Abstract
BackgroundDNA methylation and demethylation are important epigenetic regulatory mechanisms in eukaryotic cells and, so far, only partially understood. We exploit the minimalistic biological ciliate system to understand the crosstalk between DNA modification and chromatin structure. In the macronucleus of these cells, the DNA is fragmented into individual short DNA molecules, each representing a functional expression and replication unit. Therefore, long range epigenomic interaction can be excluded in this system.ResultsIn the stichotrichous ciliate Stylonychia lemnae, cytosine methylation occurs in a small subset of macronuclear nanochromosomes expressed only during sexual reproduction. Methylation pattern shows similarity to that observed in fungi and Drosophila. Cytosine methylation correlates with gene activity and chromatin structure. Upon gene activation, cytosines become demethylated and a redistribution of histone post-translational modifications (PTMs) takes place. Evidence is presented that the formation of a permissive chromatin structure in the vicinity of the 5meCs precedes cytosine methylation and is probably a necessary prerequisite for their demethylation. Shortly after demethylation of cytosines occurs, the parental macronucleus degenerates, a new macronucleus is formed from a micronuclear derivative and the specific methylation pattern is transmitted from the germline micronucleus to the new macronucleus.ConclusionsWe show that very few, or even only one, discrete methylated cytosines are required to assign regulatory functions at a specific locus. Furthermore, evidence is provided that a permissive chromatin structure is probably a necessary prerequisite for the demethylation of specific cytosines. Our results allow us to propose a mechanistic model for the biological function of cytosine methylation in the ciliate cell and its regulation during the cell cycle.
Highlights
DNA methylation and demethylation are important epigenetic regulatory mechanisms in eukaryotic cells and, so far, only partially understood
We show that H3K9me3/ K27me3, a Posttranslational modification (PTM) typical for repressed chromatin is found at the 5-′end in the silenced state but becomes removed upon activation
In the silenced status we observe an enrichment of the active marker at the 3′end similar to those observations reported for other PTMs typical for active chromatin [23], but interestingly, enrichment of H3K9me3/K27me3 could be detected on the 5′-end of both mdp1 and mdp2 nanochromosomes at a similar position to that where we find methylated cytosines (Figure 2D and E, vegetative growth phase)
Summary
DNA methylation and demethylation are important epigenetic regulatory mechanisms in eukaryotic cells and, so far, only partially understood. There is general agreement that differential molecular signatures of both the DNA and the proteinous contents of chromatin, such as histones, above the primary DNA sequence encode epigenetic information that are prerequisites for the spatiotemporal control of gene expression in a potentially heritable way. The level of epigenomic regulation of gene expression above the level of DNA modifications is the compaction of the 10 nm chromatin fiber, in which the DNA is wrapped around nucleosomes. This 10 nm fiber becomes further compacted by the interaction with linker histone H1 and other proteins [9]. Modifications found at distinct residues of the histone protein N-termini include - among others - lysine acetylation, lysine and arginine methylation as well as serine and threonine phosphorylation
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