An AT-Hook Domain in MeCP2 Determines
the Clinical Course of Rett Syndrome
and Related Disorders
Steven Andrew Baker,1,2,5LinChen,3Angela Dawn Wilkins,3Peng Yu,3Olivier Lichtarge,3,5andHudaYahya Zoghbi1,3,4,5,*
1Program in Developmental Biology
2Medical Scientist Training Program
3Department of Molecular and Human Genetics
4Department of Neuroscience and Howard Hughes Medical Institute
Baylor College of Medicine, Houston, TX 77030, USA
5Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston, TX 77030, USA
Mutations in the X-linked MECP2 cause Rett
syndrome, a devastating neurological disorder typi-
fied by a period of apparently normal development
followed by loss of cognitive and psychomotor skills.
and severity can be influenced by the location of
the mutation, with amino acids 270 and 273 marking
the difference between neonatal encephalopathy
and death, on the one hand, and survival with
deficits on the other. We therefore generated two
mouse models expressing either MeCP2-R270X or
MeCP2-G273X. The mice developed phenotypes at
strikingly different rates and showed differential
ATRX nuclear localization within the nervous system,
over time, coinciding with phenotypic progression.
We discovered that MeCP2 contains three AT-
hook-like domains over a stretch of 250 amino acids,
like HMGA DNA-bending proteins; one conserved
AT-hook is disrupted in MeCP2-R270X, lending
further support to the notion that one of MeCP2’s
key functions is to alter chromatin structure.
Rett syndrome (RTT) is caused by mutations in the X-linked gene
methyl-CpG-binding protein 2 (MECP2) (Amir et al., 1999). Girls
with RTT achieve expected milestones for the first 6–18 months,
only to experience a progressive loss of acquired linguistic,
social, and motor skills. They also develop seizures, stereoty-
pies, autonomic dysfunction, and spasticity—yet there is no
evidence of neurodegeneration (Chahrour and Zoghbi, 2007). It
is this pattern of normal development followed by functional
impairment of the nervous system that is paradigmatic of
RTT. The molecular mechanisms underlying this regression are
RTT-causing mutations have been identified throughout
the entire length of MeCP2, which contains two functional
domains: an N-terminal methyl-CpG-binding domain (MBD),
and a C-terminal transcriptional repression domain (TRD).
Despite nearly 2 decades of investigation, the function of
MeCP2 remains unclear. Its TRD can recruit an HDAC-contain-
ing complex via the Sin3A corepressor, suggesting that it
represses transcription (Nan et al., 1998). In vitro, MeCP2 asso-
ciates with nucleosomal linker DNA, compacts nucleosomal
arrays (NAs), and competes with histone H1 for chromatin
binding (Ghosh et al., 2010; Nan et al., 1997; Nikitina et al.,
2007a). More recent in vivo studies showed that MeCP2 loss
results in greater neuronal H1 content, raising the possibility
that MeCP2 serves as an alternative linker histone (Skene
et al., 2010); the same work found that MeCP2 binds throughout
the genome and might act to dampen overall transcriptional
activity. Interestingly, MeCP2 loss of function or overexpression
results in the inverse misregulation of thousands of genes in
specific brain regions (Ben-Shachar et al., 2009; Chahrour
et al., 2008; Samaco et al., 2012).
Given the clinical variability of RTT and the range of mutations,
one way to clarify MeCP2 function is to correlate mutation with
phenotype. Although this is difficult in females because of the
tions have been reported in about 60 boys (Villard, 2007) (http://
mecp2.chw.edu.au/). Seventeen are nonmosaic, karyotypically
normal males with truncating mutations. These boys can be
grouped into two broad categories: (1) severe neonatal enceph-
alopathy and death before 4 years of age, or (2) survival for
decades with either RTT-like phenotypes or neuropsychiatric
deficits. Boys in category 1 tend to have early truncating muta-
tions (e.g., G163fs, G252fs, G269fs, and R270fs) (Villard, 2007),
whereas boys in category 2 tend to have late truncating muta-
tions (e.g., G273fs, R294X, L386fs, Q406X, and E472fs) (Villard,
2007). Although there is only one male reported to have the
G273fs mutation (Ravn et al., 2003), he lived considerably longer
than males with R270fs (Kankirawatana et al., 2006; Vena ˆncio
R270 and G273, exerts a significant effect on the phenotype.
984 Cell 152, 984–996, February 28, 2013 ª2013 Elsevier Inc.
We therefore generated transgenic mice that express
either MeCP2-R270X or MeCP2-G273X from the endogenous
MECP2 locus. We characterized the mice over the course of
disease, examined molecular phenotypes associated with
ical for its role in chromatin organization.
Generation of Transgenic Mice Bearing the R270X
and G273X Alleles
We modified a human PAC containing only the endogenous
MECP2 locus with all known regulatory elements (Collins
et al., 2004) to bear either a G273X or R270X mutation by recom-
bineering (Figure 1A). We also inserted a C-terminal GFP tag
within exon 4 to monitor protein level and localization in vivo.
