Regulation and function of mammalian DNA methylation patterns: a genomic perspective

Mammalian DNA methyltransferases (DNMTs) establish and maintain genomic DNA methylation patterns that are required for proper epigenetic regulation of gene expression and maintenance of genome stability during normal development. Aberrant DNA methylation patterns are implicated in a variety of pathological conditions including cancer and neurological disorders. Rapid advances in genomic technologies have allowed the generation of high resolution whole-genome views of DNA methylation and DNA methyltransferase occupancy in pluripotent stem cells and differentiated somatic cells. Furthermore, recent identification of oxidation derivatives of cytosine methylation in mammalian DNA raises the possibility that DNA methylation patterns are more dynamic than previously anticipated. Here, we review the recent progress in our understanding of the genomic function and regulatory mechanisms of mammalian DNA methylation.     

Intergenerational genomic DNA methylation patterns in mouse hybrid strains

Background

DNA methylation is a contributing factor to both rare and common human diseases, and plays a major role in development and gene silencing. While the variation of DNA methylation among individuals has been partially characterized, the degree to which methylation patterns are preserved across generations is still poorly understood. To determine the extent of methylation differences between two generations of mice we examined DNA methylation patterns in the livers of eight parental and F1 mice from C57BL/6J and DBA/2J mouse strains using bisulfite sequencing.

Results

We find a large proportion of reproducible methylation differences between C57BL/6J and DBA/2J chromosomes in CpGs, which are highly heritable between parent and F1 mice. We also find sex differences in methylation levels in 396 genes, and 11% of these are differentially expressed between females and males. Using a recently developed approach to identify allelically methylated regions independently of genotypic differences, we identify 112 novel putative imprinted genes and microRNAs, and validate imprinting at the RNA level in 10 of these genes.

Conclusions

The majority of DNA methylation differences among individuals are associated with genetic differences, and a much smaller proportion of these epigenetic differences are due to sex, imprinting or stochastic intergenerational effects. Epigenetic differences can be a determining factor in heritable traits and should be considered in association studies for molecular and clinical traits, as we observed that methylation differences in the mouse model are highly heritable and can have functional consequences on molecular traits such as gene expression.     

Covalent genomic DNA modification patterns revealed by denaturing gradient gel blots

Several approaches are used to survey genomic DNA methylation patterns, including Southern blot, PCR, and microarray strategies. All of these methods are based on the use of methylation-sensitive isoschizomer restriction enzyme pairs and/or sodium bisulfite treatment of genomic DNA. They have many limitations, including PCR bias, lack of comprehensive assessment of methylated sites, labor-intensive protocols, and/or the need for expensive equipment. Since the presence of 5-methylcytosine alters the melting properties of DNA molecules, denaturing gradient gel blots (DGG blots), a gene scanning technique which detects differences in DNA fragments based on differential melting behavior, were used to examine genomic modification patterns in normal tissues. Variations in melting behavior, observed as restriction fragment melting polymorphisms (RFMPs), were detected in various tissues from single individuals in all human and mouse genes tested, suggesting the presence of widespread differential cell type-specific DNA modification. Additional DGG blot experiments comparing genomic DNA to unmethylated cloned DNA suggested that the melting variants were most likely caused by DNA methylation differences. The results suggest that the use of DGG blots can provide a comprehensive and rapid method for comparing complex in vivo DNA modification patterns in normal adult somatic cells.     

Decoding the genome beyond sequencing: the new phase of genomic research

While our understanding of gene-based biology has greatly improved, it is clear that the function of the genome and most diseases cannot be fully explained by genes and other regulatory elements. Genes and the genome represent distinct levels of genetic organization with their own coding systems; Genes code parts like protein and RNA, but the genome codes the structure of genetic networks, which are defined by the whole set of genes, chromosomes and their topological interactions within a cell. Accordingly, the genetic code of DNA offers limited understanding of genome functions. In this perspective, we introduce the genome theory which calls for the departure of gene-centric genomic research. To make this transition for the next phase of genomic research, it is essential to acknowledge the importance of new genome-based biological concepts and to establish new technology platforms to decode the genome beyond sequencing.     

