DNA-Methylation Patterns in Trisomy 21 Using Cells from Monozygotic Twins

DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes.     

Genome-Wide DNA Methylation Patterns and Transcription Analysis in Sheep Muscle

DNA methylation plays a central role in regulating many aspects of growth and development in mammals through regulating gene expression. The development of next generation sequencing technologies have paved the way for genome-wide, high resolution analysis of DNA methylation landscapes using methodology known as reduced representation bisulfite sequencing (RRBS). While RRBS has proven to be effective in understanding DNA methylation landscapes in humans, mice, and rats, to date, few studies have utilised this powerful method for investigating DNA methylation in agricultural animals. Here we describe the utilisation of RRBS to investigate DNA methylation in sheep Longissimus dorsi muscles. RRBS analysis of ∼1% of the genome from Longissimus dorsi muscles provided data of suitably high precision and accuracy for DNA methylation analysis, at all levels of resolution from genome-wide to individual nucleotides. Combining RRBS data with mRNAseq data allowed the sheep Longissimus dorsi muscle methylome to be compared with methylomes from other species. While some species differences were identified, many similarities were observed between DNA methylation patterns in sheep and other more commonly studied species. The RRBS data presented here highlights the complexity of epigenetic regulation of genes. However, the similarities observed across species are promising, in that knowledge gained from epigenetic studies in human and mice may be applied, with caution, to agricultural species. The ability to accurately measure DNA methylation in agricultural animals will contribute an additional layer of information to the genetic analyses currently being used to maximise production gains in these species.     

Plasticity of DNA methylation in a nerve injury model of pain

The response of the peripheral nervous system (PNS) to injury may go together with alterations in epigenetics, a conjecture that has not been subjected to a comprehensive, genome-wide test. Using reduced representation bisulfite sequencing, we report widespread remodeling of DNA methylation in the rat dorsal root ganglion (DRG) occurring within 24 h of peripheral nerve ligation, a neuropathy model of allodynia. Significant (P < 10⁻⁴) cytosine hyper- and hypo-methylation was found at thousands of CpG sites. Remodeling occurred outside of CpG islands. Changes affected genes with known roles in the PNS, yet methylome remodeling also involved genes that were not linked to neuroplasticity by prior evidence. Consistent with emerging models relying on genome-wide methylation and RNA-seq analysis of promoter regions and gene bodies, variation of methylation was not tightly linked with variation of gene expression. Furthermore, approximately 44% of the dynamically changed CpGs were located outside of genes. We compared their positions with the intergenic, tissue-specific differentially methylated CpGs (tDMCs) of an independent experimental set consisting of liver, spleen, L4 control DRG, and muscle. Dynamic changes affected those intergenic CpGs that were different between tissues (P < 10⁻¹⁵) and almost never the invariant portion of the methylome (those CpGs that were identical across all tissues). Our findings—obtained in mixed tissue—show that peripheral nerve injury leads to methylome remodeling in the DRG. Future studies may address which of the cell types found in the DRG, such as specific groups of neurons or non-neuronal cells are affected by which aspect of the observed methylome remodeling.     

DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia

The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject’s colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands—in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)—were significantly hypermethylated in tumor vs. normal tissues (P < 0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-β1 network—the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated genes, as well as hypomethylated LINE-1 sequences, which may serve as potential biomarkers for CRC in African Americans. Our discovered biomarkers were intimately linked to the insulin/TGF-B1 pathway, further strengthening the association of diabetic disorders with colon oncogenic transformation.     

Methylation analysis revealed salicylic acid affects pearl millet defense through external cytosine DNA demethylation

The cytosine DNA methylation and demethylation have a role in regulating plant responses to the environment by affecting the promoter regions of most plant defense-related genes through the CpG islands or the CCGG motifs. Salicylic acid, a defense and signaling plant hormone, is seen playing crucial role in the variation of the methylome. In this study, the effects of salicylic acid and feeding of the millet headminer (Heliocheilus albipunctella de Joannis) on pearl millet DNA methylome changes were evaluated through MSAP epigenotyping during panicle development. The results showed that millet headminer feeding increased the level of genomic methylation while application of salicylic acid caused DNA demethylation occurring mostly at external cytosine and accompanied by a decrease of the number of larvae per panicle. This suggests that hemimethylation (external cytosine methylation) has key role in regulating defense responses and conferring tolerance to pearl millet through salicylic acid application.     

