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Assessing the preservation of cytosine methylation in ancient DNA from five prehistoric Native American populations
textCytosine methylation of CpG dinucleotides is an important epigenetic mark that regulates gene expression in humans. While methylation patterns in extant populations have been widely studied, few studies have attempted to analyze methylation in ancient DNA. Indeed, it was only recently shown that methyl groups can be preserved in ancient DNA. However, it is unknown how often methylation patterns can be recovered from ancient samples with preserved nuclear DNA. If they are frequently preserved, it may ultimately be possible to infer patterns of gene activity at the population level in ancient times. In this study, I assessed the preservation of cytosine methylation in ancient DNA from the remains of 30 prehistoric Native Americans from California, Illinois, Kentucky, and Mexico. These samples were previously shown to contain endogenous mitochondrial and nuclear DNA. I analyzed the cytosine methylation states of CpG-rich retrotransposons, which are epigenetically inactivated by cytosine methylation in humans. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite. Bisulfite products were pyrosequenced, and C-to-T conversions at potentially methylated CpG dinucleotides were quantified. I found that cytosine methylation is readily recoverable from human remains with preserved nuclear DNA from various localities over the time depth tested (~6000 years). This study presents the first direct evidence of cytosine methylation in ancient human remains, and suggests that it may be possible to analyze patterns of gene activity in ancient populations.Anthropolog
Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions
Background: Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome- wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material.
Methods: We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species.
Results: As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice.
Conclusions: Plant-RRBS offers high-throughput and broad, genome- dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations
Streptomyces coelicolor: DNA methylation and differentiation
DNA cytosine methylation is an epigenetic modification regulating many biological processes in eukaryotes, including chromatin organization, genome maintenance and gene expression. The role of DNA cytosine methylation in prokaryotes has not been deeply investigated. In Escherichia coli it was recently demonstrated that cytosine methylation regulates gene expression during stationary phase [1] and that an induced state of cytosine hypermethylation leads to chromosomal DNA cleavage and cell death [2]. Streptomyces coelicolor is a mycelial soil microorganism, which exhibits a complex life cycle that includes three different cell types: unigenomic spores, a compartmentalized mycelium (MI) and a multinucleated mycelium (substrate and aerial mycelium, MII) [3]. The importance of DNA methylation was already described in Streptomycetes [4], but its biological role remains unknown.
The main objectives of this study are to analyze cytosine methylation pattern of Streptomyces coelicolor M145 during growth in liquid and on solid media, and to investigate the relationship between DNA cytosine methylation and morphological/physiological differentiation.
Cytosine methylation of total genomic DNA extracted from different developmental stages was investigated by dot-blot experiments using antibody anti-5-methylcytosine. Cytosine methylome was analyzed by BiSulphite sequencing. The biological effect of cytosine methylation was studied adding 5-aza-2\u2019-deoxycytidine (aza-dC), a hypomethylating agent, to the cultures.
Dot blot analysis revealed that the level of cytosine methylation changes during development (MI, MII and spores). Specifically, DNA methylation is higher at the MI stage than in the MII or spores. BiSulphite sequencing revealed that 30% of S. coelicolor genes contained a methylated motif in their upstream regions. Genes harbouring these motifs included genes related to differentiation (aerial mycelium formation and sporulation), genes involved in DNA repair/replication/condensation, as well as genes encoding proteins with unknown functions. Phenotypic analyses of cultures treated with aza-dC demonstrated that DNA methylation influences germination, aerial mycelium formation and sporulation on solid medium and antibiotic production both, on solid and in liquid medium.
Overall, our preliminary results suggest a role for DNA cytosine methylation in morphological and physiological differentiation of S. coelicolor. Further experiments are ongoing to demonstrate the molecular mechanisms and pathways behind the observed phenotypes
Spatial and Functional Relationships Among Pol V-Associated Loci, Pol IV-Dependent siRNAs, and Cytosine Methylation in the \u3cem\u3eArabidopsis\u3c/em\u3e Epigenome
Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%–60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites
Internuclear gene silencing in Phytophthora infestans is established through chromatin remodelling
In the plant pathogen Phytophthora infestans, nuclear integration of inf1 transgenic DNA sequences results in internuclear gene silencing of inf1. Although silencing is regulated at the transcriptional level, it also affects transcription from other nuclei within heterokaryotic cells of the mycelium. Here we report experiments exploring the mechanism of internuclear gene silencing in P. infestans. The DNA methylation inhibitor 5-azacytidine induced reversion of the inf1-silenced state. Also, the histone deacetylase inhibitor trichostatin-A was able to reverse inf1 silencing. inf1-expression levels returned to the silenced state when the inhibitors were removed except in non-transgenic inf1-silenced strains that were generated via internuclear gene silencing, where inf1 expression was restored permanently. Therefore, inf1-transgenic sequences are required to maintain the silenced state. Prolonged culture of non-transgenic inf1-silenced strains resulted in gradual reactivation of inf1 gene expression. Nuclease digestion of inf1-silenced and non-silenced nuclei showed that inf1 sequences in silenced nuclei were less rapidly degraded than non-silenced inf1 sequences. Bisulfite sequencing of the endogenous inf1 locus did not result in detection of any cytosine methylation. Our findings suggest that the inf1-silenced state is based on chromatin remodelling
Cytosine Methylation: Remaining Faithful
SummaryDNA methyltransferase-1 (DNMT1) has a higher specific activity on hemimethylated DNA than on unmethylated DNA, but this preference is too small to explain the faithful mitotic inheritance of genomic methylation patterns. New genetic studies in plants and mammals have identified a novel factor that increases the fidelity of maintenance methylation
Optimized design and data analysis of tag-based cytosine methylation assays
Genome-wide, tag-based cytosine methylation analysis is optimized
Effects of vitamin D status on global DNA mthylation
Thesis (M.A.)--Boston UniversityProblem: Vitamin D deficiency has been linked to increased risks of hypertension, multiple sclerosis, cardiovascular diseases, and various forms of cancer. Studies have investigated the role of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and vitamin D status on epigenetics of specific genes. However, there has not been any investigation on the direct relationship between vitamin D status and global DNA methylation. This study attempted to examine that relationship.
Method: Subjects were recruited with flyers around the Boston University Medical (BUMC) campus. Subjects’ blood was drawn at the General Clinical Research Unit (GCRU). Serum was isolated from whole blood by centrifugation. Buffy coat was isolated from whole blood with Accuspin System Histopaque-1077 tubes from Sigma- Aldrich. DNA was isolated from buffy coat with AllPrep DNA/RNA Mini prep from Qiagen. Subject’s serum 25(OH)D concentration was measured with automated IDS-iSYS system, and their percent DNA methylation was measured by high performance liquid chromatography (HPLC).
Results: The mean serum 25(OH)D concentration of five subjects was 32.9ng/mL. The mean percent cytosine methylation was 4.7% with standard deviation of 0.2%. One subject was vitamin D deficient, and the remaining four subjects were vitamin D sufficient. The vitamin D deficient subject had a percent cytosine methylation of 4.3%. The mean percent cytosine methylation of vitamin D sufficient subjects was 4.8% with standard deviation of 0.1%.
Conclusions: The percent cytosine methylation of the vitamin D deficient subject was lower than the mean percent cytosine methylation of four vitamin D sufficient subjects. This data suggests that there is a possible correlation between vitamin D status and global DNA methylation. However, a larger sample size is needed to make any conclusions for the study
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