL9, and HOXD9, in which significant methylation differences were confirmed in breast cancer patients through a quantitative real-time polymerase chain reaction. Differential methylation of the mentioned genes has been reported in different cancer tissues and cell-free DNA, including breast cancer. Methylation of those genes listed in the white blood cells of our young patients not only relates to their importance in the pathogenesis of breast cancer but may also highlight their potential as primary epimarkers that can warrant further evaluation in large cohort studies. It is important to note that methylation alteration in WBC, as well as genetic mutation, can be identified years before cancer development, which emphasizes this issue as a potential screening marker.

Effects of low lead exposure on sperm quality and sperm DNA methylation in adult men

InstructionLead (Pb) exposure is a risk factor for male infertility, but the epigenetic changes in sperm DNAattributable to lead exposure is poorly defined.MethodsIn this study, we investigated whether low Pb exposure (< 10 µg/dL) affects the sperm quality. Blood, urine, and semen samples of 297 men of childbearing age were analyzed for all relevant parameters. Based on the blood Pb level (BLL), participants were allocated to RL (0–2.5 µg/dL), RM (2.5–5 µg/dL), and RH (5–10 µg/dL) groups. The 5-methylcytosine and 5-hydroxymethylcytosine patterns in the sperm DNA were identified using methylated DNA immunoprecipitation and hydroxymethylated DNA immunoprecipitation sequencing.ResultsThe non-progressive motility (NP) was significantly increased and associated with global hypomethylation of sperm DNA in the RH group compared with the RL group, indicating that aberrant sperm methylation due to low Pb exposure is possibly associated with reduced sperm motility. The hypomethylated promoter regions were primarily enriched in the calcium (Ca) homeostasis pathway. Further, the interaction between Ca and Pb was associated with sperm rapid progressive motility and asthenospermia risk, although no significant methylation abnormality was observed in those with BLL < 5 µg/dL. When BLL was > 5 µg/dL or when predicting NP, no significant Pb–Ca interaction was observed.DiscussionOverall, our results indicate that aberrant DNA methylation of the Ca homeostasis pathway, induced by low Pb exposure, is the potential cause for reduced sperm velocity.

Gadd45α is involved in regulating activity-dependent and exon-specific BDNF expression in postmitotic cortical neurons.

This study aimed to explore the epigenetic regulation of activity-dependent and exon-specific brain-derived neurotrophic factor (BDNF) expression under KCl depolarization in primary cortical neurons. We investigated BDNF exon I, exon IV and the growth arrest and DNA damage-inducible protein 45 alpha (Gadd45α) transcription levels under KCl-induced neuronal activation in postmitotic neurons. Gadd45α occupancy at BDNF I and IV promoter was measured by chromatin immunoprecipitation (ChIP) followed by quantitative PCR; DNA methylation level was checked by methylated DNA immunoprecipitation (MeDIP) followed by qPCR. In addition, lentiviral shRNA targeting Gadd45α was used to knockdown Gadd45α expression. BDNF exon I and IV mRNA expressions were both highly induced by KCl depolarization. However, ChIP analysis demonstrated a significantly increased Gadd45α occupancy only at the BDNF P1 promotor, but not P4, which is associated with reducing DNA methylation within BDNF P1 promoter. Furthermore, after the lentiviral-mediated knockdown of Gadd45α, the increased Gadd45α occupancy at the BDNF P1 was inhibited, which was accompanying the complete blocking of the demethylation effect at P1. Nonetheless, the induction of BDNF exon I mRNA by KCl was only partially prevented by Gadd45α shRNA, indicting other mechanisms involved in regulating BDNF exon I expression. DNA demethylation mediated by Gadd45α protein involves promoting the regulation of activity-dependent BDNF exon I expression in neurons.

Genome-wide methylation analysis reveals differentially methylated CpG sites and altered expression of heart development-associated genes in fetuses with cardiac defects.

