Radically truncated MeCP2 rescues Rett syndrome-like neurological defects

Abstract

Heterozygous mutations in the X-linked MECP2 gene cause the neurological disorder Rett syndrome. The methyl-CpG-binding protein 2 (MeCP2) protein is an epigenetic reader whose binding to chromatin primarily depends on 5-methylcytosine. Functionally, MeCP2 has been implicated in several cellular processes on the basis of its reported interaction with more than 40 binding partners, including transcriptional co-repressors (for example, the NCoR/SMRT complex), transcriptional activators, RNA, chromatin remodellers, microRNA-processing proteins and splicing factors. Accordingly, MeCP2 has been cast as a multi-functional hub that integrates diverse processes that are essential in mature neurons. At odds with the concept of broad functionality, missense mutations that cause Rett syndrome are concentrated in two discrete clusters coinciding with interaction sites for partner macromolecules: the methyl-CpG binding domain and the NCoR/SMRT interaction domain. Here we test the hypothesis that the single dominant function of MeCP2 is to physically connect DNA with the NCoR/SMRT complex, by removing almost all amino-acid sequences except the methyl-CpG binding and NCoR/SMRT interaction domains. We find that mice expressing truncated MeCP2 lacking both the N- and C-terminal regions (approximately half of the native protein) are phenotypically near-normal; and those expressing a minimal MeCP2 additionally lacking a central domain survive for over one year with only mild symptoms. This minimal protein is able to prevent or reverse neurological symptoms when introduced into MeCP2-deficient mice by genetic activation or virus-mediated delivery to the brain. Thus, despite evolutionary conservation of the entire MeCP2 protein sequence, the DNA and co-repressor binding domains alone are sufficient to avoid Rett syndrome-like defects and may therefore have therapeutic utility.

Extended Data Figure 1. Design of the MeCP2 deletion series

a, Diagram of the genomic DNA sequences encoding WT and ΔNIC MeCP2, showing the retention of the extreme N-terminal amino acids encoded in exons 1 and 2 and the first 10 bp of exon 3, the deletion of the N- and C-terminal regions, the replacement of the intervening region with a linker and SV40 NLS, and the addition of the C-terminal EGFP tag. Colour key: 5’UTR=white, MBD=blue, NID=pink, other MeCP2 coding regions=grey, SV40 NLS=orange, linkers=dark grey and EGFP=green. b, The N-terminal ends of the sequences of all three truncated proteins (e1 and e2 isoforms) showing the fusion of the extreme N-terminal amino acids to the MBD (starting with P72). c, d, Protein sequence alignment of the MBD (c) and NID (d) regions using ClustalWS, shaded according to BLOSUM62 score. Both alignments are annotated with RTT-causing missense mutations (red), activity-dependent phosphorylation sites,, (orange), sequence conservation, interaction domains and known/predicted structure. Interaction sites: methyl-DNA binding (residues 78-16213), AT hook 1 (residues 183-19536), AT hook 2 (residues 257-27228), NCoR/SMRT binding (residues 285-3095). The bipartite nuclear localisation signal (NLS) is also shown (residues 253-256 and 266-271). The regions retained in ΔNIC are: MBD resides 72-173 (highlighted by the blue shading in panel c) and NID resides 272-312 (highlighted by the pink shading panel d). Residue numbers correspond to that of mammalian e2 isoforms.

Extended Data Figure 2. Truncated MeCP2 proteins retain the ability to bind methylated DNA and the NCoR/SMRT complex

a, EGFP-tagged truncated proteins immunoprecipitate components of the NCoR/SMRT co-repressor complex: NCoR, HDAC3 and TBL1XR1. WT and R306C were used as positive and negative controls for binding, respectively. ‘In’ = input, ‘IP’ = immunoprecipiate. For gel source data, see Supplementary Information. b, EGFP-tagged truncated MeCP2 proteins localise to mCpG-rich heterochromatic foci when overexpressed in mouse fibroblasts (NIH-3T3 cells). WT and R111G were used as controls to show focal and diffuse localisation, respectively. Scale bars indicate 10 µm. c, EGFP-tagged truncated proteins recruit TBL1X-mCherry to heterochromatin when co-overexpressed in NIH-3T3 cells. WT and R306C were used as positive and negative controls for TBL1X-mCherry recruitment, respectively. scale bars indicate 10 µm. Quantification (right) shows the percentage of cells with focal TBL1X-mCherry localisation, evaluated relative to WT using Fisher’s exact tests: R306C **** P<0.0001, ΔN P=0.071, ΔNC P=0.604, ΔNIC P=0.460. Total numbers of cells counted: WT n=117, R306C n=119, ΔN n=113, ΔNC n=119, ΔNIC n=125.

