Journal: Communications Biology
Article Title: MeCP2-driven chromatin organization controls nuclear stiffness
doi: 10.1038/s42003-025-09328-6
Figure Lengend Snippet: a Scheme of the main domains of the MeCP2 protein (MBD: methyl binding domain; TRD: transcription repression domain) and the location of the mutations studied. b Clustering analysis using 3D confocal microscopy on fixed C2C12 cells, untransfected or transfected with plasmids containing wt or mutant MeCP2 cDNA. Cells were fixed with formaldehyde and stained with DAPI (n number used for each condition: mb = 35, wt MeCP2 = 40; P101H = 15; R106W = 13; R133C = 20; A140V = 14; T158M = 14; R168X = 7; R255X = 20; R270X = 29; R294X = 19). Only transfected cells were analyzed; however, their expression levels were heterogeneous, which is reflected in the variability observed in the atomic force microscopy results. The mean and 95% confidence interval of the number of heterochromatin compartments and the average volume of the compartments per cell are represented. The violin plots containing the individual data are shown in Supplementary Fig. . c Representative histogram of the elastic modulus with Gaussian mixture model (GMM) fits. Nuclei were purified from untransfected C2C12 cells (mb ut), seeded on an agarose pad, and analyzed by atomic force microscopy to determine their elastic modulus. A three-component GMM was applied, showing the overall model fit (black line) and the individual subpopulations (cyan, blue, and magenta lines). Histograms of the elastic modulus values for wild type MeCP2 and mutant nuclei are provided in Supplementary Fig. . d Dendrogram showing the effect of Rett mutations on myoblasts based on elastic modulus values distribution. Elastic modulus histograms were fitted with a GMM as described above to obtain the mean modulus values of each subpopulation (Supplementary Fig. ). In addition, in order to make the populations comparable, all data were pulled together and fitted to 3 populations, followed by the assignment of the individual data to the three populations based on k-means to obtain the weight of the populations (see Supplementary Fig. ). All population means and weights were normalized using z-scores, and Euclidean distances were calculated and represented in a dendrogram. The mutants were classified into mild (gray) and severe (bold) based on previous publications. R270X is variably classified as mild or severe in the literature, likely due to differences in clinical scoring parameters and diagnostic criteria applied across cohorts. e Relationship between heterochromatin organization and stiffness contributions of nuclear subpopulations. Linear regressions showing the relationship between the heterochromatin organization index (HOI, log₁₀-transformed) and the weighted stiffness of the soft (left), mid (center), and stiff (right) nuclear fractions across MeCP2 mutants. Weighted stiffness values were calculated by multiplying the proportion of each population by its representative elastic modulus acquired from k-means clustering (1.7, 7.4, and 40.7 kPa for soft, mid, and stiff, respectively). The analysis revealed a negative trend for the soft fraction ( β = –0.68, R 2 = 0.20), a weak positive trend for the mid fraction ( β = 1.50, R 2 = 0.09), and a significant positive relationship for the stiff fraction ( β = 7.91, R 2 = 0.40, p = 0.037), indicating that increased heterochromatin organization (higher HOI) is associated with greater mechanical stiffening of nuclei. f . Dendrogram of the Rett mutant rescue of NSC MeCP2 KO based on the elastic modulus distribution. The procedure was done as described for d , based on GMM populations (Supplementary Fig. ) and the k-means weights (Supplementary Fig. ).
Article Snippet: This was achieved by transfecting the J1 ESC MIN-MeCP2 with a plasmid coding for the Bxb1 recombinase (pCAG-NLS-Bxb1, Addgene #65625, Supplementary Table ) together with a plasmid containing the attB site, followed by mCherry cDNA and a stop codon (pattB-Cherry-Stop-puro, Addgene #65529, Supplementary Table ).
Techniques: Binding Assay, Confocal Microscopy, Transfection, Mutagenesis, Staining, Expressing, Microscopy, Purification, Diagnostic Assay, Transformation Assay
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