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.     

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.

     

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.     

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。     

Transgenerational maintenance of transgene body CG but not CHG and CHH methylation

In plants, RNA-directed DNA methylation (RdDM) can target both transgene promoters and coding regions/gene bodies. RdDM leads to methylation of cytosines in all sequence contexts: CG, CHG and CHH. Upon segregation of the RdDM trigger, at least CG methylation can be maintained at promoter regions in the progeny. So far, it is not clear whether coding region methylation can be also maintained. We showed that the body of Potato spindle tuber viroid (PSTVd) transgene constructs became densely de novo methylated at CG, CHG and CHH sites upon PSTVd infection. In this study, we demonstrate that in viroid-free progeny plants, asymmetric CHH and CHG methylation was completely lost. However, symmetric CG methylation was stably maintained for at least two generations. Importantly, the presence of transgene body methylation did not lead to an increase of dimethylation of histone H3 lysine 9 or a decrease of acetylation of H3. Our data supports the view that CG methylation can be maintained not only in promoters but also in the body of transgenes. They further suggest that maintenance of methylation may occur independently of tested chromatin modifications.     

Transgenerational maintenance of transgene body CG but not CHG and CHH methylation

In plants, RNA-directed DNA methylation (RdDM) can target both transgene promoters and coding regions/gene bodies. RdDM leads to methylation of cytosines in all sequence contexts: CG, CHG and CHH. Upon segregation of the RdDM trigger, at least CG methylation can be maintained at promoter regions in the progeny. So far, it is not clear whether coding region methylation can be also maintained. We showed that the body of Potato spindle tuber viroid (PSTVd) transgene constructs became densely de novo methylated at CG, CHG and CHH sites upon PSTVd infection. In this study, we demonstrate that in viroid-free progeny plants, asymmetric CHH and CHG methylation was completely lost. However, symmetric CG methylation was stably maintained for at least two generations. Importantly, the presence of transgene body methylation did not lead to an increase of dimethylation of histone H3 lysine 9 or a decrease of acetylation of H3. Our data supports the view that CG methylation can be maintained not only in promoters but also in the body of transgenes. They further suggest that maintenance of methylation may occur independently of tested chromatin modifications.