Extended Data Figure 3. Generation of ΔN and ΔNC mice

Diagrammatic representation of ΔN (a) and ΔNC (b) knock-in mouse line generation. The endogenous Mecp2 allele was targeted in male ES cells. The site of Cas9 cleavage in the WT sequence is shown by the scissors symbol (used for production of ΔN knock-in ES cells). The selection cassette was removed in vivo by crossing chimaeras with deleter (CMV-Cre) transgenic mice. Southern blot analysis shows correct targeting of ES cells and successful cassette deletion in the knock-in mice. The solid black line represents the sequence encoded in the targeted vector and the dotted lines indicate the flanking regions of mouse genomic DNA. For gel source data, see Supplementary Information.

Extended Data Figure 4. ΔN and ΔNC knock-in mice express truncated proteins at approximately WT levels and display minimal phenotypes

a, Western blot analysis of whole brain extract showing protein sizes and abundance of MeCP2 in ΔN and ΔNC mice and WT-EGFP controls, detected using a GFP antibody. Histone H3 was used as a loading control. *denotes a non-specific band detected by the GFP antibody. For gel source data, see Supplementary Information. b, Flow cytometry analysis of protein levels in nuclei from whole brain (‘All’) and the high-NeuN subpopulations (‘Neurons’) in WT-EGFP (n=3), ΔN (n=3) and ΔNC (n=3) mice, detected using EGFP fluorescence. Graph shows mean ± S.E.M. and genotypes were compared to WT-EGFP controls by t-test: ‘All’ ΔN P=0.338, ΔNC ** P=0.003; and ‘Neurons’ ΔN P=0.672, ΔNC * P=0.014. c, Flow cytometry analysis of protein levels in WT (n=3) and WT-EGFP (n=3) mice, detected using an MeCP2 antibody. Graph shows mean ± S.E.M. and genotypes were compared by t-test: ‘All’ P=0.214; and ‘Neurons’ P=0.085. d, Example histogram (of one WT-EGFP sample) showing how the ‘Neuronal’ subpopulation was defined according to NeuN-AF647 staining. ‘au’ = arbitrary units. e, f, g, Growth curves of the backcrossed scoring cohorts (e and f; see Fig. 2a-d) and an outbred (g; 75% C57BL/6J) cohort of ΔNC mice (n=7) and WT littermates (n=9). Graphs show mean values ± S.E.M. Genotypes were compared using repeated measures ANOVA: ΔN P=0.362, ΔNC **** P<0.0001, ΔNC (outbred) P=0.739. Mecp2-null data (n=20) is shown as a comparator for the backcrossed cohorts. h, Behavioural analysis of ΔN (n=10) and ΔNC mice (n=10) compared to their WT littermates (n=10) at 20 weeks of age (see Fig. 2e-g). Total distance travelled in the Open Field test was measured during a 20 minute trial. Graphs show individual values and medians. Genotypes were compared using t-tests: ΔN P=0.691; ΔNC P=0.791. ‘n.s.’ = not significant.

Extended Data Figure 5. Generation of ΔNIC and STOP mice

Diagrammatic representation of ΔNIC and STOP mouse line generation. The endogenous Mecp2 allele was targeted in male ES cells. The site of Cas9 cleavage in the WT sequence is shown by the scissors symbol. The selection cassette was removed in vivo by crossing chimaeras with deleter (CMV-Cre) transgenic mice to produce constitutively expressing ΔNIC mice, or retained to produce STOP mice. Southern blot analysis shows correct targeting of ES cells and successful cassette deletion in the ΔNIC knock-in mice. The solid black line represents the sequence encoded in the targeted vector and the dotted lines indicate the flanking regions of mouse genomic DNA. For gel source data, see Supplementary Information.

Extended Data Figure 6. ΔNIC mice have a normal lifespan and no activity phenotype but decreased body weight

a, Kaplan-Meier plot showing survival of an outbred (75% C57BL/6J) cohort of ΔNIC mice (n=10) and their WT littermate (n=1). b, Growth curve of the backcrossed cohort used for phenotypic scoring (see Fig. 3d-e). Graph shows mean ± S.E.M. Genotypes were compared using repeated measures ANOVA **** P<0.0001. Mecp2-null data (n=20) is shown as a comparator. c, Behavioural analysis of ΔNIC mice (n=10) compared to their WT littermates (n=10) at 20 weeks of age (see Fig. 3f-h). Total distance travelled the Open Field test was measured during a 20 minute trial. Graph shows individual values and medians. Genotypes were compared using a t-test P=0.333. ‘n.s.’ = not significant.

Extended Data Figure 7. ΔNIC mice have a less severe phenotype than the mildest mouse model of RTT, R133C

a, b, c, Repeat presentation of phenotypic analysis of ΔNIC mice and WT littermates in Fig. 3d-e and Extended Data Fig. 6b, this time including EGFP-tagged R133C mice (n=10) as a comparator.

