DNA methylation of retrotransposons,DNA transposons and genes in sugar beet (Beta vulgaris L.)

The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome‐wide cytosine methylation in the sugar beet genome was studied in leaves and leaf‐derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome‐wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves.     

Kismeth: Analyzer of plant methylation states through bisulfite sequencing

Background  

There is great interest in probing the temporal and spatial patterns of cytosine methylation states in genomes of a variety of organisms. It is hoped that this will shed light on the biological roles of DNA methylation in the epigenetic control of gene expression. Bisulfite sequencing refers to the treatment of isolated DNA with sodium bisulfite to convert unmethylated cytosine to uracil, with PCR converting the uracil to thymidine followed by sequencing of the resultant DNA to detect DNA methylation. For the study of DNA methylation, plants provide an excellent model system, since they can tolerate major changes in their DNA methylation patterns and have long been studied for the effects of DNA methylation on transposons and epimutations. However, in contrast to the situation in animals, there aren't many tools that analyze bisulfite data in plants, which can exhibit methylation of cytosines in a variety of sequence contexts (CG, CHG, and CHH).     

铜胁迫对不同抗性种群海州香薷酸性转化酶基因启动子甲基化的影响

采用亚硫酸氢盐测序技术,检测了Cu胁迫对海州香薷（Elsholtzia haichowensis Sun） Cu抗性和非抗性种群酸性转化酶基因启动子甲基化的影响。结果表明,海州香薷液泡转化酶基因（vINV）和细胞壁转化酶基因（cwINV）的启动子在CG位点分别表现出超甲基化和低甲基化的现象。酸性转化酶基因启动子CHG和CHH位点的甲基化状态受Cu胁迫影响较大。Cu胁迫下,vINV和cwINV启动子分别有1个CHG和6个CHH位点的甲基化状态在Cu抗性种群和非抗性种群之间表现出较大差异。抗Cu种群中这些甲基化差异位点对Cu胁迫不敏感,但在非抗性种群中这些位点的甲基化水平在Cu胁迫后出现大幅上升或下降。有些甲基化差异位点位于或者临近预测的启动子顺式作用元件区域,可能参与Cu胁迫下酸性转化酶基因的表达调控。     

Virus‐induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two patterns of transgene body methylation

We used bisulfite sequencing to study the methylation of a viral transgene whose expression was silenced upon plum pox virus infection of the transgenic plant and its subsequent recovery as a consequence of so‐called virus‐induced gene silencing (VIGS). VIGS was associated with a general increase in the accumulation of small RNAs corresponding to the coding region of the viral transgene. After VIGS, the transgene promoter was not methylated and the coding region showed uneven methylation, with the 5′ end being mostly unmethylated in the recovered tissue or mainly methylated at CG sites in regenerated silenced plants. The methylation increased towards the 3′ end, which showed dense methylation in all three contexts (CG, CHG and CHH). This methylation pattern and the corresponding silenced status were maintained after plant regeneration from recovered silenced tissue and did not spread into the promoter region, but were not inherited in the sexual offspring. Instead, a new pattern of methylation was observed in the progeny plants consisting of disappearance of the CHH methylation, similar CHG methylation at the 3′ end, and an overall increase in CG methylation in the 5′ end. The latter epigenetic state was inherited over several generations and did not correlate with transgene silencing and hence virus resistance. These results suggest that the widespread CG methylation pattern found in body gene bodies located in euchromatic regions of plant genomes may reflect an older silencing event, and most likely these genes are no longer silenced.     

Endogenous pararetrovirus sequences associated with 24 nt small RNAs at the centromeres of Fritillaria imperialis L. (Liliaceae), a species with a giant genome

