FIG. 2.
Genomic hypermethylation induces a delay in cell cycle progression. (A) Imaginal discs and brains were isolated from third-instar larvae and used for the determination of cell numbers and the mitotic index, respectively. White bars represent results from flies overexpressing Dnmt3a, and black bars represent results from flies overexpressing the catalytically inactive protein. Overexpression of Dnmt3a caused a significant (P < 0.01, as determined by a standard t test) reduction in cell numbers and also resulted in a slightly increased mitotic index. (B) Isolated brains were cultured in vitro in the presence of BrdU. BrdU incorporation was visualized by immunodetection. Bars, 100 μm. Overexpression of Dnmt3a caused dramatically reduced BrdU incorporation. (C) Overexpression of the catalytically inactive protein had no effect on BrdU incorporation. (D) In vivo BrdU uptake into larval neuroblasts. Larvae were fed BrdU-containing medium for 1 h, followed by preparation of brain neuroblasts. Staining with a BrdU-specific antibody revealed reduced levels of BrdU incorporation in hypermethylated metaphase (M) and anaphase (A) chromosomes. Black fractions represent complete staining, grey fractions partial staining, and white fractions no staining. Because most of the interphase (I) nuclei were presumably in G1 phase, only a small percentage incorporated BrdU. The data are based on the examination of several hundred nuclei from several independent preparations.