Extended Data Figure 8. ‘STOP’ mice with transcriptionally silenced ΔNIC resemble Mecp2-nulls

a, Western blot analysis of whole brain extract showing protein sizes and abundance of MeCP2 in STOP mice and WT-EGFP and ΔNIC controls, detected using a GFP antibody. Histone H3 was used as a loading control. *denotes a non-specific band detected by the GFP antibody. For gel source data, see Supplementary Information. b, Flow cytometry analysis of protein levels in nuclei from whole brain (‘All’) and the high-NeuN subpopulation (‘Neurons’) in WT-EGFP (n=3), ΔNIC (n=3) and STOP (n=3) mice, detected using EGFP fluorescence. Graph shows mean ± S.E.M. and genotypes were compared using t-tests: **** denotes a P value <0.0001. ‘au’ = arbitrary units. c, Phenotypic scoring of STOP mice (n=22) compared to published Mecp2-null data (n=12). Graph shows mean scores ± S.E.M. d, Kaplan-Meier plot showing survival of STOP mice (n=14) compared to Mecp2-null data (n=24).

Extended Data Figure 9. Successful activation of ΔNIC in Tamoxifen-injected STOP CreER^T mice led to symptom reversal

a, Southern blot analysis of genomic DNA to determine the level of recombination mediated by CreER^T in Tamoxifen-injected (+Tmx) STOP CreER^T animals. WT, WT CreER^T, ΔNIC and STOP samples, with or without Tamoxifen injection, were included as controls. (Bsu36I digestion, see restriction map in Extended Data Fig. 5.) b, Protein levels in Tamoxifen-injected STOP CreER^T animals were determined using western blotting (upper, n=5) and flow cytometry (lower, n=3). Constitutively expressing ΔNIC mice (n=3) were used as a comparator. Graphs show mean values ± S.E.M. (quantification by western blotting is shown normalised to ΔNIC). Genotypes were compared using t-tests: western blotting P=0.434; flow cytometry ‘All’ nuclei P=0.128 and ‘Neuronal’ nuclei * P=0.016. ‘au’ = arbitrary units. For gel source data, see Supplementary Information. c, Heatmap of the phenotypic scores of the Tamoxifen-injected STOP CreER^T (upper; n=9) and STOP (lower; n=9 until 8 weeks of age, see survival plot in Fig. 4c) animals (see Fig. 4b), divided into the six categories. The plot is shaded according to the mean score for each category.

Extended Data Figure 10. Virus-encoded ΔNIC is expressed in brain and does not have adverse consequences in WT mice

a, b, Representative confocal images from thalamus and brainstem of scAAV-injected Mecp2-null (a) and WT (b) mice; scale bars indicate 20 µm visualised using an antibody against the Myc epitope (red) and the neuronal marker NeuN (green). Nuclei are stained with DAPI (blue). Graphs show transduction efficiency (mean ± SEM) in different brain regions (n=3 mice per genotype, 27 fields from each brain region). c, Phenotypic scoring (mean ± SEM) of scAAV-injected mice from 5-30 weeks: WT + vehicle (n=15), Mecp2-null + vehicle (n=20) and WT + hΔNIC (n=14). d, Kaplan-Meier plot showing survival of the cohort shown in panel c. One WT + hΔNIC animal was culled due to injuries at 28 weeks of age (shown by a tick). An arrow indicates the timing of the viral injection.

Figure 1. Stepwise truncation of MeCP2 protein to retain only the MBD and NID

a, Diagram of human MeCP2 protein sequence with the Methyl-CpG Binding Domain (MBD) and the NCoR/SMRT Interaction Domain (NID); annotated to show single nucleotide polymorphisms (SNPs) in males in the general population (black lines) and RTT-causing missense mutations (red lines). Sequence identity between human and other vertebrate MeCP2 proteins is shown by purple bars and insertions by dark lines. b, Schematic diagram of the deletion series based on the mouse e2 isoform that were generated in this study, compared with WT-EGFP.

Figure 2. Deletion of the MeCP2 N- and C-terminal regions has minimal phenotypic consequence

a, b, Phenotypic severity scores of hemizygous male (a) ΔN mice (n=10) and (b) ΔNC mice (n=10), compared to their WT littermates (n=10) over one year. Graphs show mean scores ± S.E.M. Published Mecp2-null data (n=12) is shown as a comparator. c, d, Kaplan-Meier plots showing survival of the cohorts shown in panels a and b. Mecp2-null data (n=24) is shown as a comparator. e,f,g, Behavioural analysis of separate cohorts performed at 20 weeks of age: ΔN (n=10) and ΔNC mice (n=10 for Open Field/Rotarod; 11 for Elevated Plus Maze), each compared to WT littermates (n=10). Graphs show individual values and medians, and statistical significance as follows : not significant (‘n.s.’) P>0.05, * P<0.05. e, Time spent in the closed and open arms of the Elevated Plus Maze during a 15 min trial. Genotypes were compared using KS tests: ΔN closed arms P=0.988 and open arms P=0.759; ΔNC closed arms P=0.950 and open arms P=0.932. f, Time spent in the central region of the Open Field test was measured during a 20 minute trial. Genotypes were compared using t-tests: ΔN P=0.822; ΔNC * P=0.020. g, Average latency to fall from the Accelerating Rotarod in four trials was calculated for each of the three days of the experiment. Genotypes were compared using KS tests: ΔN day 1 P=0.759, day 2 P=0.401 and day 3 P=0.055; ΔNC day 1 P=0.988, day 2 P=0.401 and day 3 P=0.759.

