Sexually divergent DNA methylation patterns with hippocampal aging

DNA methylation is a central regulator of genome function, and altered methylation patterns are indicative of biological aging and mortality. Age‐related cellular, biochemical, and molecular changes in the hippocampus lead to cognitive impairments and greater vulnerability to neurodegenerative disease that varies between the sexes. The role of hippocampal epigenomic changes with aging in these processes is unknown as no genome‐wide analyses of age‐related methylation changes have considered the factor of sex in a controlled animal model. High‐depth, genome‐wide bisulfite sequencing of young (3 month) and old (24 month) male and female mouse hippocampus revealed that while total genomic methylation amounts did not change with aging, specific sites in CG and non‐CG (CH) contexts demonstrated age‐related increases or decreases in methylation that were predominantly sexually divergent. Differential methylation with age for both CG and CH sites was enriched in intergenic and intronic regions and under‐represented in promoters, CG islands, and specific enhancer regions in both sexes, suggesting that certain genomic elements are especially labile with aging, even if the exact genomic loci altered are predominantly sex‐specific. Lifelong sex differences in autosomal methylation at CG and CH sites were also observed. The lack of genome‐wide hypomethylation, sexually divergent aging response, and autosomal sex differences at CG sites was confirmed in human data. These data reveal sex as a previously unappreciated central factor of hippocampal epigenomic changes with aging. In total, these data demonstrate an intricate regulation of DNA methylation with aging by sex, cytosine context, genomic location, and methylation level.     

Enforcement of a lifespan‐sustaining distribution of Sir2 between telomeres,mating‐type loci,and rDNA repeats by Rif1

Telomere dysfunction is linked with genome instability and premature aging. Roles for sirtuin proteins at telomeres are thought to promote lifespan in yeast and mammals. However, replicative lifespan of the budding yeast Saccharomyces cerevisiae shortens upon deletion of Rif1, a protein that limits the recruitment of the sirtuin histone deacetylase Sir2 to telomeres. Here we show that Rif1 maintains replicative lifespan by ultimately stabilizing another age‐related chromosomal domain harboring the ribosomal DNA (rDNA) repeats. Deletion of Rif1 increases Sir2 localization to telomeres and the silent mating‐type loci, while releasing a pool of the histone deacetylase from the intergenic spacer 1 (IGS1) of rDNA. This is accompanied by a disruption of IGS1 silent chromatin assembly and increases in aberrant recombination within rDNA repeats. Lifespan defects linked with Rif1 deletion are abolished if rDNA repeats are forcibly stabilized via deletion of the replication fork‐blocking protein Fob1. In addition, Sir2 overexpression prevents Rif1 deletion from disrupting Sir2 at IGS1 and shortening lifespan. Moreover, subjecting cells lacking Rif1 to caloric restriction increases IGS1 histone deacetylation and lifespan, while uncovering novel genetic interactions between RIF1 and SIR2. Our data indicate that Rif1 maintains lifespan‐sustaining levels of Sir2 at rDNA by preventing excessive recruitment of the histone deacetylase to telomeric and silent mating‐type loci. As sirtuin histone deacetylases, such as Sir2 or mammalian SIRT6, each operate at multiple age‐related loci, we propose that factors limiting the localization of sirtuins to certain age‐related loci can promote lifespan‐sustaining roles of these sirtuins elsewhere in the genome.     

Epigenetic heterogeneity of developmentally important genes in human sperm: Implications for assisted reproduction outcome

The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de)methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated GTL2 gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% GTL2 epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1–14% GTL2 epimutations.     

The CHH motif in sugar beet satellite DNA: a modulator for cytosine methylation

Methylation of DNA is important for the epigenetic silencing of repetitive DNA in plant genomes. Knowledge about the cytosine methylation status of satellite DNAs, a major class of repetitive DNA, is scarce. One reason for this is that arrays of tandemly arranged sequences are usually collapsed in next‐generation sequencing assemblies. We applied strategies to overcome this limitation and quantified the level of cytosine methylation and its pattern in three satellite families of sugar beet (Beta vulgaris) which differ in their abundance, chromosomal localization and monomer size. We visualized methylation levels along pachytene chromosomes with respect to small satellite loci at maximum resolution using chromosome‐wide fluorescent in situ hybridization complemented with immunostaining and super‐resolution microscopy. Only reduced methylation of many satellite arrays was obtained. To investigate methylation at the nucleotide level we performed bisulfite sequencing of 1569 satellite sequences. We found that the level of methylation of cytosine strongly depends on the sequence context: cytosines in the CHH motif show lower methylation (44–52%), while CG and CHG motifs are more strongly methylated. This affects the overall methylation of satellite sequences because CHH occurs frequently while CG and CHG are rare or even absent in the satellite arrays investigated. Evidently, CHH is the major target for modulation of the cytosine methylation level of adjacent monomers within individual arrays and contributes to their epigenetic function. This strongly indicates that asymmetric cytosine methylation plays a role in the epigenetic modification of satellite repeats in plant genomes.     

