Rett-causing mutations reveal two domains critical for MeCP2 function and for toxicity in MECP2 duplication syndrome mice
- Baylor College of Medicine, United States
- Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, United States
- Harvard Medical School, United States
- Howard Hughes Medical Institute, Baylor College of Medicine, United States
Figures
Figure 1
Generation of transgenic lines.
(A) Schematic representation of MeCP2-EGFP fusion protein, showing the methyl-CpG binding domain (MBD, blue), transcriptional repression domain (TRD, red), a cluster of basic amino acids (yellow), and enhanced green fluorescent protein (EGFP, green). The location of the residues arginine 111 (R111) and arginine 306 (R306) are shown. (B) Western blot analyses of whole brain lysates show that two independent lines of MeCP2-R111G and MeCP2-R306C express the transgene at levels similar to endogenous MeCP2 as judged by the similar intensities of WT and transgenic bands using an anti-MeCP2 antibody. Quantification of total MeCP2 is shown to the right for each mutant. An anti-GAPDH antibody was used to detect GAPDH as a loading control. (C) Immunofluorescence (IF) of midsagittal brain sections (2 months, upper panel) shows that the expression pattern of the transgenes throughout the whole brain (anti-GFP antibody, green) parallels that of endogenous MeCP2 (anti-MeCP2 antibody, red). Colocalization is visible as yellow in the merged image, with 4',6-diamidino-2-phenylindole (DAPI, blue) as a general marker for the nucleus. Closer examination of the cortex and cerebellum (lower panel) mirrors this.
Figure 2
R111G mice phenocopy the null mice whereas R111G Tg mice are indistinguishable from WT mice.
(A) Using a Kaplan–Meier survival curve, both WT (black, n = 11) and R111G Tg (blue, n = 10) have a normal lifespan, whereas R111G mice (green, n = 10) phenocopy the premature lethality of the null mice (red, n = 11), with a median lifespan of 11 weeks. (B–I) Behavioral assays performed on WT (n = 7) and R111G Tg (n = 7) mice at 9 months of age show that R111G Tg mice are indistinguishable from WT mice in a variety of assays. The open field assay reveals that both lines travel the same distance (B) and have the same percentage of time traveled in the center (C), indicating that they are not anxious, which is confirmed by the percentage of time spent in the dark in the light/dark assay (D). Motor function and coordination were unchanged as measured by time spent on the rod (for four trials a day for four days) in rotarod (E) and footslips per centimeter traveled in parallel rod footslip (F). Purposeful paw movement is also normal, as assayed by nest building after 24 hr (G). R111G Tg mice also have no learning and memory deficits, as assayed by their freezing in the contextual (H) and cued (I) conditioned fear test. Data were analyzed by an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. Results were plotted as the mean ± SEM. n.s. not significant.
Figure 3
R111G abolishes MeCP2 binding to methyl-CpGs but does not affect its ability to interact with known binding partners.
(A) MeCP2-R111G does not bind to methyl-CpGs in vivo. Immunofluorescence for endogenous MeCP2 (anti-MeCP2 antibody, red), the transgene (anti-GFP antibody, green), and heterochromatic foci (puncta within the nucleus visualized by DAPI, blue), shows that endogenous MeCP2 localizes to the heterochromatic foci and can therefore bind methyl-CpGs, whereas MeCP2-R111G is diffuse within the nucleus. (B) MeCP2-R111G retains the ability to bind known interactors in vivo. Immunoprecipitation (IP) with anti-GFP antibody was used to determine if MeCP2-R111G binds known interactors. Using WT mice lacking GFP as a negative control and MeCP2-EGFP as a positive control, we show that MeCP2-R111G retains binding to Sin3a, HDAC1, and HDAC2 of the Sin3a complex and HDAC3 and Tbl1 of the NCoR complex. I = input, E = eluate.
Figure 4
R306C mice have a milder phenotype than null mice.
(A) A Kaplan–Meier survival curve shows that WT (black, n = 28) and R306C Tg (blue, n = 22) mice have a normal lifespan, while R306C (green, n = 21) mice have a shortened lifespan with 50% lethality at 18 weeks, but not as severely as the null mice (red, n = 26), with 50% survival at 11 weeks. (B) A weight curve shows that WT and R306C Tg mice have normal weights whereas R306C mice have a milder obesity phenotype. R306C mice are more overweight than WT and R306C Tg mice, but not as severely affected as the null mice. (C) R306C mice have smaller brains. At 7 weeks of age, WT and R306C Tg mice have similar brain sizes, whereas R306C and null mice have brains that are about 85% the normal weight. n = 7 per genotype. (D) ATRX localization to heterochromatic foci is used as a marker of disease severity. At 7 weeks of age, the number of ATRX-positive foci per cell (quantification to the upper right) and focus intensity (quantification to the lower right) is indistinguishable in WT and R306C Tg mice, while they are decreased in R306C, and even more decreased in null mice. The circled cell (white) in the merge of the left panel is shown at increased magnification in the center panel. Immunofluorescence was performed using DAPI (blue) and an anti-ATRX antibody (red). n = 3 per genotype. Data were analyzed by an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. Results were plotted as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.