Plasmid profiles and genomic DNA restriction endonuclease patterns of 30 independent Staphylococcus lugdunensis strains

Extrachromosomal DNA analysis and restriction endonuclease analysis of whole cellular DNA were used to characterize 30 Staphylococcus lugdunensis strains isolated from 13 different hospitals from 1977 to 1988. All the strains were susceptible to most of the antibiotics tested, including penicillin G. A single 3.2 kilobase plasmid was detected in 13 strains and one or two plasmids, ranging from 2.3 to 6.6 kilobases, were found in 7 strains. EcoRI, PstI and PvuII restriction patterns of total cellular DNA were identical for 23 isolates, indicating strong conservation of endonuclease sites in this species. One or two additional DNA bands occurred in seven isolates. Molecular markers show rather little variations between different S. lugdunensis isolates suggesting that they are closely related.     

Defence on demand: mechanisms behind optimal defence patterns

Background

The optimal defence hypothesis (ODH) predicts that tissues that contribute most to a plant''s fitness and have the highest probability of being attacked will be the parts best defended against biotic threats, including herbivores. In general, young sink tissues and reproductive structures show stronger induced defence responses after attack from pathogens and herbivores and contain higher basal levels of specialized defensive metabolites than other plant parts. However, the underlying physiological mechanisms responsible for these developmentally regulated defence patterns remain unknown.

Scope

This review summarizes current knowledge about optimal defence patterns in above- and below-ground plant tissues, including information on basal and induced defence metabolite accumulation, defensive structures and their regulation by jasmonic acid (JA). Physiological regulations underlying developmental differences of tissues with contrasting defence patterns are highlighted, with a special focus on the role of classical plant growth hormones, including auxins, cytokinins, gibberellins and brassinosteroids, and their interactions with the JA pathway. By synthesizing recent findings about the dual roles of these growth hormones in plant development and defence responses, this review aims to provide a framework for new discoveries on the molecular basis of patterns predicted by the ODH.

Conclusions

Almost four decades after its formulation, we are just beginning to understand the underlying molecular mechanisms responsible for the patterns of defence allocation predicted by the ODH. A requirement for future advances will be to understand how developmental and defence processes are integrated.     

Challenges in studying genomic structural variant formation mechanisms: the short-read dilemma and beyond

Next-generation sequencing (NGS) technologies have revolutionised the analysis of genomic structural variants (SVs), providing significant insights into SV de novo formation based on analyses of rearrangement breakpoint junctions. The short DNA reads generated by NGS, however, have also created novel obstacles by biasing the ascertainment of SVs, an aspect that we refer to as the 'short-read dilemma'. For example, recent studies have found that SVs are often complex, with SV formation generating large numbers of breakpoints in a single event (multi-breakpoint SVs) or structurally polymorphic loci having multiple allelic states (multi-allelic SVs). This complexity may be obscured in short reads, unless the data is analysed and interpreted within its wider genomic context. We discuss how novel approaches will help to overcome the short-read dilemma, and how integration of other sources of information, including the structure of chromatin, may help in the future to deepen the understanding of SV formation processes.     

DNA sequencing: bench to bedside and beyond

Fifteen years elapsed between the discovery of the double helix (1953) and the first DNA sequencing (1968). Modern DNA sequencing began in 1977, with development of the chemical method of Maxam and Gilbert and the dideoxy method of Sanger, Nicklen and Coulson, and with the first complete DNA sequence (phage ϕX174), which demonstrated that sequence could give profound insights into genetic organization. Incremental improvements allowed sequencing of molecules >200 kb (human cytomegalovirus) leading to an avalanche of data that demanded computational analysis and spawned the field of bioinformatics. The US Human Genome Project spurred sequencing activity. By 1992 the first ‘sequencing factory’ was established, and others soon followed. The first complete cellular genome sequences, from bacteria, appeared in 1995 and other eubacterial, archaebacterial and eukaryotic genomes were soon sequenced. Competition between the public Human Genome Project and Celera Genomics produced working drafts of the human genome sequence, published in 2001, but refinement and analysis of the human genome sequence will continue for the foreseeable future. New ‘massively parallel’ sequencing methods are greatly increasing sequencing capacity, but further innovations are needed to achieve the ‘thousand dollar genome’ that many feel is prerequisite to personalized genomic medicine. These advances will also allow new approaches to a variety of problems in biology, evolution and the environment.     