Epigenetic heterogeneity of developmentally important genes in human sperm: Implications for assisted reproduction outcome

The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de)methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated GTL2 gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% GTL2 epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1–14% GTL2 epimutations.     

Linkage disequilibrium analysis of allelic heterogeneity in DNA methylation

Heterogeneity of DNA methylation status among alleles is observed in various cell types and is involved in epigenetic gene regulation and cancer biology. However, the individual methylation profile within each allele has not yet been examined at the whole-genome level. In the present study, we applied linkage disequilibrium analysis to the DNA methylation data obtained from whole-genome bisulfite sequencing studies in mouse germline and other types of cells. We found that the methylation status of 2 consecutive CpG sites showed deviation from equilibrium frequency toward concordant linkage (both methylated or both unmethylated) in germline cells. In the imprinting loci where methylation of constituent alleles is known, our analysis detected the deviation toward the concordant linkage as expected. In addition, we applied this analysis to the transitional zone between methylated and unmethylated regions and to the cells undergoing epigenetic reprogramming. In both cases, deviation to the concordant-linked alleles was conspicuous, indicating that the methylation pattern is not random but rather concordant within each allele. These results will provide the key to understanding the mechanism underlying allelic heterogeneity.     

A novel imprinted transgene located near a repetitive element that exhibits allelic imbalance in DNA methylation during early development

A mouse line carrying a lacZ transgene driven by the human EEF1A1/EF1alpha promoter was established. Although the promoter is known to show ubiquitous activity, only paternal transgene alleles were expressed, resulting in a transgene imprinting. At mid‐gestation, the promoter sequence was differentially methylated, hypomethylated for paternal and hypermethylated for maternal alleles. In germline, the promoter was a typical differentially methylated region. After fertilization, however, both alleles were hypermethylated. Thus, the differential methylation of the promoter required for transgene imprinting was re‐established during later embryonic development independently of the germline differential methylation. Furthermore, also a retroelement promoter closely‐flanking imprinted transgene and its wild type counterpart displayed similar differential methylation during early development. The retroelement promoter was methylated differentially also in germline, but in an opposite pattern to the embryonic differential methylation. These results suggest that there might be an unknown epigenetic regulation inducing transgene imprinting independently of DNA methylation in the transgene insertion site. Then, besides CpG dinucleotides, non‐CpG cytosines of the retroelement promoter were highly methylated especially in the transgene‐active mid‐gestational embryos, suggesting that an unusual epigenetic regulation might protect the active transgene against de novo methylation occurring generally in mid‐gestational embryo.     

Short‐term calorie restriction ameliorates genomewide,age‐related alterations in DNA methylation

DNA methylation plays major roles in many biological processes, including aging, carcinogenesis, and development. Analyses of DNA methylation using next‐generation sequencing offer a new way to profile and compare methylomes across the genome in the context of aging. We explored genomewide DNA methylation and the effects of short‐term calorie restriction (CR) on the methylome of aged rat kidney. Whole‐genome methylation of kidney in young (6 months old), old (25 months old), and OCR (old with 4‐week, short‐term CR) rats was analyzed by methylated DNA immunoprecipitation and next‐generation sequencing (MeDIP‐Seq). CpG islands and repetitive regions were hypomethylated, but 5′‐UTR, exon, and 3′‐UTR hypermethylated in old and OCR rats. The methylation in the promoter and intron regions was decreased in old rats, but increased in OCR rats. Pathway enrichment analysis showed that the hypermethylated promoters in old rats were associated with degenerative phenotypes such as cancer and diabetes. The hypomethylated promoters in old rats related significantly to the chemokine signaling pathway. However, the pathways significantly enriched in old rats were not observed from the differentially methylated promoters in OCR rats. Thus, these findings suggest that short‐term CR could partially ameliorate age‐related methylation changes in promoters in old rats. From the epigenomic data, we propose that the hypermethylation found in the promoter regions of disease‐related genes during aging may indicate increases in susceptibility to age‐related diseases. Therefore, the CR‐induced epigenetic changes that ameliorate age‐dependent aberrant methylation may be important to CR's health‐ and life‐prolonging effects.     