Figure 5
R306C mice recapitulate many RTT phenotypes.
(A–E) Behavioral tests were performed at 5 weeks of age to examine the R306C phenotype. At this age, WT (black, n = 18) and R306C Tg (blue, n = 17) mice are indistinguishable. Like the null mice (red, n = 14), R306C mice (green, n = 16) have increased anxiety, as measured by increased time spent in the dark in the light/dark assay (A), purposeful paw movement deficits as measured by their ability to build nests (B), motor dysfunction as measured by increased footslips per cm traveled in parallel rod footslip (C), and learning and memory deficits in contextual (D) and cued (E) fear conditioning. (F–J) Behavioral tests were performed on a new cohort of mice at 11 weeks of age, when most of the null mice have succumbed to disease, to examine any changes in the observed phenotypes. WT (n = 17) and R306C Tg (n = 15) mice remain indistinguishable at this age. R306C (n = 15) mice were hypoactive in the open field assay (F) and continue to exhibit increased anxiety as measured by decreased time spent in the center in the open field assay (G). Additionally, their motor dysfunction and learning and memory deficits have both worsened (H–J). The motor coordination of older R306C Tg mice was tested with a new cohort of 5 month old WT (n = 12) and R306C Tg (n = 11) mice subjected to rotarod (four trials a day for 4 days), and no difference was observed in their time spent on the rod (K). Data were analyzed by an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. Results were plotted as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 6
R306C does not affect methyl-CpG binding, but alters binding to a subset of known interactors.
(A) MeCP2-R306C binds methyl-CpGs in vivo. Immunofluorescence for endogenous MeCP2 (anti-MeCP2 antibody, red), the transgene (anti-GFP antibody, green), and heterochromatic foci (puncta within the nucleus visualized by DAPI, blue) shows that both endogenous MeCP2 and MeCP2-R036C localize to the heterochromatic foci and can therefore bind methyl-CpGs. (B) MeCP2-R306C has altered binding to a subset of known interactors. Immunoprecipitation (IP) with anti-GFP antibody was used to determine if MeCP2-R306C binds known interactors. Using WT mice lacking GFP as a negative control and MeCP2-EGFP as a positive control, we show that MeCP2-R306C retains binding to Sin3a, HDAC1, and HDAC2 of the Sin3a complex, but not to HDAC3 and Tbl1 of the NCoR complex. I = input, E = eluate.
Figure 7
R306C decreases the affinity of the C-terminus of MeCP2 to DNA and reduces its DNA occupancy.
(A) An overview of the lifespans of RTT mouse models emphasizes the fact that the R306C missense mutation is more detrimental than an earlier truncation at G273X. Full-length MeCP2 (gray) with the MBD (blue), TRD (red), and basic cluster (yellow) is depicted. The top shows truncating mutations, while the bottom shows the missense mutations we generated. The median lifespans of mice carrying the mutations are shown in weeks. (B) Chromatin immunoprecipitation (ChIP) using an anti-GFP antibody followed by quantitative PCR (qPCR) to determine enrichment of the following amplicons: Major Satellite repetitive sequences, L1 retrotransposons (L1), somatostatin (Sst), afamin (Afm), corticotropin releasing hormone (Crh), and Gapdh shows that MeCP2-R306C has decreased binding to sequences known to be bound by MeCP2 in vivo. Comparing MeCP2-R306C (green, n = 7) to MeCP2-G273X (orange, n = 7) shows that the differences observed are due to an additional effect of the C-terminus that is absent in the full-length MeCP2-EGFP (black, n = 4). WT mice with no GFP (gray, n = 3) were used as a negative control. (C) An electrophoretic mobility shift assay (EMSA) using increasing amounts of N-terminally GST-tagged C-terminal human MeCP2 recombinant protein (amino acids 274–340, from left to right: WT, R306C, R306H, K304E) shows that the WT C-terminal fragment of MeCP2 can bind DNA and that RTT-causing mutations altering the charge of basic residues in the fragment abolish this binding. Data were analyzed by an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. Results were plotted as the mean ± SEM. *p<0.05.
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Rett-causing mutations reveal two domains critical for MeCP2 function and for toxicity in MECP2 duplication syndrome mice
eLife 3:e02676.
https://doi.org/10.7554/eLife.02676
