Fig 2: MeCP2 binds mCG and mCAC, and to a lesser extent, mCAT(A) Bio-layer interferometry (BLI) analysis of the interaction between the MBD of MeCP2 and methylated DNA probes containing a single mCG, mCAC, or mCAT site. KD values: mCG 22.25 ± 3.12 nM; mCAC 13.90 ± 0.64 nM; mCAT 63.71 ± 6.94 nM (means ± SEMs). Binding affinity to mCG and mCAC is similar (not significant [NS] p = 0.06); binding to mCAT is significantly weaker than mCG and mCAC (**p = 0.006 and **p = 0.002, respectively), t tests.(B) Western blot analysis of MeCP2 following pull-down from rat brain nuclear extracts using immobilized DNA containing unmethylated and methylated CG, CAC, and CAT (top) and unbound MeCP2 in the supernatant (bottom).(C) Whole-genome bisulfite sequencing analysis in the mouse hypothalamus (Lagger et al., 2017) showing the distribution of methylation at each CN dinucleotide (top) and each CAN trinucleotide (bottom). Sites were binned by level of methylation.(D–F) ATAC-seq footprinting analysis of MeCP2 over methylated (top) and unmethylated (bottom) CG (D), CAC (E), and CAT (F) sites in the mouse hypothalamus.

Fig 3: Human MECP2‐KO neurons exhibit alterations in synapse‐relevant genes and pathways Pluripotent stem cells (PSCs) with one of two distinct exonic loss‐of‐function mutations (Q83X nonsense or K82 frameshift (K82fs)) at the MECP2 locus were generated and differentiated into neurons via a neural progenitor cell (NPC) intermediate.Immunofluorescent staining confirmed the absence of MeCP2 in MECP2‐KO PSCs, NPCs, and neurons. Scale bar = 10 µm.RT–qPCR array revealed differential gene expression between control and MECP2‐KO 28‐day neurons. The P values are calculated based on a Student’s t‐test of the replicate 2−ΔΔ C t values for each gene in the control group and KO groups (WT83/Q83X cell lines were used; N = 3 clones from each genotype).Overtime quantitative gene expression profile of many of the involved genes relevant to synaptic function (days 14 and 28 post‐differentiation; normalized by control NPC expression).Single‐cell RT–qPCR analysis distinguished populations of control and KO 28‐day neurons. Principal component analysis (PCA) of 441 cells projected onto the first two components. Overlaid populations of WT83/Q83X and WT82/K82fs neurons are shown.Violin plots of selected genes showing the comparison between control and MECP2‐KO neurons from the single‐cell analyses (log2(expression) values) that overlapped with the results obtained via RT–PCR array.Volcano plot illustrates differences in expression patterns of target genes of neurons from the qPCR single‐cell analyses. The dotted lines represent differentially expressed genes between the groups at P < 0.05 (unpaired Student’s t‐test).Left: Schematic of laminar cortical markers. Right: Top: Quantitation of laminar cell markers based on the single‐cell RT–qPCR analysis (two‐way analysis of variance (ANOVA), N = 3 replicates per genotype, F 3,24 = 3.31, *P = 0.04, with difference in the proportion of cortical layers V and VI, 95% confidence interval (CI) [8.31, 42.41]); Bottom: altered proportions of neurotransmitter markers (two‐way ANOVA, N = 3 replicates per genotype, F 5,24 = 27.33, ***P < 0.0001, with differences in the proportions of glutamatergic, 95% CI [22.33, 33.60], and cholinergic, 95% CI [0.166, 11.43], neurons).Gene ontology (GO) analysis of targeted genes relevant to synaptic function displaying the differences between MECP2‐KO and control neurons. For the full list of markers used see Dataset EV1. Data information: Note that asterisks signify statistically significant difference from MECP2‐KO. Data are presented as mean ± standard error of the mean (s.e.m.).

Fig 4: Normal survival and absence of RTT-like signs in peripheral KO mice. (A) Survival plot showing normal survival of WT (blue circles, n = 17) and PKO mice (green squares, n = 7) compared to reduced lifespan in KO mice (red triangles, n = 8). The median survival period was significantly reduced in KO mice (***P < 0.001, log-rank test). (B) Plot showing aggregate phenotype severity (mean ± SEM) based on an established observational scoring system. There was a highly significant difference in score between WT and PKO animals from 8 weeks onwards when compared to KO (***P < 0.001, Kruskal-Wallis test with Dunn’s post hoc analysis). No significant difference was seen between WT and PKO at the time of behavioural testing (15 weeks) but these groups differed significantly at 1 year (*P < 0.05). (C) Plot showing body weight changes over time (mean ± SEM). No significant differences were seen between genotypes at the time of behavioural testing (15 weeks; P > 0.05, one-way ANOVA) but PKO mice had significantly lower bodyweight than WT mice when compared at 1 year (***P < 0.001, student’s unpaired t-test). In B) and C) group sizes at the start of the experiment are as given in A).

Fig 5: MeCP2G273X-EGFP Mice Appear Unaffected by a Mutation Inactivating the MeCP2 NLS(A) Survival curves for Mecp2G273X-EGFP (n = 8) and Mecp2G273XΔNLS-EGFP mice (n = 9), as well as wild-type controls for each group (n = 12 and n = 10, respectively). No significant difference between survival of Mecp2G273X-EGFP and Mecp2G273XΔNLS-EGFP mice was observed (p = 0.78; Kolmogorov-Smirnov test). An illustrative survival curve for Mecp2-null mice (based on data from previous studies) is shown as a broken line.(B) Average phenotypic severity score of surviving animals plotted against age for each of the groups. Data for each group are plotted up to the last point, where there are at least three surviving animals.(C) Expression levels relative to Gapdh of genes that are upregulated (Flrt3 and Arhgap10) or downregulated (Sst, Tspan17, and Gabbr1) in brains of Mecp2 mutant mice as assessed by qPCR. Data are presented as means ± SD.(D) Brain weights for Mecp2G273X-EGFP and Mecp2G273XΔNLS-EGFP mice (n = 4 for each group) as well as wild-type controls (n = 5 and n = 3). Indicated statistical significance was calculated using the t test. Data are presented as means ± SD.(E) Cortical thickness plotted for Mecp2G273X-EGFP and Mecp2G273XΔNLS-EGFP mice (n = 4 each) and wild-type control animals (n = 5 and n = 3). Data are presented as means ± SD. Indicated statistical significance was calculated using the t test. The image depicts the area of the primary motor cortex (M1) measured. The scale bar represents 1,000 μm.See also Figure S2.