FIG. 6.

DNMTs relocalize in the presence of 5-azadC, and DNMT1 colocalizes with γ-H2AX DNA damage foci in 5-azadC-treated cells. (A) HeLa cells were transfected with GFP-DNMT1 or GFP-Dnmt3b1. Cells were then mock treated (untreated) or were treated with 10 μM 5-azadC for 48 h, fixed, and then examined for localization of GFP signal. Both GFP-DNMTs localize diffusely throughout the nucleoplasm and are also concentrated in DAPI-dense regions corresponding to heterochromatin in untreated cells (particularly Dnmt3b; green staining), as has been shown previously by us along with other investigators. Upon 5-azadC treatment, DNMT1 and Dnmt3b1 relocalize, becoming aggregated into more discrete foci and absent from DAPI-dense heterochromatin regions. 5-azadC-treated cells were also stained for γ-H2AX (red staining), showing the formation of characteristic foci in cells with damaged DNA. Overlaid images (far right) of the red and green signals show that DNMT1 is highly colocalized with γ-H2AX (yellow signal). (B) DNA staining of untreated and 5-azadC-treated HeLa cells to demonstrate that there is no gross disruption of heterochromatin or nuclear morphology in 5-azadC-treated cells. (C) Colocalization of activated ATM (phosphorylated at serine 1981) and γ-H2AX in 5-azadC-treated HeLa cells. Bar, 5 μm.