Novel Epigenetic Changes Unveiled by Monozygotic Twins Discordant for Smoking Habits

Exposure to cigarette smoking affects the epigenome and could increase the risk of developing diseases such as cancer and cardiovascular disorders. Changes in DNA methylation associated with smoking may help to identify molecular pathways that contribute to disease etiology. Previous studies are not completely concordant in the identification of differentially methylated regions in the DNA of smokers. We performed an epigenome-wide DNA methylation study in a group of monozygotic (MZ) twins discordant for smoking habits to determine the effect of smoking on DNA methylation. As MZ twins are considered genetically identical, this model allowed us to identify smoking-related DNA methylation changes independent from genetic components. We investigated the whole blood genome-wide DNA methylation profiles in 20 MZ twin pairs discordant for smoking habits by using the Illumina HumanMethylation450 BeadChip. We identified 22 CpG sites that were differentially methylated between smoker and non-smoker MZ twins by intra-pair analysis. We confirmed eight loci already described by other groups, located in AHRR, F2RL3, MYOG1 genes, at 2q37.1 and 6p21.33 regions, and also identified several new loci. Moreover, pathway analysis showed an enrichment of genes involved in GTPase regulatory activity. Our study confirmed the evidence of smoking-related DNA methylation changes, emphasizing that well-designed MZ twin models can aid the discovery of novel DNA methylation signals, even in a limited sample population.     

Variant Histone H2afv reprograms DNA methylation during early zebrafish development

The DNA methylome is re-patterned during discrete phases of vertebrate development. In zebrafish, there are 2 waves of global DNA demethylation and re-methylation: the first occurs before gastrulation when the parental methylome is changed to the zygotic pattern and the second occurs after formation of the embryonic body axis, during organ specification. The occupancy of the histone variant H2A.Z and regions of DNA methylation are generally anti-correlated, and it has been proposed that H2A.Z restricts the boundaries of highly methylated regions. While many studies have described the dynamics of methylome changes during early zebrafish development, the factors involved in establishing the DNA methylation landscape in zebrafish embryos have not been identified. We test the hypothesis that the zebrafish ortholog of H2A.Z (H2afv) restricts DNA methylation during development. We find that, in control embryos, bulk genome methylation decreases after gastrulation, with a nadir at the bud stage, and peaks during mid-somitogenesis; by 24 hours post -fertilization, total DNA methylation levels return to those detected in gastrula. Early zebrafish embryos depleted of H2afv have significantly more bulk DNA methylation during somitogenesis, suggesting that H2afv limits methylation during this stage of development. H2afv deficient embryos are small, with multisystemic abnormalities. Genetic interaction experiments demonstrate that these phenotypes are suppressed by depletion of DNA methyltransferase 1 (Dnmt1). This work demonstrates that H2afv is essential for global DNA methylation reprogramming during early vertebrate development and that embryonic development requires crosstalk between H2afv and Dnmt1.     

Repression of DERL3 via DNA methylation by Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is Epstein-Barr virus (EBV)-associated invasive malignancy. Increasing evidence indicates that epigenetic abnormalities, including DNA methylation, play important roles in the development of NPC. In particular, the EBV principal oncogene, latent membrane protein 1 (LMP1), is considered a key factor in inducing aberrant DNA methylation of several tumour suppressor genes in NPC, although the mechanism remains unclear. Herein, we comprehensively analysed the methylome data of Infinium BeadArray from 51 NPC and 52 normal nasopharyngeal tissues to identify LMP1-inducible methylation genes. Using hierarchical clustering analysis, we classified NPC into the high-methylation, low-methylation, and normal-like subgroups. We defined high-methylation genes as those that were methylated in the high-methylation subgroup only and common methylation genes as those that were methylated in both high- and low-methylation subgroups. Subsequently, we identified 715 LMP1-inducible methylation genes by observing the methylome data of the nasopharyngeal epithelial cell line with or without LMP1 expression. Because high-methylation genes were enriched with LMP1-inducible methylation genes, we extracted 95 high-methylation genes that overlapped with the LMP1-inducible methylation genes. Among them, we identified DERL3 as the most significantly methylated gene affected by LMP1 expression. DERL3 knockdown in cell lines resulted in significantly increased cell proliferation, migration, and invasion. Lower DERL3 expression was more frequently detected in the advanced T-stage NPC than in early T-stage NPC. These results indicate that DERL3 repression by DNA methylation contributes to NPC tumour progression.     

