A CpG-rich RNA and an RNA helicase tightly associated with the DNA demethylation complex are present mainly in dividing chick embryo cells

In the developing chicken embryo, active DNA demethylation requires both RN A and proteins (Nucleic Acids Res. 25, 2375-2380, 1997; ibid. 25, 4545-4550 , 1997, FEBS Lett. 449, 251-254, 1999a). In vitro assays indicate that in t he 5- and 12-day-old embryos the highest specific activity of 5-methylcytos ine DNA glycosylase is found in the brain, the eyes and the skin. In situ h ybridization with antisense CpG-rich RNA tightly associated to the DNA deme thylation complex shows a restricted expression pattern only in proliferati ng tissues such as the neuroepithelia of the brain in 5-day-old embryos. Th e RNA is absent in differentiated tissues like the skeletal and heart muscl e, liver and the crystallin-producing cells in the lens. The CpG-rich RNA i s transcribed in a developmental stage-specific rather than in a cell-speci fic manner. In contrast transcripts of DNA methyltransferase are found in d ividing and quiescent cells. In situ hybridization with a probe of a RNA he licase which is also associated with the DNA demethylation complex shows a very similar localization in mitotically active tissues as the CpG-rich RNA . The content of 5-methylcytosine in individual cells was determined with a specific monoclonal antibody and cytometric analysis on tissue sections. T he results indicate that proliferating cells have on the average 15% more m ethylated cytosines than non-dividing cells. This represents roughly 3 x 10 (6) more methylation sites per haploid genome.