FIGURE 3.

The chromatin binding ability of MeCP2 is elevated in PARP-1^−/− cells. A–D, image analysis protocol for quantification of chromocenter accumulation. All steps necessary for analysis were performed using Volocity 5.5 built-in functions such as image processing, object segmentation, and measurements. Z stacks of cells transfected with a GFP fusion protein were cropped to obtain single nuclei per image. Thresholds for segmentation were set individually for each nucleus. The chromocenter and nucleoplasm were segmented utilizing the built-in thresholding function, which uses the percentage of the overall image intensity. A, nucleus of an MeCP2-GFP-expressing cell before segmentation. Bottom right panel, the nucleus in x-y axis view. Top right and bottom left panels, the nucleus in x-z and y-z axis views, respectively. Scale bars = 5 μm. B, histogram of the frequency of fluorescence intensities plotted on a logarithmic scale. The thresholds for chromocenter (CC) and nucleoplasm (NP) segmentation are displayed in red and blue, respectively. Care was taken to exclude chromocenters from nucleoplasm segmentation and vice versa. C and D, visualization of chromocenter (red) and nucleoplasm (blue) segmentation according to the thresholds displayed in B. E, WT or PARP-1^−/− MEF cells were transfected with expression vectors coding GFP- or YFP-tagged MeCP2 constructs as indicated. Z stacks of images were taken from cells expressing comparable levels of the GFP-fused construct. Experiments were repeated at least twice with as many as 30 cells analyzed per construct each time. The graphs show the accumulation of the MeCP2 constructs at heterochromatin in PARP-1^−/− cells normalized to wild-type mouse fibroblasts. *, p < 0.05; **, p < 0.001. Error bars represent 95% confidence interval. F, FRAP analysis of full-length GFP-tagged MeCP2 and mutant proteins. Top panel, an example of MeCP2G protein recovery after photobleaching. Circles indicate the bleached region. Scale bars = 5 μm. Center panel, fluorescence recovery curve for each construct. The experiment was repeated twice with 7–15 cells used each time for analysis. Results were averaged, and the mean value was plotted. Bottom panel, the half-time of each construct and p value for WT and PARP-1^−/− cell line are calculated using a t test. The cell number and median are indicated beside the box plot. G, in situ extraction of full-length GFP-tagged MeCP2 and mutant proteins. Top panel, representative images of MeCP2G and MeCP2Y.1 extraction after 0.5% Triton X-100 treatment. Scale bars = 5 μm. Bottom panel, normalized fluorescence intensity before and after treatment. For each construct, the normalized intensity was averaged, and the mean value was plotted. The experiment was repeated twice, and each time 5–10 cells were used for analysis.