DNA methylation, as an epigenetic mechanism, has a vital role in heart development. An increasing number of studies have investigated aberrant DNA methylation in pediatric or adult heart samples from patients with congenital heart defects (CHD). Placenta tissue, umbilical cord blood, or newborn blood have also been used to detect DNA methylation biomarkers for CHD. However, few studies have compared the methylation levels in fetal heart tissue with cardiac defects with that in normal controls. The present study conducted an integrative whole-genome and CpG site-specific DNA methylation analysis of fetal heart samples from 17 isolated cardiac defect cases, 14 non-isolated cardiac defect cases, and 22 controls with normal hearts, using methylated DNA immunoprecipitation microarray and MassARRAY EpiTYPER assays. Expression of genes adjacent to differentially methylated regions (DMRs) was measured by RT-qPCR and western blot analysis. The results revealed that fetuses with cardiac defects presented global hypomethylation. Genomic analysis of DMRs revealed that a proportion of DMRs were located in exons (12.4%), distal intergenic regions (11.14%), and introns (8.97%). Only 55.7% of DMRs were observed at promoter regions. Functional enrichment analysis for genes adjacent to these DMRs revealed that hypomethylated genes were involved in embryonic heart tube morphogenesis and immune-related regulation functions. Intergenic hypermethylation of EGFR and solute carrier family 19 member 1 (SLC19A1), and intragenic hypomethylation of NOTCH1 were validated in fetal heart tissues with cardiac defects. Only SLC19A1 expression was significantly decreased at the mRNA level, while EGFR, NOTCH1, and SLC19A1 expression were all significantly decreased at the protein level. In conclusion, the present study demonstrated that fetal cardiac defects may be associated with alterations in regional and single CpG site methylation outside of promoter regions, resulting in differentiated expression of corresponding genes associated with heart development. These results present new insights into the epigenetic mechanisms underlying abnormal heart development.

Genome Wide MeDIP-Seq Profiling of Wild and Cultivated Olives Trees Suggests DNA Methylation Fingerprint on the Sensory Quality of Olive Oil.

Secondary metabolites are particularly important to humans due to their pharmaceutical properties. Moreover, secondary metabolites are key compounds in climate change adaptation in long-living trees. Recently, it has been described that the domestication of Olea subspecies had no major selection signature on coding variants and was mainly related to changes in gene expression. In addition, the phenotypic plasticity in Olea subspecies was linked to the activation of transposable elements in the genes neighboring. Here, we investigated the imprint of DNA methylation in the unassigned fraction of the phenotypic plasticity of the Olea subspecies, using methylated DNA immuno-precipitation sequencing (MeDIP-seq) for a high-resolution genome-wide DNA methylation profiling of leaves and fruits during fruit development in wild and cultivated olives from Turkey. Notably, the methylation profiling showed a differential DNA methylation in secondary metabolism responsible for the sensory quality of olive oil. Here, we highlight for the first time the imprint of DNA methylation in modulating the activity of the Linoleate 9S lipoxygenase in the biosynthesis of volatile aromatic compounds. Unprecedently, the current study reveals the methylation status of the olive genome during fruit ripening.

Abstract 2102: Development and validation of Predicine methylation assay towards early cancer detection

Abstract DNA methylation is one of the earliest signatures during cancer development. DNA methylation patterns from different cancer types can provide distinct epigenetic signatures. Several methods are available for sequencing DNA methylation throughout the genome, including whole genome bisulfite sequencing (WGBS) and antibody-dependent DNA immunoprecipitation (MeDIP). As a non-invasive approach, plasma cfDNA and urinary cfDNA (ucfDNA) have been widely explored for clinical applications. More recently, in virtue of its cancer-specific methylation signatures, cfDNA has been adopted for early cancer detection and further identification of cancer tissue of origin. Predicine has developed a liquid biopsy methylation assay that can robustly detect methylation using genomic and circulating cell-free DNA in body fluids. Predicine's methylation assay has great improvement over WGBS in reducing DNA damage, GC bias and increasing NGS reads mapping rate, and mapping quality score. In this study, we report the clinical utility of Predicine's methylation assay, which efficiently distinguishing healthy donors from patients of various types of cancer. Combined with its genomics NGS counterpart, Predicine's methylation platform is a powerful tool for early cancer detection and real-time monitoring of cancer progression. Citation Format: Binggang Xiang, Chao Dai, Ming Wang, Zhixin Zhao, Amy Xiaohong Wang, Kemin Zhou, Shidong Jia, Shujun Luo, Pan Du. Development and validation of Predicine methylation assay towards early cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2102.

Tumor-Naïve Multimodal Profiling of Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma.

