Genome-wide DNA methylation changes in skeletal muscle between young and middle-aged pigs

Background

Age-related physiological, biochemical and functional changes in mammalian skeletal muscle have been shown to begin at the mid-point of the lifespan. However, the underlying changes in DNA methylation that occur during this turning point of the muscle aging process have not been clarified. To explore age-related genomic methylation changes in skeletal muscle, we employed young (0.5 years old) and middle-aged (7 years old) pigs as models to survey genome-wide DNA methylation in the longissimus dorsi muscle using a methylated DNA immunoprecipitation sequencing approach.

Results

We observed a tendency toward a global loss of DNA methylation in the gene-body region of the skeletal muscle of the middle-aged pigs compared with the young group. We determined the genome-wide gene expression pattern in the longissimus dorsi muscle using microarray analysis and performed a correlation analysis using DMR (differentially methylated region)-mRNA pairs, and we found a significant negative correlation between the changes in methylation levels within gene bodies and gene expression. Furthermore, we identified numerous genes that show age-related methylation changes that are potentially involved in the aging process. The methylation status of these genes was confirmed using bisulfite sequencing PCR. The genes that exhibited a hypomethylated gene body in middle-aged pigs were over-represented in various proteolysis and protein catabolic processes, suggesting an important role for these genes in age-related muscle atrophy. In addition, genes associated with tumorigenesis exhibited aged-related differences in methylation and expression levels, suggesting an increased risk of disease associated with increased age.

Conclusions

This study provides a comprehensive analysis of genome-wide DNA methylation patterns in aging pig skeletal muscle. Our findings will serve as a valuable resource in aging studies, promoting the pig as a model organism for human aging research and accelerating the development of comparative animal models in aging research.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-653) contains supplementary material, which is available to authorized users.     

Prenatal stress decreases Bdnf expression and increases methylation of Bdnf exon IV in rats

There is ample evidence that exposure to stress during gestation increases the risk of the offspring to develop mood disorders. Brain-derived neurotrophic factor (Bdnf) plays a critical role during neuronal development and is therefore a prime candidate to modulate neuronal signaling in adult offspring of rat dams that were stressed during gestation. In the current study, we tested the hypothesis that alterations in Bdnf expression in prenatally stressed (PNS) offspring are mediated by changes in DNA methylation in exons IV and VI of the Bdnf gene. We observed decreased Bdnf expression in the amygdala and hippocampus of prenatally stressed rats both at weaning and in adulthood. This decrease in Bdnf expression was accompanied by increased DNA methylation in Bdnf exon IV in the amygdala and hippocampus, suggesting that PNS-induced reduction in Bdnf expression may, at least in part, be mediated by increased DNA methylation of Bdnf exon IV. Expression of DNA methyltransferases (Dnmt) 1 and 3a was increased in PNS rats in the amygdala and hippocampus. Our data suggest that PNS induces decreases in Bdnf expression that may at least in part be mediated by increased DNA methylation of Bdnf exon IV.     

A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR

Assessment of DNA methylation has become a critical factor for the identification, development and application of methylation based biomarkers. Here we describe a systematic comparison of a quantitative high-resolution mass spectrometry-based approach (MassARRAY), pyrosequencing and the broadly used methylation-specific PCR (MSP) technique analyzing clinically relevant epigenetically silenced genes in acute myeloid leukemia (AML). By MassARRAY and pyrosequencing, we identified significant DNA methylation differences at the ID4 gene promoter and in the 5′ region of members of the SFRP gene family in 62 AML patients compared with healthy controls. We found a good correlation between data obtained by MassARRAY and pyrosequencing (correlation coefficient R² = 0.88). MSP-based assessment of the identical samples showed less pronounced differences between AML patients and controls. By direct comparison of MSP-derived and MassARRAY-based methylation data as well as pyrosequencing, we could determine overestimation of DNA methylation data by MSP. We found sequence-context dependent highly variable cut-off values of quantitative DNA methylation values serving as discriminator for the two MSP methylation categories. Moreover, good agreements between quantitative methods and MSP could not be achieved for all investigated loci. Significant correlation of the quantitative assessment but not of MSP-derived methylation data with clinically important characteristics in our patient cohort demonstrated clinical relevance of quantitative DNA methylation assessment. Taken together, while MSP is still the most commonly applied technique for DNA methylation assessment, our data highlight advantages of quantitative approaches for precise characterization and reliable biomarker use of aberrant DNA methylation in primary patient samples, particularly.     

