DNA Methylation Dynamics in Eisenia andrei Regeneration: The Effects of Time, Region, and a Hypomethylating Agent.

The impact of epigenetic mechanisms on molecular and cellular processes of regeneration remains a less-investigated field, especially concerning earthworm segment restoration. To evaluate distinct methylated cytosines under different conditions, earthworm segments were collected: decitabine-treated and controls; anterior and posterior amputation; intact and regenerated (2- and 4-week). 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) distribution in tissues was assessed by immunohistochemistry (IHC), and in genomic DNA by dot blot. DNA-methyltransferase (DNMT) and ten-eleven translocation (TET) dioxygenase activity was determined by immuno-based colorimetry. DNMT1 and TET gene expressions were verified with real-time PCR. Decitabine treatment reduced 5mC levels in most tissues, persisting in the anterior coelomic cavity (intact and blastemas). 5hmC remained elevated in most tissues, remarkably in 2-week blastemas. In the same period, DNMT and TET presented the highest activity in anterior treated samples compared to other treated periods. Concurrently, DNMT1 gene expression increased prominently in anterior segments, while TET showed the highest expression in both anterior and posterior 2-week treated groups. Thus far, our observations indicate that, despite decitabine's effect on the DNA methylation machinery of the earthworm model, the region of amputation and the regeneration period also interfere with activity and expression of epigenetic enzymes, affecting 5mC and 5hmC distribution.