Circulating tumor DNA (ctDNA) enables personalized treatment strategies in oncology by providing a noninvasive source of clinical biomarkers. In patients with low ctDNA abundance, tumor-naïve methods are needed to facilitate clinical implementation. Here, using locoregionally confined head and neck squamous cell carcinoma (HNSCC) as an example, we demonstrate tumor-naïve detection of ctDNA by simultaneous profiling of mutations and methylation. We conducted CAncer Personalized Profiling by deep Sequencing (CAPP-seq) and cell-free Methylated DNA ImmunoPrecipitation and high-throughput sequencing (cfMeDIP-seq) for detection of ctDNA-derived somatic mutations and aberrant methylation, respectively. We analyzed 77 plasma samples from 30 patients with stage I-IVA human papillomavirus-negative HNSCC as well as plasma samples from 20 risk-matched healthy controls. In addition, we analyzed leukocytes from patients and controls. CAPP-seq identified mutations in 20 of 30 patients at frequencies similar to that of The Tumor Genome Atlas (TCGA). Differential methylation analysis of cfMeDIP-seq profiles identified 941 ctDNA-derived hypermethylated regions enriched for CpG islands and HNSCC-specific methylation patterns. Both methods demonstrated an association between ctDNA abundance and shorter fragment lengths. In addition, mutation- and methylation-based ctDNA abundance was highly correlated (r > 0.85). Patients with detectable pretreatment ctDNA by both methods demonstrated significantly worse overall survival (HR = 7.5; P = 0.025) independent of clinical stage, with lack of ctDNA clearance post-treatment strongly correlating with recurrence. We further leveraged cfMeDIP-seq profiles to validate a prognostic signature identified from TCGA samples. Tumor-naïve detection of ctDNA by multimodal profiling may facilitate biomarker discovery and clinical use in low ctDNA abundance applications.

Paternal exposure to microcystin-LR triggers developmental neurotoxicity in zebrafish offspring via an epigenetic mechanism involving MAPK pathway

Microcystin-LR (MCLR) induced impairment to male reproductive system and revealed the effects of transgenerational toxicity on offspring. But very little is known about the inheritance of these effects to offspring and the mechanisms involved. Here, we used methylated DNA immunoprecipitation sequencing (MeDIP-Seq) and microarray to characterize whole-genome DNA methylation and mRNA expression patterns in zebrafish testis after 6-week exposure to 5 and 20 μg/L MCLR. Accompanied with these analyses it revealed that MAPK pathway and ER pathway significantly enriched in zebrafish testes. Apoptosis and testicular damage were also observed in testis. Next, we test the transmission of effects to compare control-father and MCLR exposure-father progenies. DNA methylation analyses (via reduced representation bisulfite sequencing) reveal that the enrichment of differentially methylated regions on neurodevelopment after paternal MCLR exposure. Meanwhile, several genes associated with neurodevelopment were markedly downregulated in zebrafish larvae, and swimming speed was also reduced in the larvae. Interestingly, paternal MCLR exposure also triggered activation the phosphorylation of mitogen-activated protein kinase (MAPK) pathway which is also associated with neurodevelopmental disorders. These results demonstrated the significant effect that paternal MCLR exposure may have on gene-specific DNA methylation patterns in testis. Inherited epigenetic alterations through the germline may be the mechanism leading to developmental neurotoxicity in the offspring.

RASAL2 Deficiency Attenuates Hepatic Steatosis by Promoting Hepatic VLDL Secretion via the AKT/TET1/MTTP Axis.

RAS protein activator like 2 (RASAL2) is a newly discovered metabolic regulator involved in energy homeostasis and adipogenesis. However, whether RASAL2 is involved in hepatic lipid metabolism remains undetermined. This study explored the function of RASAL2 and elucidated its potential mechanisms in nonalcoholic fatty liver disease (NAFLD). NAFLD models were established either by feeding mice a high-fat diet or by incubation of hepatocytes with 1 mM free fatty acids (oleic acid:palmitic acid=2:1). Pathological changes were observed by hematoxylin and eosin staining. Lipid accumulation was assessed by Oil Red O staining, BODIPY493/503 staining, and triglyceride quantification. The in vivo secretion rate of very low-density lipoprotein was determined by intravenous injection of tyloxapol. Gene regulation was analyzed by chromatin immunoprecipitation assays and hydroxymethylated DNA immunoprecipitation combined with real-time polymerase chain reaction. RASAL2 deficiency ameliorated hepatic steatosis both in vivo and in vitro. Mechanistically, RASAL2 deficiency upregulated hepatic TET1 expression by activating the AKT signaling pathway and thereby promoted MTTP expression by DNA hydroxymethylation, leading to increased production and secretion of very low-density lipoprotein, which is the major carrier of triglycerides exported from the liver to distal tissues. Our study reports the first evidence that RASAL2 deficiency ameliorates hepatic steatosis by regulating lipid metabolism through the AKT/TET1/MTTP axis. These findings will help understand the pathogenesis of NAFLD and highlight the potency of RASAL2 as a new molecular target for NAFLD.