Drugging the methylome: DNA methylation and memory

Over the past decade, since epigenetic mechanisms were first implicated in memory formation and synaptic plasticity, dynamic DNA methylation reactions have been identified as integral to long-term memory formation, maintenance, and recall. This review incorporates various new findings that DNA methylation mechanisms are important regulators of non-Hebbian plasticity mechanisms, suggesting that these epigenetic mechanisms are a fundamental link between synaptic plasticity and metaplasticity. Because the field of neuroepigenetics is so young and the biochemical tools necessary to probe gene-specific questions are just now being developed and used, this review also speculates about the direction and potential of therapeutics that target epigenetic mechanisms in the central nervous system and the unique pharmacokinetic and pharmacodynamic properties that epigenetic therapies may possess. Mapping the dynamics of the epigenome in response to experiential learning, even a single epigenetic mark in isolation, remains a significant technical and bioinformatic hurdle facing the field, but will be necessary to identify changes to the methylome that govern memory-associated gene expression and effectively drug the epigenome.     

Non-referenced genome assembly from epigenomic short-read data

Current computational methods used to analyze changes in DNA methylation and chromatin modification rely on sequenced genomes. Here we describe a pipeline for the detection of these changes from short-read sequence data that does not require a reference genome. Open source software packages were used for sequence assembly, alignment, and measurement of differential enrichment. The method was evaluated by comparing results with reference-based results showing a strong correlation between chromatin modification and gene expression. We then used our de novo sequence assembly to build the DNA methylation profile for the non-referenced Psammomys obesus genome. The pipeline described uses open source software for fast annotation and visualization of unreferenced genomic regions from short-read data.     

A reassessment of semiquantitative analytical procedures for DNA methylation: comparison of bisulfite- and HpaII polymerase-chain-reaction-based methods

Two techniques in particular are used to study site-specific DNA methylation: genomic sequencing after bisulfite modification and polymerase chain reaction after digestion by a methylation-sensitive endonuclease (usually HpaII). Only the former methodology assesses the methylation status of all the cytosine residues in the DNA sequence, but it is so complex and time consuming that the latter procedure, though limited to the restriction sites recognized by the endonuclease(s) used, is often preferred at least for a first analysis. In this work we investigate differences between these two techniques in the assessment of DNA methylation and offer some suggestions on how to avoid uncorrected results. Although there is substantial accordance in the results obtained using these different techniques, we observed a general overestimate for methylation levels above 30% and a general underestimate for methylation levels below this value using the HpaII/PCR technique in the study of methylation of the 5'-flanking region of the mouse myogenin gene in cultured muscle cells and mouse tissues.     

Constitutional promoter methylation and risk of familial melanoma

Constitutional epigenetic changes detected in blood or non-disease involving tissues have been associated with disease susceptibility. We measured promoter methylation of CDKN2A (p16 and p14ARF) and 13 melanoma-related genes using bisulfite pyrosequencing of blood DNA from 114 cases and 122 controls in 64 melanoma-prone families (26 segregating CDKN2A germline mutations). We also obtained gene expression data for these genes using microarrays from the same blood samples. We observed that CDKN2A epimutation is rare in melanoma families, and therefore is unlikely to cause major susceptibility in families without CDKN2A mutations. Although methylation levels for most gene promoters were very low (<5%), we observed a significantly reduced promoter methylation (odds ratio = 0.63, 95% confidence interval = 0.50, 0.80, P < 0.001) and increased expression (fold change = 1.27, P = 0.048) for TNFRSF10C in melanoma cases. Future research in large prospective studies using both normal and melanoma tissues is required to assess the significance of TNFRSF10C methylation and expression changes in melanoma susceptibility.     