Figure 3. Additional deletion of the intervening region leads to protein instability and mild RTT-like symptoms

a, Western blot analysis of whole brain extract showing protein sizes and abundance of MeCP2 in ΔNIC mice and WT-EGFP controls, detected using a GFP antibody. Histone H3 was used as a loading control. *denotes a non-specific band detected by the GFP antibody. For gel source data, see Supplementary Information. b, Flow cytometry analysis of protein levels in nuclei from whole brain (‘All’) and the high-NeuN subpopulation (‘Neurons’) in ΔNIC mice (n=3) and WT-EGFP controls (n=3), detected using EGFP fluorescence. Graph shows mean ± S.E.M. and genotypes were compared by t-test: ‘All’ *** P=0.0002 and ‘Neurons’ *** P=0.0001. ‘au’ = arbitrary units. c, Quantitative PCR analysis of mRNA prepared from whole brain of ΔNIC mice (n=3) and WT-EGFP controls (n=3). Mecp2 transcript levels were normalised to Cyclophilin A mRNA. Graph shows mean ± S.E.M. (relative to WT-EGFP) and genotypes were compared by t-test: P=0.110. d, Phenotypic severity scores of ΔNIC mice (n=10) compared to WT littermates (n=10) over one year. Graph shows mean scores ± S.E.M. Mecp2-null data (n=12) is shown as a comparator. e, Kaplan-Meier plot showing survival of the cohort shown in panel d. One ΔNIC animal died at 43 weeks, after receiving phenotypic scores of ≤2.5. Mecp2-null data (n=24) is shown as a comparator. f, g, h, Behavioural analysis of a separate cohort performed at 20 weeks of age: ΔNIC (n=10) compared to WT littermates (n=10). Graphs show individual values and medians, and statistical significance as follows: not significant (‘n.s.’) P>0.05, * P<0.05, ** P<0.01. f, Time spent in the closed and open arms and centre of the Elevated Plus Maze during a 15 minute trial. Genotypes were compared using KS tests: closed arms ** P=0.003, open arms P=0.055 and centre * P=0.015. g, Time spent in the central region of the Open Field measured during a 20 minute trial. Genotypes were compared using a t-test: P=0.402. h, Average latency to fall from the Accelerating Rotarod in four trials was calculated for each of the three days of the experiment. Genotypes were compared using KS tests: day 1 P=0.164, day 2 P=0.055 and day 3 ** P=0.003. Changed performance (learning/worsening) over the three day period was determined using Friedman tests: wild-type animals P=0.601, ΔNIC animals ** P=0.003.

Figure 4. Activation or viral transduction of ΔNIC ameliorates neurological phenotypes in MeCP2-deficient mice

a, Timeline of Cre-mediated activation of ΔNIC induced by Tamoxifen injections. b, Phenotypic severity scores (mean ± SEM) of mice injected with Tamoxifen (arrows) from 4-28 weeks: WT (n=4), WT CreER^T (n=4), STOP (n=9) and STOP CreER (n=9). c, Kaplan-Meier plot showing survival of the cohort shown in panel b. d, Diagram of the DNA sequence inserted into an scAAV viral vector, comprising a 426 nt Mecp2 promoter driving the human ΔNIC coding sequence plus a C-terminal Myc tag and 3’ UTR. A vector containing full-length human MECP2 is shown for comparison. e, Timeline of the scAAV-mediated gene therapy experiment. f, Phenotypic severity scores (mean ± SEM) of scAAV-injected mice from 5-30 weeks: WT + vehicle (n=15), Mecp2-null + vehicle (n=20) and Mecp2-null + hΔNIC (n=17). g, Kaplan-Meier plot showing survival of the cohort shown in panel f. Four Mecp2-null + hΔNIC animals reached their humane end-point. Five Mecp2-null + ΔNIC animals were culled due to injuries unrelated to RTT-like phenotypes at 16, 23, 25, 26 and 29 weeks of age (data shown as ticks). Survival of Mecp2-null + ΔNIC animals was compared to Mecp2-null + vehicle controls using the Mantel-Cox test: P=<0.0001.