Increased PARylation impacts the DNA methylation process in type 2 diabetes mellitus

BackgroundEpigenetic modifications, such as DNA methylation, can influence the genetic susceptibility to type 2 diabetes mellitus (T2DM) and the progression of the disease. Our previous studies demonstrated that the regulation of the DNA methylation pattern involves the poly(ADP-ribosyl)ation (PARylation) process, a post-translational modification of proteins catalysed by the poly(ADP-ribose) polymerase (PARP) enzymes. Experimental data showed that the hyperactivation of PARylation is associated with impaired glucose metabolism and the development of T2DM. Aims of this case–control study were to investigate the association between PARylation and global and site-specific DNA methylation in T2DM and to evaluate metabolic correlates.ResultsData were collected from 61 subjects affected by T2DM and 48 healthy individuals, recruited as controls. Global levels of poly(ADP-ribose) (PAR, a surrogate of PARP activity), cytosine methylation (5-methylcytosine, 5mC) and de-methylation intermediates 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were determined in peripheral blood cells by ELISA-based methodologies. Site-specific DNA methylation profiling of SOCS3, SREBF1 and TXNIP candidate genes was performed by mass spectrometry-based bisulfite sequencing, methyl-sensitive endonucleases digestion and by DNA immuno-precipitation. T2DM subjects presented higher PAR levels than controls. In T2DM individuals, increased PAR levels were significantly associated with higher HbA1c levels and the accumulation of the de-methylation intermediates 5hmC and 5fC in the genome. In addition, T2DM patients with higher PAR levels showed reduced methylation with increased 5hmC and 5fC levels in specific SOCS3 sites, up-regulated SOCS3 expression compared to both T2DM subjects with low PAR levels and controls.ConclusionsThis study demonstrates the activation of PARylation processes in patients with T2DM, particularly in those with poor glycaemic control. PARylation is linked to dysregulation of DNA methylation pattern via activation of the DNA de-methylation cascade and may be at the basis of the differential gene expression observed in presence of diabetes.

Disruption of YY1-EZH2 Interaction Using Synthetic Peptides Inhibits Breast Cancer Development.

Simple SummaryBoth Yin Yang 1 (YY1) and enhancer of zeste homolog 2 (EZH2) are oncogenes with overexpressed statuses in cancers. As a transcription factor, YY1 recruits EZH2 through its oncoprotein binding (OPB) domain to repress gene expression. In this study, we identified the interaction domain of YY1 on EZH2 protein with amino acids 493–519, named the YY1 protein binding (YPB) domain. Synthetic peptides using YPB and OPB domain sequences effectively blocked endogenous YY1-EZH2 interaction. Functionally, YPB and OPB peptides could efficiently inhibit the proliferation of breast cancer cells, promote their apoptosis, and reduce tumor growth in a xenograft mouse model. Using chromatin immunoprecipitation DNA sequencing (ChIP-seq) analysis, we discovered that YPB and OPB peptides could interfere with H3K27 trimethylation of multiple genes. Eventually, we identified that YPB and OPB peptides primarily targeted the PTENP1 gene and validated its importance in the anticancer activity of the two peptides.Enhancer of zeste homolog 2 (EZH2) is a methyltransferase to mediate lysine 27 trimethylation in histone H3 (i.e., H3K27me3) and repress gene expression. In solid tumors, EZH2 promotes oncogenesis and is considered a therapeutic target. As a transcription factor, Yin Yang 1 (YY1) recruits EZH2 through its oncoprotein binding (OPB) domain to establish gene repression. In this study, we mapped the YY1 protein binding (YPB) domain on EZH2 to a region of 27 amino acids. Both YPB and OPB domain synthetic peptides could disrupt YY1EZH2 interaction, markedly reduce breast cancer cell viability, and efficiently inhibit tumor growth in a xenograft mouse model. We analyzed MDA-MB-231 cells treated with YPB, OPB, and control peptides by chromatin immunoprecipitation DNA sequencing (ChIP-seq) using an antibody against H3K27me3. YPB and OPB treatments altered H3K27me3 on 465 and 1137 genes, respectively, compared to the control. Of these genes, 145 overlapped between the two peptides. Among them, PTENP1, the PTEN pseudogene, showed reduced H3K27me3 signal when treated by either YPB or OPB peptide. Consistently, the two peptides enhanced both PTENP1 and PTEN expression with concomitantly reduced AKT activation. Further studies validated PTENP1′s contribution to the anticancer activity of YPB and OPB peptides.