Demethylation of CpG islands in the 5' upstream regions mediates the expression of the human testis-specific gene MAGEB16 and its mouse homolog Mageb16

Tissue-specific gene expression is regulated by epigenetic modification involving trans-acting factors. Here, we identified that the human MAGEB16 gene and its mouse homolog, Mageb16, are only expressed in the testis. To investigate the mechanism governing their expression, the promoter methylation status of these genes was examined in different samples. Two CpG islands (CGIs) in the 5'' upstream region of MAGEB16 were highly demethylated in human testes, whereas they were methylated in cells without MAGEB16 expression. Similarly, the CGI in Mageb16 was hypomethylated in mouse testes but hypermethylated in other tissues and cells without Mageb16 expression. Additionally, the expression of these genes could be activated by treatment with the demethylation agent 5''-aza-2''-deoxycytidine (5''-aza-CdR). Luciferase assays revealed that both gene promoter activities were inhibited by methylation of the CGI regions. Therefore, we propose that the testis-specific expression of MAGEB16 and Mageb16 is regulated by the methylation status of their promoter regions. [BMB Reports 2014; 47(2): 86-91]     

Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase

Cytosine methylation of genomic DNA controls gene expression and maintains genome stability. How a specific DNA sequence is targeted for methylation by a methyltransferase is largely unknown. Here, we show that histone H3 tails lacking lysine 4 (K4) methylation function as an allosteric activator for methyltransferase Dnmt3a by binding to its plant homeodomain (PHD). In vitro, histone H3 peptides stimulated the methylation activity of Dnmt3a up to 8-fold, in a manner reversely correlated with the level of K4 methylation. The biological significance of allosteric regulation was manifested by molecular modeling and identification of key residues in both the PHD and the catalytic domain of Dnmt3a whose mutations impaired the stimulation of methylation activity by H3 peptides but not the binding of H3 peptides. Significantly, these mutant Dnmt3a proteins were almost inactive in DNA methylation when expressed in mouse embryonic stem cells while their recruitment to genomic targets was unaltered. We therefore propose a two-step mechanism for de novo DNA methylation - first recruitment of the methyltransferase probably assisted by a chromatin- or DNA-binding factor, and then allosteric activation depending on the interaction between Dnmt3a and the histone tails - the latter might serve as a checkpoint for the methylation activity.     

γ-Glutamyl hydrolase modulation significantly influences global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells

γ-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0444-0) contains supplementary material, which is available to authorized users.     

Spatiotemporal expression of Prdm genes during Xenopus development

Epigenetic regulation is known to be important in embryonic development, cell differentiation and regulation of cancer cells. Molecular mechanisms of epigenetic modification have DNA methylation and histone tail modification such as acetylation, phosphorylation and ubiquitination. Until now, many kinds of enzymes that modify histone tail with various functional groups have been reported and regulate the epigenetic state of genes. Among them, Prdm genes were identified as histone methyltransferase. Prdm genes are characterized by an N-terminal PR/SET domain and C-terminal some zinc finger domains and therefore they are considered to have both DNA-binding ability and methylation activity. Among vertebrate, fifteen members are estimated to belong to Prdm genes family. Even though Prdm genes are thought to play important roles for cell fate determination and cell differentiation, there is an incomplete understanding of their expression and functions in early development. Here, we report that Prdm genes exhibit dynamic expression pattern in Xenopus embryogenesis. By whole mount in situ hybridization analysis, we show that Prdm genes are expressed in spatially localized manners in embryo and all of Prdm genes are expressed in neural cells in developing central nervous systems. Our study suggests that Prdm genes may be new candidates to function in neural cell differentiation.     

Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.