Endogenous pararetroviral sequences are the most commonly found virus sequences integrated into angiosperm genomes. We describe an endogenous pararetrovirus (EPRV) repeat in Fritillaria imperialis, a species that is under study as a result of its exceptionally large genome (1C = 42 096 Mbp, approximately 240 times bigger than Arabidopsis thaliana). The repeat (FriEPRV) was identified from Illumina reads using the RepeatExplorer pipeline, and exists in a complex genomic organization at the centromere of most, or all, chromosomes. The repeat was reconstructed into three consensus sequences that formed three interconnected loops, one of which carries sequence motifs expected of an EPRV (including the gag and pol domains). FriEPRV shows sequence similarity to members of the Caulimoviridae pararetrovirus family, with phylogenetic analysis indicating a close relationship to Petuvirus. It is possible that no complete EPRV sequence exists, although our data suggest an abundance that exceeds the genome size of Arabidopsis. Analysis of single nucleotide polymorphisms revealed elevated levels of C→T and G→A transitions, consistent with deamination of methylated cytosine. Bisulphite sequencing revealed high levels of methylation at CG and CHG motifs (up to 100%), and 15–20% methylation, on average, at CHH motifs. FriEPRV's centromeric location may suggest targeted insertion, perhaps associated with meiotic drive. We observed an abundance of 24 nt small RNAs that specifically target FriEPRV, potentially providing a signature of RNA‐dependent DNA methylation. Such signatures of epigenetic regulation suggest that the huge genome of F. imperialis has not arisen as a consequence of a catastrophic breakdown in the regulation of repeat amplification.     

Identification of differential genomic DNA Methylation in the hypothalamus of pubertal rat using reduced representation Bisulfite sequencing

Background

There are many variables affecting the onset of puberty in animals, including genetic, nutritional, and environmental factors. Recent studies suggest that epigenetic regulation, especially DNA methylation, plays a majorrole in the regulation of puberty. However, there have been no reports on DNA methylation of the pubertal genome.

Methods

We investigated DNA methylation in the female rat hypothalamus at prepuberty and puberty using reduced representation bisulfite sequencing technology. The identified genes and signaling pathways exhibiting changes to DNA methylation in pubertal rats were determined by Gene Ontogeny and Kyoto Encyclopedia of Genes and Genomes analysis.

Results

The distribution of the three types of methylated C bases in promoter and CpG island (CGI) regions in the hypothalamus was as follows: 87.79% CG, 3.05% CHG, 9.16% CHH for promoters, and 88.35% CG, 3.21% CHG, 88.35% CHH for CGI in prepubertal rats; and 90.78% CG, 2.13% CHG, 7.09% CHH for promoters, and 88.59% CG, 88.59% CHG, 8.35% CHH for CGI in pubertal animals. CG showed the highest percentage of methylation, and was the highest methylation state in CGI. Compared to prepubertal hyoyhalamus samples, we identified ten genes with altered methylation in promoter regions in the pubertal hypothalamus samples, and 43 genes with altered methylation in the CGI. Changes in DNA methylation were found in gonadotropin-releasing hormone signaling pathways, and the oocyte meiosis pathway.

Conclusion

Our results demonstrate changes in DNA methylation occur in female rats from prepuberty to puberty suggestng DNA methylation may play a crucial role in the regulation of puberty onset. This study provides essential information for future studies on the role of epigenetics in the regulation of puberty.

     

Roles,and establishment,maintenance and erasing of the epigenetic cytosine methylation marks in plants

Heritable information in plants consists of genomic information in DNA sequence and epigenetic information superimposed on DNA sequence. The latter is in the form of cytosine methylation at CG, CHG and CHH elements (where H = A, T or C) and a variety of histone modifications in nucleosomes. The epialleles arising from cytosine methylation marks on the nuclear genomic loci have better heritability than the epiallelic variation due to chromatin marks. Phenotypic variation is increased manifold by epiallele comprised methylomes. Plants (angiosperms) have highly conserved genetic mechanisms to establish, maintain or erase cytosine methylation from epialleles. The methylation marks in plants fluctuate according to the cell/tissue/organ in the vegetative and reproductive phases of plant life cycle. They also change according to environment. Epialleles arise by gain or loss of cytosine methylation marks on genes. The changes occur due to the imperfection of the processes that establish and maintain the marks and on account of spontaneous and stress imposed removal of marks. Cytosine methylation pattern acquired in response to abiotic or biotic stress is often inherited over one to several subsequent generations. Cytosine methylation marks affect physiological functions of plants via their effect(s) on gene expression levels. They also repress transposable elements that are abundantly present in plant genomes. The density of their distribution along chromosome lengths affects meiotic recombination rate, while their removal increases mutation rate. Transposon activation due to loss of methylation causes rearrangements such that new gene regulatory networks arise and genes for microRNAs may originate. Cytosine methylation dynamics contribute to evolutionary changes. This review presents and discusses the available evidence on origin, removal and roles of cytosine methylation and on related processes, such as RNA directed DNA methylation, imprinting, paramutation and transgenerational memory in plants.     