Genome-wide CpG density and DNA methylation analysis method (MeDIP, RRBS, and WGBS) comparisons

ABSTRACT Genome-wide DNA methylation analysis is one of the most common epigenetic processes analysed for genome characterization and differential DNA methylation assessment. Previous genome-wide analysis has suggested an important variable in DNA methylation methods involves CpG density. The current study was designed to investigate the CpG density in a variety of different species genomes and correlate this to various DNA methylation analysis data sets. The majority of all genomes had >90% of the genome in the low density 1–3 CpG/100 bp category, while <10% of the genome was in the higher density >5 CpG/100 bp category. Similar observations with human, rat, bird, and fish genomes were observed. The methylated DNA immunoprecipitation (MeDIP) procedure uses the anti-5-methylcytosine antibody immunoprecipitation followed by next-generation sequencing (MeDIP-Seq). The MeDIP procedure is biased to lower CpG density of <5 CpG/100 bp, which corresponds to >95% of the genome. The reduced representation bisulphite (RRBS) protocol generally identifies DMRs in higher CpG density regions of ≥3 CpG/100 bp which corresponds to approximately 20% of the genome. The whole-genome bisulphite (WGBS) analyses resulted in higher CpG densities, often greater than 10 CpG/100bp. WGBS generally identifies ≥2 CpG/100bp, which corresponds to approximately 50% of the genome. Limitations and potential optimization approaches for each method are discussed. None of the procedures can provide complete genome-wide assessment of the genome, but MeDIP-Seq provides coverage of the highest percentage. Observations demonstrate that CpG density is a critical variable in DNA methylation analysis, and different molecular techniques focus on distinct genomic regions.

DNA methylation variations in familial female and male breast cancer.

In total, ~25% of familial breast cancer (BC) is attributed to germline mutations of the BRCA1 and BRCA2 genes, while the rest of the cases are included in the BRCAX group. BC is also known to affect men, with a worldwide incidence of 1%. Epigenetic alterations, including DNA methylation, have been rarely studied in male breast cancer (MBC) on a genome-wide level. The aim of the present study was to examine the global DNA methylation profiles of patients with BC to identify differences between familial female breast cancer (FBC) and MBC, and according to BRCA1, BRCA2 or BRCAX mutation status. The genomic DNA of formalin-fixed paraffin-embedded tissues from 17 women and 7 men with BC was subjected to methylated DNA immunoprecipitation and hybridized on human promoter microarrays. The comparison between FBC and MBC revealed 2,846 significant differentially methylated regions corresponding to 2,486 annotated genes. Gene Ontology enrichment analysis revealed molecular function terms, such as the GTPase superfamily genes (particularly the GTPase Rho GAP/GEF and GTPase RAB), and cellular component terms associated with cytoskeletal architecture, such as ‘cytoskeletal part’, ‘keratin filament’ and ‘intermediate filament’. When only FBC was considered, several cancer-associated pathways were among the most enriched KEGG pathways of differentially methylated genes when the BRCA2 group was compared with the BRCAX or BRCA1+BRCAX groups. The comparison between the BRCA1 and BRCA2+BRCAX groups comprised the molecular function term ‘cytoskeletal protein binding’. Finally, the functional annotation of differentially methylated genes between the BRCAX and BRCA1+BRCA2 groups indicated that the most enriched molecular function terms were associated with GTPase activity. In conclusion, to the best of our knowledge, the present study was the first to compare the global DNA methylation profile of familial FBC and MBC. The results may provide useful insights into the epigenomic subtyping of BC and shed light on a possible novel molecular mechanism underlying BC carcinogenesis.

