Triticale Green Plant Regeneration Is Due to DNA Methylation and Sequence Changes Affecting Distinct Sequence Contexts in the Presence of Copper Ions in Induction Medium

Metal ions in the induction medium are essential ingredients allowing green plant regeneration. For instance, Cu(II) and Ag(I) ions may affect the mitochondrial electron transport chain, influencing the Yang cycle and synthesis of S-adenosyl-L-methionine, the prominent donor of the methylation group for all cellular compounds, including cytosines. If the ion concentrations are not balanced, they can interfere with the proper flow of electrons in the respiratory chain and ATP production. Under oxidative stress, methylated cytosines might be subjected to mutations impacting green plant regeneration efficiency. Varying Cu(II) and Ag(I) concentrations in the induction medium and time of anther culture, nine trials of anther culture-derived regenerants of triticale were derived. The methylation-sensitive AFLP approach quantitative characteristics of tissue culture-induced variation, including sequence variation, DNA demethylation, and DNA de novo methylation for all symmetric-CG, CHG, and asymmetric-CHH sequence contexts, were evaluated for all trials. In addition, the implementation of mediation analysis allowed evaluating relationships between factors influencing green plant regeneration efficiency. It was demonstrated that Cu(II) ions mediated relationships between: (1) de novo methylation in the CHH context and sequence variation in the CHH, (2) sequence variation in CHH and green plant regeneration efficiency, (3) de novo methylation in CHH sequences and green plant regeneration, (4) between sequence variation in the CHG context, and green plant regeneration efficiency. Cu(II) ions were not a mediator between de novo methylation in the CG context and green plant regeneration. The latter relationship was mediated by sequence variation in the CG context. On the other hand, we failed to identify any mediating action of Ag(I) ions or the moderating role of time. Furthermore, demethylation in any sequence context seems not to participate in any relationships leading to green plant regeneration, sequence variation, and the involvement of Cu(II) or Ag(I) as mediators.

Dnmt1 has de novo activity targeted to transposable elements

DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.

Induction of Promoter DNA Methylation Upon High-Pressure Spraying of Double-Stranded RNA in Plants

Exogenous application of RNA molecules is a potent method to trigger RNA interference (RNAi) in plants in a transgene-free manner. So far, all exogenous RNAi (exo-RNAi) applications have aimed to trigger mRNA degradation of a given target. However, the issue of concomitant epigenetic changes was never addressed. Here, we report for the first time that high-pressure spraying of dsRNAs can trigger de novo methylation of promoter sequences in plants.

QSER1 protects DNA methylation valleys from de novo methylation

DNA methylation is essential to mammalian development, and dysregulation can cause serious pathological conditions. Key enzymes responsible for deposition and removal of DNA methylation are known, but how they cooperate to regulate the methylation landscape remains a central question. Using a knockin DNA methylation reporter, we performed a genome-wide CRISPR-Cas9 screen in human embryonic stem cells to discover DNA methylation regulators. The top screen hit was an uncharacterized gene, QSER1, which proved to be a key guardian of bivalent promoters and poised enhancers of developmental genes, especially those residing in DNA methylation valleys (or canyons). We further demonstrate genetic and biochemical interactions of QSER1 and TET1, supporting their cooperation to safeguard transcriptional and developmental programs from DNMT3-mediated de novo methylation.

Inference of DNA methylation patterns in molluscs

Mollusca are the second largest and arguably most diverse phylum of the animal kingdom. This is in sharp contrast to our very limited knowledge concerning epigenetic mechanisms including DNA methylation in this invertebrate group. Here, we inferred DNA methylation patterns by analysing the normalized dinucleotide CG content in protein-coding sequences and identified DNA methyltransferases (DNMT1 and 3) in published transcriptomes and genomes of 140 species across all eight classes of molluscs. Given the evolutionary age and morphological diversity of molluscs, we expected to find evidence for diverse methylation patterns. Our inferences suggest that molluscs possess substantial levels of DNA methylation in gene bodies as a rule. Yet, we found deviations from this general picture with regard to (i) the CpG observed/expected distributions indicating a reduction in DNA methylation in certain groups and (ii) the completeness of the DNMT toolkit. Reductions were evident in Caudofoveata, Solenogastres, Polyplacophora, Monoplacophora, as well as Scaphopoda. Heterobranchia and Oegopsida were remarkable as they lacked DNMT3, usually responsible for de novo methylation, yet showed signs of DNA methylation. Our survey may serve as guidance for direct empirical analyses of DNA methylation in molluscs. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