Epigenetic profiling of heterochromatic satellite DNA

Sugar beet (Beta vulgaris) chromosomes consist of large heterochromatic blocks in pericentromeric, centromeric, and intercalary regions comprised of two different highly abundant DNA satellite families. To investigate DNA methylation at single base resolution at heterochromatic regions, we applied a method for strand-specific bisulfite sequencing of more than 1,000 satellite monomers followed by statistical analyses. As a result, we uncovered diversity in the distribution of different methylation patterns in both satellite families. Heavily methylated CG and CHG (H=A, T, or C) sites occur more frequently in intercalary heterochromatin, while CHH sites, with the exception of CAA, are only sparsely methylated, in both intercalary and pericentromeric/centromeric heterochromatin. We show that the difference in DNA methylation intensity is correlated to unequal distribution of heterochromatic histone H3 methylation marks. While clusters of H3K9me2 were absent from pericentromeric heterochromatin and restricted only to intercalary heterochromatic regions, H3K9me1 and H3K27me1 were observed in all types of heterochromatin. By sequencing of a small RNA library consisting of 6.76 million small RNAs, we identified small interfering RNAs (siRNAs) of 24 nucleotides in size which originated from both strands of the satellite DNAs. We hypothesize an involvement of these siRNAs in the regulation of DNA and histone methylation for maintaining heterochromatin.     

Climate‐dependent variation in cold tolerance of weedy rice and rice mediated by OsICE1 promoter methylation

The mechanisms by which weedy rice (Oryza sativa f. spontanea) has adapted to endure low‐temperature stress in northern latitudes remain unresolved. In this study, we assessed cold tolerance of 100 rice varieties and 100 co‐occurring weedy rice populations, which were sampled across a broad range of climates in China. A parallel pattern of latitude‐dependent variation in cold tolerance was detected in cultivated rice and weedy rice. At the molecular level, differential cold tolerance was strongly correlated with relative expression levels of CBF cold response pathway genes and with methylation levels in the promoter region of OsICE1, a regulator of this pathway. Among all methylated cytosine sites of the OsICE1 promoter, levels of CHG and CHH methylation were found to be significantly correlated with cold tolerance among accessions. Furthermore, within many of the collection locales, weedy rice shared identical or near‐identical OsICE1 methylation patterns with co‐occurring cultivated rice. These findings provide new insights on the possible roles that methylation variation in the OsICE1 promoter may play in cold tolerance, and they suggest that weedy rice can rapidly acquire cold tolerance via methylation patterns that are shared with co‐occurring rice cultivars.     

Genome-wide evidence for local DNA methylation spreading from small RNA-targeted sequences in Arabidopsis

Transposable elements (TEs) and their relics play major roles in genome evolution. However, mobilization of TEs is usually deleterious and strongly repressed. In plants and mammals, this repression is typically associated with DNA methylation, but the relationship between this epigenetic mark and TE sequences has not been investigated systematically. Here, we present an improved annotation of TE sequences and use it to analyze genome-wide DNA methylation maps obtained at single-nucleotide resolution in Arabidopsis. We show that although the majority of TE sequences are methylated, ∼26% are not. Moreover, a significant fraction of TE sequences densely methylated at CG, CHG and CHH sites (where H = A, T or C) have no or few matching small interfering RNA (siRNAs) and are therefore unlikely to be targeted by the RNA-directed DNA methylation (RdDM) machinery. We provide evidence that these TE sequences acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, we show that although both methylated and unmethylated TE sequences located in euchromatin tend to be more abundant closer to genes, this trend is least pronounced for methylated, siRNA-targeted TE sequences located 5′ to genes. Based on these and other findings, we propose that spreading of DNA methylation through promoter regions explains at least in part the negative impact of siRNA-targeted TE sequences on neighboring gene expression.