Aberrant methylation modifications reflect specific drug responses in small cell lung cancer

In the study, Methylated DNA immunoprecipitation sequencing, RNA sequencing, and whole-exome sequencing were employed to clinical small cell lung cancer (SCLC) patients. Then, we verified the therapeutic predictive effects of differentially methylated genes (DMGs) in 62 SCLC cell lines. Of 4552 DMGs between chemo-sensitive and chemo-insensitive group, coding genes constituted the largest percentage (85.08%), followed by lncRNAs (10.52%) and miRNAs (3.56%). Both two groups demonstrated two methylation peaks near transcription start site and transcription end site. Two lncRNA-miRNA-mRNA networks suggested the extensive genome connection between chemotherapy efficacy-related non-coding RNAs (ncRNAs) and mRNAs. Combing miRNAs and lncRNAs could effectively predict chemotherapy response in SCLC. In addition, we also verified the predictive values of mutated genes in SCLC cell lines. This study was the first to evaluate multiple drugs efficacy-related ncRNAs and mRNAs which were modified by methylation in SCLC. DMGs identified in our research might serve as promising therapeutic targets to reverse drugs-insensitivity by complex lncRNA-miRNA-mRNA mechanisms in SCLC.

Sperm Methylome Profiling Can Discern Fertility Levels in the Porcine Biomedical Model.

A combined Genotyping By Sequencing (GBS) and methylated DNA immunoprecipitation (MeDIP) protocol was used to identify—in parallel—genetic variation (Genomic-Wide Association Studies (GWAS) and epigenetic differences of Differentially Methylated Regions (DMR) in the genome of spermatozoa from the porcine animal model. Breeding boars with good semen quality (n = 11) and specific and well-documented differences in fertility (farrowing rate, FR) and prolificacy (litter size, LS) (n = 7) in artificial insemination programs, using combined FR and LS, were categorized as High Fertile (HF, n = 4) or Low Fertile (LF, n = 3), and boars with Unknown Fertility (UF, n = 4) were tested for eventual epigenetical similarity with those fertility-proven. We identified 165,944 Single Nucleotide Polymorphisms (SNPs) that explained 14–15% of variance among selection lines. Between HF and LF individuals (n = 7, 4 HF and 3 LF), we identified 169 SNPs with p ≤ 0.00015, which explained 58% of the variance. For the epigenetic analyses, we considered fertility and period of ejaculate collection (late-summer and mid-autumn). Approximately three times more DMRs were observed in HF than in LF boars across these periods. Interestingly, UF boars were clearly clustered with one of the other HF or LF groups. The highest differences in DMRs between HF and LF experimental groups across the pig genome were located in the chr 3, 9, 13, and 16, with most DMRs being hypermethylated in LF boars. In both HF and LF boars, DMRs were mostly hypermethylated in late-summer compared to mid-autumn. Three overlaps were detected between SNPs (p ≤ 0.0005, n = 1318) and CpG sites within DMRs. In conclusion, fertility levels in breeding males including FR and LS can be discerned using methylome analyses. The findings in this biomedical animal model ought to be applied besides sire selection for andrological diagnosis of idiopathic sub/infertility.

Differentially methylated genes in proliferative verrucous leukoplakia reveal potential malignant biomarkers for oral squamous cell carcinoma

ObjectivesTo explore the pathophysiology of proliferative verrucous leucoplakia (PVL) through a methylated DNA immunoprecipitation and high-throughput sequencing (MeDIP-seq) case-control study. Materials and MethodsOral biopsies from ten PVL patients and five healthy individuals were obtained and used to compare their epigenetic patterns. Network biology methods and integrative analyses of MeDIP-seq and RNAseq data were applied to investigate functional relations among differentially methylated genes (DMGs). The value of selected genes as malignant biomarkers was evaluated in a large cohort of oral squamous cell carcinoma (OSCC) patients from TCGA. ResultsA total of 4647 differentially methylated regions were found, with a prominent state of hypermethylation in PVL patients. At the gene level, differentially methylated regions (DMRs) covered 826 genes with distinct roles, including transcription factors and binding proteins with functions in cell adhesion, migration, proliferation, regulation of transcription, bone morphogenesis, and cell signalling. Network analysis revealed three major hubs, two of them collecting proteins related to the response of the patients to PVL and treatment and one hub collecting proteins related to PVL and cancer. The integrative analysis revealed 8 genes (ARTN, CD8A, GATA3, HOXD10, MYO7A, OSR2, PLCB1, and SPOCK2) significantly upregulated in PVL compared to control and 5 genes (ANKRD6, DLG2, GPX3, PITX2, and ZNF736) significantly downregulated. The status of de-regulation found for PVL patients was concordant with what was found for OSCC samples compared to normal adjacent tissue. ConclusionOur findings show the potential of methylation markers in PVL and suggest novel OSCC diagnostic biomarkers which may boost the development of novel epigenetic-based therapies.

