Physarum polycephalum is a lower eukaryotic organism which stands out as a promising model for epigenetic studies because of its very unique life and cell cycles. Preliminary analysis of its DNA methylation levels showed similar content to that of vertebrates, and reports suggested that this epigenetic modification could be involved in its sporulation. In this short paper, authors showed that this is indeed the case, and proved that methylation targeted specifically a sporulation- related gene, spherulin-4.
After transference to the starvation medium, cultures undergo a metabolic transition which results in the formation of spherules. To see if DNA methylation regulation is involved in this transition, authors added azaC to the starvation medium, and assayed the presence of spherules after a period of time (72 hrs). They found that azaC inhibited the formation of spherules when used at concentrations over 50 uM, and this inhibition was presumed to be complete [and not only slowed down], since longer incubations (up to 168 hrs) did not produce spherules either; the inhibition also was not due to unspecific toxicity, since cultures resumed growth promptly after transfer to medium without azaC. Complete inhibition of spherule formation was seen after continuous presence during six or more hours. On the other hand, germination of spherules in absence of azaC was not influenced, which suggests that DNA methylation is involved in the formation of spherules, but not in their later germination (although authors recognized that this might require further investigations). Other methylation inhibitors assayed (5-aza- 2-deoxycytidine and L-ethionine) also produce similar effects.
To assess the DNA methylation content during spherulation, cultures were pulse- labeled with methyl- 3H-methionine, and the incorporation of 3H was measured; these experiments showed however that the methylation remained almost unchanged. Later they measured the DNA methylation by HPLC, and although the levels were not statistically significative, important differences were seen when individual culture time- points were compared. This supported the hypothesis that changes in DNA methylation patterns were occurring indeed.
To observe DNA methylation of specific sequences, several spherulation genes were analyzed by Southern blotting, using methylation- sensitive restrictases. Here, only a probe corresponding to the spherulin-4 gene showed differential patterns, which indicated changes in DNA methylation, and although several independent cultures displayed similar band patterns, some gave more complex hybridizations, suggesting that the methylation changes could be transient.
Our studies with inhibitors of DNA methylation indicated that for P.polycephalum to differentiate into spherules methylation of DNA must take place at least during the second half of spherulation [...]. Two classes of inhibitors were used: (i) nucleoside analogues [...], and (ii) L-ethionine [...]. Since these two classes of inhibitors operate by distinct mechanisms their influence on spherulation is likely to be caused via the feature they have in common, i.e. by inhibition of DNA methylation. [...] We have also shown that inhibiting methylation of DNA does not lead to significant overall growth retardation of P. polycephalum, thus indicating the specific role of DNA methylation in spherulation.Fronk J, and R.Magiera. 1994. Biochem J 304(Pt 1):101-104
[...A previous] study [...] failed to detect any changes in the methylation pattern of several genes during differentiation of P. polycephalum. The most likely explanation of this discrepancy is the fact that not all developmentally regulated genes undergo differential methylation detectable by the method used in the study in question as well as in this work [...].
epigenetics