The use of DNA methylation clock in aging research

One of the key characteristics of aging is a progressive loss of physiological integrity, which weakens bodily functions and increases the risk of death. A robust biomarker is important for the assessment of biological age, the rate of aging, and a person''s health status. DNA methylation clocks, novel biomarkers of aging, are composed of a group of cytosine-phosphate-guanine dinucleotides, the DNA methylation status of which can be used to accurately measure subjective age. These clocks are considered accurate biomarkers of chronological age for humans and other vertebrates. Numerous studies have demonstrated these clocks to quantify the rate of biological aging and the effects of longevity and anti-aging interventions. In this review, we describe the purpose and use of DNA methylation clocks in aging research.     

A simple and universal ligation mediated fusion of genes based on hetero-staggered PCR for generating immunodominant chimeric proteins

A potential relationship between transposon-derived repeats (TDR) and human germline methylation is of biological importance since many genes are flanked by TDR and methylation could affect the expression of nearby genes. Furthermore, DNA methylation has been suggested as a global defense mechanism against genome instability threatened by TDR. We studied the correlation between the density of HapMap methyl-associated SNPs (mSNPs), a marker of germline methylation, and proportion of TDR.After correcting for confounding variables, we found a negative correlation between proportion of Alu repeats and mSNP density for 125-1000 kb windows. Similar results were found for the most active subgroup of repeats. In contrast, a negative correlation between proportion of L1 repeats and mSNP density was found only in the larger 1000 kb windows.Using methylation data on germ cells (sperm) from the Human Epigenome Project, we found a lower proportion of Alu repeats adjacent (3-15 kb) to hypermethylated amplicons. On the contrary, there was a higher proportion of L1 repeats in the 3-5 kb of sequence flanking hypermethylated amplicons but not in the 10-15 kb flanks.Our data indicate a differential response to the major repeat families and that DNA methylation is unlikely to be a uniform global defense system against all TDR. It appears to play a role for the L1 subgroup, with sequences adjacent to L1 repeats methylated in response to their proximity. In contrast, sequences adjacent to Alu repeats appear to be hypomethylated, arguing against a role of methylation in germline defense against those elements.     

Influences of array size and homogeneity on minisatellite mutation.

Unstable minisatellites display high frequencies of spontaneous gain and loss of repeats in the human germline. Most length changes arise through complex recombination events including intra-allelic duplications/deletions and inter-allelic transfers of repeats. Definition of the factors modulating instability requires both measurement of mutation rate and detailed analysis of mutant structures at the level of individual alleles. We have measured mutation rates in sperm for a wide range of alleles of the highly unstable human minisatellite CEB1. Instability varies by three orders of magnitude between alleles and increases steadily with the size of the tandem array. Structural analysis of mutant molecules derived from six alleles revealed that it is the rate of intra-allelic rearrangements which increases with array size and that intra-allelic duplication events tend to cluster within homogeneous segments of alleles; both phenomena resemble features of trinucleotide repeat instability. In contrast, inter-allelic transfers occur at a fairly constant rate, irrespective of array length, and show a mild polarity towards one end of the minisatellite, suggesting the possible influence of flanking DNA on these conversion-like events.     

Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.     

Satellite 2 methylation patterns in normal and ICF syndrome cells and association of hypomethylation with advanced replication

Mutation in the DNMT3B DNA methyltransferase gene is a common cause of ICF (immunodeficiency, centromeric heterochromatin, facial anomalies) immunodeficiency syndrome and leads to hypomethylation of satellites 2 and 3 in pericentric heterochromatin. This hypomethylation is associated with centromeric decondensation and chromosomal rearrangements, suggesting that these satellite repeats have an important structural role. In addition, the satellite regions may have functional roles in modifying gene expression. The extent of satellite hypomethylation in ICF cells is unknown because methylation status has only been determined with restriction enzymes that cut infrequently at these loci. We have therefore developed a bisulfite conversion-based method to determine the detailed cytosine methylation patterns at satellite 2 sequences in a quantitative manner for normal and ICF samples. From our sequence analysis of unmodified DNA, the internal repeat region analyzed for methylation contains an average of 17 CpG sites. The average level of methylation in normal lymphoblasts and fibroblasts is 69% compared with 20% in such cells from ICF patients with DNMT3B mutations and 29% in normal sperm. Although the mean satellite 2 methylation values for these groups do not overlap, there is considerable overlap at the level of individual DNA strands. Our analysis has also revealed a pattern of methylation specificity, suggesting that some CpGs in the repeat are more prone to methylation than other sites. Variation in satellite 2 methylation among lymphoblasts from different ICF patients has prompted us to determine the frequency of cytogenetic abnormalities in these cells. Although our data suggest that some degree of hypomethylation is necessary for pericentromeric decondensation, factors other than DNA methylation appear to play a major role in this phenomenon. Another such factor may be altered replication timing because we have discovered that the hypomethylation of satellite 2 in ICF cultures is associated with advanced replication.