Integrative analysis of chloroplast DNA methylation in a marine alga-Saccharina japonica.

We applied an integrative approach using multiple methods to verify cytosine methylation in the chloroplast DNA of the multicellular brown alga Saccharina japonica. Cytosine DNA methylation is a heritable process which plays important roles in regulating development throughout the life cycle of an organism. Although methylation of nuclear DNA has been studied extensively, little is known about the state and role of DNA methylation in chloroplast genomes, especially in marine algae. Here, we have applied an integrated approach encompassing whole-genome bisulfite sequencing, methylated DNA immunoprecipitation, gene co-expression networks and photophysiological analyses to provide evidence for the role of chloroplast DNA methylation in a marine alga, the multicellular brown alga Saccharina japonica. Although the overall methylation level was relatively low in the chloroplast genome of S. japonica, gametophytes exhibited higher methylation levels than sporophytes. Gene-specific bisulfite-cloning sequencing provided additional evidence for the methylation of key photosynthetic genes. Many of them were highly expressed in sporophytes whereas genes involved in transcription, translation and biosynthesis were strongly expressed in gametophytes. Nucleus-encoded photosynthesis genes were co-expressed with their chloroplast-encoded counterparts potentially contributing to the higher photosynthetic performance in sporophytes compared to gametophytes where these co-expression networks were less pronounced. A nucleus-encoded DNA methyltransferase of the DNMT2 family is assumed to be responsible for the methylation of the chloroplast genome because it is predicted to possess a plastid transit peptide.

AD7c-NTP Impairs Adult Striatal Neurogenesis by Affecting the Biological Function of MeCP2 in APP/PSl Transgenic Mouse Model of Alzheimer's Disease.

The processes by which neural stem cells (NSCs) and neural precursor cells (NPCs) transform into the characteristic lineages observed in Alzheimer's disease (AD) are poorly characterized. Understanding these processes is of critical importance due to the increased prevalence of AD and the lack of effective AD strategies. Here, we used immunohistochemistry and Western blot to find out if MeCP2 was phosphorylated at a specific amino acid residue, Serine 421 (S421), and activated in response to AD-induced damage in amyloid precursor protein (APP)/PSl transgenic mice, altering its nuclear to cytoplasmic shuttling. Epigenetic examinations combined with chromatin immunoprecipitation and methylated DNA immunoprecipitation revealed that the translocation of MeCP2 from the nucleus to cytoplasm led to the loss of lineage-specific gene promoters (such as Gfap, Nestin, and Dcx), decreased transcriptional repression, and the activation of gene expression. Immunofluorescence data demonstrated that neurogenic progenitors with high levels of active phosphorylated MeCP2 at S421 (MeCP2 pS421) possessed a high probability of development into doublecortin (DCX)-expressing cells. AD7c-NTP will control neurogenic progenitor regeneration through its effects on MeCP2 pS421, leading to altered lineage-specific gene expression. This adds to the growing list of biological effects of AD7c-NTP in the brain and highlights MeCP2 as relevant to the plasticity of neural cells in the AD mice striatum.

Maternal folic acid impacts DNA methylation profile in male rat offspring implicated in neurodevelopment and learning/memory abilities

BackgroundPericonceptional folic acid (FA) supplementation not only reduces the incidence of neural tube defects, but also improves cognitive performances in offspring. However, the genes or pathways that are epigenetically regulated by FA in neurodevelopment were rarely reported.MethodsTo elucidate the underlying mechanism, the effect of FA on the methylation profiles in brain tissue of male rat offspring was assessed by methylated DNA immunoprecipitation chip. Differentially methylated genes (DMGs) and gene network analysis were identified using DAVID and KEGG pathway analysis.ResultsCompared with the folate-normal diet group, 1939 DMGs were identified in the folate-deficient diet group, and 1498 DMGs were identified in the folate-supplemented diet group, among which 298 DMGs were overlapped. The pathways associated with neurodevelopment and learning/memory abilities were differentially methylated in response to maternal FA intake during pregnancy, and there were some identical and distinctive potential mechanisms under FA deficiency or FA-supplemented conditions.ConclusionsIn conclusion, genes and pathways associated with neurodevelopment and learning/memory abilities were differentially methylated in male rat offspring in response to maternal FA deficiency or supplementation during pregnancy.