MeCP2-induced chromatin unfolding acts independently of the methyl-cytosine-binding domain.

<p>(A) The images show AO3_1 cells (CHO-derived clone containing an amplified chromosomal region consisting of the DHFR cDNA transgene and 256 lac operator repeats) that were transfected (green signal) with EGFP-lacR (control) or EGFP-lacR-tagged full-length MeCP2, -VP16 and -MeCP2 separate domains (i.e. MBD, ΔC-terminus, TRD, C-terminus and R133C Rett syndrome mutation). The images show a typical representation of cells transfected for 48 hours. For quantitative analysis 30 nuclei per condition were measured with comparable microscopical set-up. The images represent examples of individual optical sections. Bar = 5 μm. (B) The figure shows the changes in lacO array large-scale chromatin structure measured with a 3D image analysis parameter, i.e. the surface factor. The surface factor determines the surface of a given chromosomal domain/object normalized to the surface of a sphere with an equal volume <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069347#pone.0069347-Rottach1" target="_blank">[43]</a>. The distribution of the surface factor measurements is plotted as a box-plot. The second and third quartiles of the observed values are within the box, the median value is shown by the white horizontal line, the whiskers show the first and fourth quartiles of the observed values, dots are the outliers. The dotted line represents the ∼20% of cells in the EGFP-lacR control population that exhibit a mildly decondensed array (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069347#pone.0069347-Li1" target="_blank">[69]</a>). Considering a population (20%) of control cells exhibiting a decondensed configuration as the threshold of MeCP2-induced decondensation, the following percentages are found for lacR control 18%, VP16 68%, MeCP2 full length 47%, MBD 23%, ΔC-terminus 23%, C-terminus 47% and R133C 50%). We scored the chromatin structure based on the surface factor in the full cell population. Based on our statistical analysis the EGFP-lacR tagged VP16, MeCP2 (full length) and the R133C population are significantly different from EGFP-lacR control, whereas EGFP-lacR tagged MBD, TRD, C-terminus and ΔC-terminus, are not significantly different from EGFP-lacR (for p values see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069347#pone-0069347-t001" target="_blank">Table 1</a>). (C) The figure shows the intensity of the transfected constructs measured within the lacO chromsomal array and plotted versus the chromatin surface factor measurements of the respective cells (I: Control, VP16 and MeCP2, II: MBD, TRD, C-terminus, ΔC-terminus and R133C). The Pearson correlation coefficient (Ρ) of the intensity of the transfected constructs and the determined surface factors are given.