Different genomic DNA methylation patterns between male and female adults of white-backed planthoppers Sogatella furcifera

DNA methylation plays a key role in gene regulation and phenotype variation in many organisms. The aim of this study was to survey the frequency and variation of cytosine methylation at CCGG sequences in adult male and female planthoppers Sogatella furcifera, a major rice pest in Asia, and to determine the occurrence of methylation changes associated with sexual dimorphism using methylation-sensitive amplification polymorphism. 1131 DNA fragments including CCGG sites were amplified using 36 pairs of selective primers: about 191 methylated bands were identified. In male planthoppers, we got a total of 581 bands, including 40 fully-methylated bands, 65 hemi-methylated bands and 476 none-methylated bands, so the fully-methylated ratio, hemi-methylated ratio and total methylated ratio were 6.88%, 11.19% and 18.07%, respectively. In the female planthopper, there were a total of 550 bands, including 44 fully-methylated bands, 42 hemi-methylated bands and 464 none-methylated bands. The fully-methylated ratio was 7.64% in female planthoppers, which was slightly higher than in the male planthoppers, however, the hemi-methylated ratio was lower (8.00%) in the female compared with the male planthopper. Altogether, 46 DNA bands displayed variable cytosine methylation patterns between male and female samples: 20 of them occurred only in male samples and 26 only in female samples. Thus, the genome methylation patterns are different between male and female adults. The results suggest that DNA methylation might be related to sexual differentiation and development in S. furcifera.     

Plant epigenetics: phenotypic and functional diversity beyond the DNA sequence

Phenotypic variation determines the capacity of plants to adapt to changing environments and to colonize new habitats. Deciphering the mechanisms contributing to plant phenotypic variation and their effects on plant ecological interactions and evolutionary dynamics is thus central to all biological disciplines. In the past few decades, research on plant epigenetics is showing that (1) epigenetic variation is related to phenotypic variation and that some epigenetic marks drive major phenotypic changes in plants; (2) plant epigenomes are highly diverse, dynamic, and can respond rapidly to a variety of biotic and abiotic stimuli; (3) epigenetic variation can respond to selection and therefore play a role in adaptive evolution. Yet, current information in terms of species, geographic ranges, and ecological contexts analyzed so far is too limited to allow for generalizations about the relevance of epigenetic regulation in phenotypic innovation and plant adaptation across taxa. In this report, we contextualize the potential role of the epigenome in plant adaptation to the environment and describe the latest research in this field presented during the symposium “Plant epigenetics: phenotypic and functional diversity beyond the DNA sequence” held within the Botany 2020 conference framework in summer 2020.     

