A permissive chromatin structure is adopted prior to site-specific DNA demethylation of developmentally expressed genes involved in macronuclear differentiation

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.

Beyond transcriptional silencing: Is methylcytosine a widely conserved eukaryotic DNA elimination mechanism?

“…It is important to note that no delineation in this study between hydroxymethylcytosine and methylcytosine was performed, and bisulfite sequencing techniques cannot distinguish the two modifications . In contrast to our Oxytricha study, these authors found that the methylation was not induced de novo – it was stably maintained in the MIC precursor DNA of vegetative cells, then passively inherited in the MAC through the process of nuclear development (Table ). The evidence for methylcytosine in Paramecium is very indirect.…”

“…The recently proposed models of methylcytosine function in Oxytricha and Stylonychia are near inverses of each other (Table ). While large‐scale evolutionary innovation in methylation pathways between these species cannot be ruled out, it is possible that the conflicting models are in fact simply describing two very different modalities for methylated cytosine in different parts of the ciliate lifecycle – an idea that finds precedent in the dual functions ascribed to histone methylation in Tetrahymena (Table ).…”

“…Contrary to initial reports, methylcytosine may be present in vegetative Stylonychia and Paramecium cells, though at levels below the experimental detection limit. In a recent Stylonychia study, cytosine methylation was detected via bisulfite sequencing the promoter region of two genes whose expression is repressed during vegetative growth but then rapidly induced during the genome rearrangement process (Table ). The induction of these genes correlates with loss of the methylcytosine from their promoters , though it would be interesting to test whether methyltransferase‐inhibiting drugs such as azacitidine or decitabine re‐induce vegetative expression of these genes (Table ).…”

“…In a recent Stylonychia study, cytosine methylation was detected via bisulfite sequencing the promoter region of two genes whose expression is repressed during vegetative growth but then rapidly induced during the genome rearrangement process (Table ). The induction of these genes correlates with loss of the methylcytosine from their promoters , though it would be interesting to test whether methyltransferase‐inhibiting drugs such as azacitidine or decitabine re‐induce vegetative expression of these genes (Table ). It is important to note that no delineation in this study between hydroxymethylcytosine and methylcytosine was performed, and bisulfite sequencing techniques cannot distinguish the two modifications .…”

Introduction to Genome Biology and Diversity

“…Thus, without those RNA molecules, the DNA genome of the stichotrichous ciliates is an incomplete informational entity [4]. Furthermore, emerging work on both Oxytricha and Stylonychia suggests that epigenetic modification of their DNA may play a role in the production of active macronuclear DNA [166,[169][170][171] Complex microbial communities live in close association with the human body and have a strong impact on human health and disease. Host genetic variation is known to influence the composition of those communities [172], and, conversely, microbial variability is thought to influence various host disease states [173].…”