Assessment of epigenetic methylation changes in hop (Humulus lupulus) plants obtained by meristem culture

In vitro meristem cultures have been used for the production of hop (Humulus lupulus L.) virus-free rootstocks worldwide, because multipropagation is considered to preserve the genetic stability of the produced plantlet. Nevertheless, in vitro tissue cultures can cause genetic and epigenetic changes. Therefore, we studied the genetic and epigenetic variability of Saaz Osvald’s clones, Sládek and Premiant cultivars on the DNA methylation level by methylation-sensitive amplification polymorphism (MSAP). In vitro propagated plants, acclimatised glasshouse rootstocks as well as derived mericlones and control plants under field conditions were used for the analyses. A total of 346 clearly and highly reproducible amplified products were detected in the MSAP analyses within the studied hop plants. We found 16 polymorphic products (4.6% of products) and 64 products with methylation changes (18.5% of products) in the analyses. The demethylation events were comparable to the de novo methylation events. Most demethylation changes were found in the in vitro plants, but only a few of them were found in the derived mericlones under field conditions. In contrast, the de novo methylation changes persisted in the acclimatised plants under glasshouse or field conditions. A hierarchical cluster analysis was used for the evaluation of the molecular genetic variability within the individual samples. The dendrogram showed that the individual samples of the same variety, more or less, clustered together. Because the methylation status varied during the virus-free rootstock production process, we suppose that de/methylation process is a natural tool of epigenetics and evolution in vegetatively propagated plants.

Methylation of the Suppressor Gene p16INK4a: Mechanism and Consequences

Tumor suppressor genes in the CDKN2A/B locus (p15INK4b, p16INK4a, and p14ARF) function as biological barriers to transformation and are the most frequently silenced or deleted genes in human cancers. This gene silencing frequently occurs due to DNA methylation of the promoter regions, although the underlying mechanism is currently unknown. We present evidence that methylation of p16INK4a promoter is associated with DNA damage caused by interference between transcription and replication processes. Inhibition of replication or transcription significantly reduces the DNA damage and CpGs methylation of the p16INK4a promoter. We conclude that de novo methylation of the promoter regions is dependent on local DNA damage. DNA methylation reduces the expression of p16INK4a and ultimately removes this barrier to oncogene-induced senescence.

Dynamic regulation of chromatin topology and transcription by inverted repeat-derived small RNAs in sunflower

Transposable elements (TEs) are extremely abundant in complex plant genomes. siRNAs of 24 nucleotides in length control transposon activity in a process that involves de novo methylation of targeted loci. Usually, these epigenetic modifications trigger nucleosome condensation and a permanent silencing of the affected loci. Here, we show that a TE-derived inverted repeat (IR) element, inserted near the sunflower HaWRKY6 locus, dynamically regulates the expression of the gene by altering chromatin topology. The transcripts of this IR element are processed into 24-nt siRNAs, triggering DNA methylation on its locus. These epigenetic marks stabilize the formation of tissue-specific loops in the chromatin. In leaves, an intragenic loop is formed, blocking HaWRKY6 transcription. While in cotyledons (Cots), formation of an alternative loop, encompassing the whole HaWRKY6 gene, enhances transcription of the gene. The formation of this loop changes the promoter directionality, reducing IR transcription, and ultimately releasing the loop. Our results provide evidence that TEs can act as active and dynamic regulatory elements within coding loci in a mechanism that combines RNA silencing, epigenetic modification, and chromatin remodeling machineries.