Relating fish kills to upwellings and wind patterns in the Salton Sea

Awareness of pond conservation value is growing all over Europe. Ponds are recognized as important ecosystems supporting large numbers of species and several rare and threatened aquatic plants, macroinvertebrates and amphibians. Notwithstanding ponds, particularly temporary ones, are still neglected in Italy. There are some gaps in our understanding of the macrophyte ecology and the conservation value of Mediterranean small still waters. Therefore, this study investigated the macrophyte communities and physico-chemical characteristics of 8 permanent and 13 temporary ponds along the Tyrrhenian coast near Rome, with the aim to relate the distribution of aquatic plants to environmental variables, and to define the botanical conservation value of ponds. Throughout the study period (Spring 2002), Principal Component Analysis performed on abiotic variables clearly discriminated temporary ponds, smaller and more eutrophic, from permanent ponds, larger and with higher pH and oxygen concentration. A total of 73 macrophyte taxa were collected in the study ponds. Temporary waters hosted a smaller number of plant species than permanent ones. Besides hydroperiod length, the environmental factors related to plant richness were maximum depth, surface area, dissolved oxygen and nitrogen concentration in the water. Moreover, the Non-metric Multidimensional Scaling showed a high dissimilarity in the taxonomic composition of aquatic plants between temporary and permanent ponds. The former contained more annual fast-growing species (Callitriche sp. pl. and Ranunculus sp. pl.), while in the latter species with long life-cycles (i.e. Potamogeton sp. pl.) were more abundant. Our results highlighted that temporary and permanent ponds in central Italy have different macrophyte assemblages, with aquatic species (including some of conservation interest at regional scale) exclusively found in each pond type. This suggested that both type of ponds could give an irreplaceable contribution to the conservation of aquatic plant diversity of these freshwater ecosystems. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guest editors: R. Céréghino, J. Biggs, B. Oertli & S. Declerck The ecology of European ponds: defining the characteristics of a neglected freshwater habitat     

Moving beyond genome sequencing into personalized genomic medicine: biological and computing challenges

A report of the second annual Beyond the Genome conference held on the 19-22 September 2011 at The Universities at Shady Grove, Rockville, Maryland, USA, where increases in computing that may help make personal genomics a reality were a major focus.     

Maintenance of genomic methylation patterns during preimplantation development requires the somatic form of DNA methyltransferase 1

DNA methylation at cytosine residues in CpG dinucleotides is a component of epigenetic marks crucial to mammalian development. In preimplantation stage embryos, a large part of genomic DNA is extensively demethylated, whereas the methylation patterns are faithfully maintained in certain regions. To date, no enzymes responsible for the maintenance of DNA methylation during preimplantation development have been identified except for the oocyte form of DNA (cytosine-5)-methyltransferase 1 (Dnmt1o) at the 8-cell stage. Herein, we demonstrate that the somatic form of Dnmt1 (Dnmt1s) is present in association with chromatin in MII-stage oocytes as well as in the nucleus throughout preimplantation development. At the early one-cell stage, Dnmt1s is asymmetrically localized in the maternal pronuclei. Thereafter, Dnmt1s is recruited to the paternal genome during pronuclear maturation. During the first two cell cycles after fertilization, Dnmt1s is exported from the nucleus in the G2 phase in a CRM1/exportin-dependent manner. Antibody microinjection and small interfering RNA-mediated knock-down decreases methylated CpG dinucleotides in repetitive intracisternal A-type particle (IAP) sequences and the imprinted gene H19. These results indicate that Dnmt1s is responsible for the maintenance methylation of particular genomic regions whose methylation patterns must be faithfully maintained during preimplantation development.     

Relating independent measures of DNA curvature: electrophoretic anomaly and cyclization efficiency

Electrophoretic methods are often used to measure DNA curvature and protein-induced DNA bending. Though convenient and widely-applied, quantitative analyses are generally limited to assays for which empirical calibration standards have been developed. Alternatively, solution-based cyclization of short DNA duplexes allows analysis of DNA curvature and bending from first principles, but a detailed understanding of this assay is still lacking. In this work, we demonstrate that calibration with an independent electrophoretic assay of DNA curvature permits interpretation of cyclization assay results in a quantitatively meaningful way. We systematically measure intrinsic DNA curvature in short duplexes using a well-established empirical ligation ladder assay. We then compare the results to those obtained from the analysis of the distribution of circular products obtained in simple enzymatic cyclization assays of the same duplexes when polymerized. A strong correlation between DNA curvature estimates from these two assays is obtained for DNA fragments between 150-300 bp in length. We discuss how this result might be used to improve quantitative analysis of protein-mediated bending events evaluated by cyclization methods. Our results suggest that measurements of DNA curvature obtained under similar conditions, in solution and in an acrylamide gel matrix, can be compared directly. The ability to correlate results of these simple assays may prove convenient in monitoring DNA curvature and flexibility.