Genome-wide DNA methylation variability in adolescent monozygotic twins followed since birth

DNA methylation patterns are characterized by highly conserved developmental programs, but allow for divergent gene expression resulting from stochastic epigenetic drift or divergent environments. Genome-wide methylation studies in monozygotic (MZ) twins are providing insight into the extent of epigenetic variation that occurs, irrespective of genotype. However, little is known about the variability of DNA methylation patterns in adolescence, a period involving significant and rapid physical, emotional, social, and neurodevelopmental change. Here, we assessed genome-wide DNA methylation using the 450 K Illumina BeadChip in a sample of 37 MZ twin pairs followed longitudinally since birth to investigate: 1) the extent of variation in DNA methylation in identical genetic backgrounds in adolescence and; 2) whether these variations are randomly distributed or enriched in particular functional pathways. We also assessed stability of DNA methylation over 3–6 months to distinguish stable trait-like and variable state-like genes. A pathway analysis found high within-pair variability in genes associated with development, cellular mechanisms, tissue and cell morphology, and various disorders. Test-retest analyses performed in a sub-sample of 8 twin pairs demonstrated enrichment in gene pathways involved in organismal development, cellular growth and proliferation, cell signaling, and particular disorders. The variability found in functional gene pathways may plausibly underlie phenotypic differences in this adolescent MZ twin sample. Furthermore, we assessed stability of methylation over 3–6 months and found that some genes were stable while others were unstable, suggesting that the methylome remains dynamic in adolescence and that dynamic sites tend to be organized in certain gene pathways.     

Genome-wide mapping of DNA methylation in Nile Tilapia

Cytosine DNA methylation is crucial for gene regulation and maintenance of genome stability. However, the detailed nile tilapia methylome remains uncharacterized. In this study, we present the first high-resolution methylome of tilapia gonad generated using methylated DNA immunoprecipitation (MeDIP) and high-throughput sequencing. In the ovary, 265 and 56 methylation peaks were identified in the genebody and promoter region of 145 genes, respectively. In the testis, 293 and 80 methylation peaks were identified in the genebody and promoter region of 144 genes. Furthermore, 8 and 49 genes showed differentially higher and lower promoter-region methylation rates, respectively, in the ovary relative to those of the testis. Quantitative PCR results revealed that the expression level of fibroblast growth factor 16 (fgf16), sialidase-3-like, fibroblast growth factor 20, aromatase (cyp19a), estrogen receptor, and gonadotropin receptor II precursor were negatively correlated to their methylation levels in the ovary and testis. The methylated levels of cyp19a and fgf16 were validated by bisulfite sequencing PCR technology, and the results were consistent with the MeDIP results. Thus, apart from generating the first methylation map, this study produced a candidate gene repository that provides additional options to explore the relationship between DNA methylation and sex differentiation or maintenance.     

Inter-individual variability contrasts with regional homogeneity in the human brain DNA methylome

The possibility that alterations in DNA methylation are mechanistic drivers of development, aging and susceptibility to disease is widely acknowledged, but evidence remains patchy or inconclusive. Of particular interest in this regard is the brain, where it has been reported that DNA methylation impacts on neuronal activity, learning and memory, drug addiction and neurodegeneration. Until recently, however, little was known about the ‘landscape’ of the human brain methylome. Here we assay 1.9 million CpGs in each of 43 brain samples representing different individuals and brain regions. The cerebellum was a consistent outlier compared to all other regions, and showed over 16 000 differentially methylated regions (DMRs). Unexpectedly, the sequence characteristics of hypo- and hypermethylated domains in cerebellum were distinct. In contrast, very few DMRs distinguished regions of the cortex, limbic system and brain stem. Inter-individual DMRs were readily detectable in these regions. These results lead to the surprising conclusion that, with the exception of cerebellum, DNA methylation patterns are more homogeneous between different brain regions from the same individual, than they are for a single brain region between different individuals. This finding suggests that DNA sequence composition, not developmental status, is the principal determinant of the human brain DNA methylome.