Maintenance DNA methyltransferase (Met1) and silencing of CpG-methylated foreign DNA in Volvox carteri.

DNA methylation plays an important role in the gene-silencing network of higher eukaryotes. We have analyzed the 21.5-kb maintenance methyltransferase (M-MTase) gene, met1, of the multicellular green alga Volvox carteri. The met1 transcript was detected only during the period when DNA replication and cell division are taking place. It encodes a 238 kDa protein containing eight C-terminal activity domains typical of M-MTases, plus upstream DNA-binding domains including the ProDom domain PD003757, which experimental analyses in animal systems have indicated is required for targeting the enzyme to DNA-replication foci. Several insertions of unknown function make Volvox Met1 the largest known member of the Met1/Dnmt1 family. Here we also show that several endogenous transposon families are CpG-methylated in Volvox, which we think causes them to be inactive. This view is supported by the observation that an in vitro CpG-methylated gene introduced into Volvox was maintained in the methylated and silent state over >100 generations. Thus, we believe that Met1 recognizes and perpetuates the in vitro methylation signal, and that the silencing machinery is then able to transduce such a methylation-only signal into a stable heterochromatic (and silent) state.

A link between DNA methylation and epigenetic silencing in transgenic Volvox carteri.

Epigenetic silencing of foreign genes introduced into plants poses an unsolved problem for transgenic technology. Here we have used the simple multicellular green alga VOLVOX: carteri as a model to analyse the relation of DNA methylation to transgenic silencing. VOLVOX: DNA contains on average 1.1% 5-methylcytosine and 0.3% N6-methyladenine, as revealed by electrospray mass spectrometry and phosphoimaging of chromatographically separated (32)P-labelled nucleotides. In two nuclear transformants of V.carteri, produced in 1993 by biolistic bombardment with a foreign arylsulphatase gene (C-ars), the transgene is still expressed in one (Hill 181), but not in the other (Hill 183), after an estimated 500-1000 generations. Each transformant clone contains multiple intact copies of C-ars, most of them integrated into the genome as tandem repeats. When the bisulphite genomic sequencing protocol was applied to examine two select regions of transgenic C-ars, we found that the inactivated copies (Hill 183) exhibited a high-level methylation (40%) of CpG dinucleotides, whereas the active copies (Hill 181) displayed low-level (7%) CpG methylation. These are average values from 40 PCR clones sequenced from each DNA strand in the two portions of C-ars. The observed correlation of CpG methylation and transgene inactivation in a green alga will be discussed in the light of transcriptional silencing.