We injected the constructs into wild-type (WT) FVB embryos,
generating four R270X and two G273X transgenic lines.
We chose two R270X lines (termed ‘‘A’’ and ‘‘B’’) and one
G273X line that exhibited approximately 13 expression of the
transgene product in brain compared to WT (Figure 1B; Fig-
ure S1A available online). MeCP2 levels were stable across
multiple generations in all lines (Figure S1A). Using immunofluo-
rescence and confocal imaging, we found the localization of the
MeCP2-R270X and MeCP2-G273X proteins to mirror that of
endogenous MeCP2 in the cortex, hippocampus, cerebellum,
hypothalamus, and brainstem (Figures S1B–S1F). High-resolu-
tion images from the cortex showed that both MeCP2-R270X
and MeCP2-G273X localized entirely to the nucleus and
concentrated at heterochromatic foci, just like the endogenous
protein (Figure 1C). In individual nuclei, the overlap coefficient
and Manders’ coefficient of MeCP2-R270X or MeCP2-G273X
with endogenous MeCP2 were indistinguishable (k1xk2 =
0.9215, 0.9258, p = 0.55 Student’s t test; M2 = 0.6093,
0.6256, p = 0.81 Student’s t test; n = 18 and n = 25 nuclei,
respectively). All three transgenic lines thus faithfully reproduce
the distribution, abundance, and subnuclear localization of
MeCP2 in the brain.
To generate mice expressing MeCP2-R270X or MeCP2-
G273X in the absence of MeCP2-WT, we crossed transgenic
male mice to Mecp2+/?heterozygous females (Guy et al.,
2001). The resulting male progenies were of four classes: (1)
Mecp2+/y(WT); (2) Mecp2+/y;R270XTgor G273XTg(WT;R270X
or WT;G273X); (3) Mecp2?/y(knockout or KO); and (4) Mecp2?/y;
R270XTgor G273XTg(R270X or G273X). All four classes were
born at expected Mendelian ratios and appeared healthy at
weaning; the fourth class serves as a model for male patients
who express only the mutant form of MeCP2.
Both WT;R270X and WT;G273X mice appeared identical to
their WT littermates (Figure S2A) and were indistinguishable
from WT mice in a number of assays (Figures S2B–S2E). As
previously reported (Chen et al., 2001; Guy et al., 2001), the
KO mice began to develop phenotypes between 4 and 6 weeks
of age and, by 8 weeks, were readily distinguished from their WT
KOs, but the G273X mice were leaner and displayed better
habitus than either age-matched R270X mice or their KO litter-
mates (Figure 2A).
G273X Mice Manifest Disease Later and Survive Longer
than KO or R270X Mice
KO and R270X mice died prematurely, with a median lifespan of
76 and 85 days, respectively (no significant difference, Gehan-
Breslow-Wilcoxon test) (Figure 2B). The G273X mice lived signif-
icantly longer and had a median lifespan of 201 days (p < 0.0001
compared with either KO or R270X mice, Gehan-Breslow-
Wilcoxon test). MeCP2loss of functionhas been shown to cause
in the hypothalamus (Fyffe et al., 2008). KO animals became
overweight compared to WT at postnatal week 6 and reached
a maximum weight at 8 weeks of age (Figure 2C). R270X mice
exhibited a similar but more gradual pattern during weeks 7
and 8. G273X mice did not begin to gain excessive weight until
postnatal week 12 and thereafter slowly gained weight until
17 weeks, when their weight reached a level equal to the
maximum KO weight (Figure 2C). R270X and G273X mice thus
achieve aweight gainsimilarto KO animals,but thetimecourses
are very different.
One featureoften observedinboyswith earlytruncating muta-
tions is brain atrophy (Schu ¨le et al., 2008). Males with later trun-
cating mutations, however, are normocephalic or show brain
growth deceleration leading to acquired microcephaly. We
measured brain weights of our mice at 4, 7, and 9 weeks of
age. WT mouse brains increase in weight until around 9 weeks
of age, then stabilize. In contrast, the KO and R270X brain
weight of G273X mice also peaked at 7 weeks but did not
diminish afterward, even at 13.5 weeks (Figure 2D). These data
show a striking difference in the pattern of brain growth between
R270X and G273X: even though the latter never achieves normal
brain weight, the growth pattern is similar to WT.
We used a published severity scale (see Experimental Proce-
dures)thatmeasurestremor, gaitabnormalities, and othermotor
features to evaluate a cohort of mice at the early (4–6 weeks old),
middle (7–9 weeks old), and late (10–12 weeks old) symptomatic
periods. The premature mortality of the KO and R270X lines
precluded comparison at later ages. As expected, the KO mice
had significantly higher severity scores than WT animals at all
time points (Figure 2E). The average severity scores of both
symptomatic periodbutmoreslowlyasthemiceentered theend
stage of disease (Figure 2E). All features observed in KO mice
were apparent in R270X mice and eventually G273X mice (e.g.,
hindlimb clasping) (Figure S2K). The severity scores of G273X
mice were significantly higher than those of WT but lower (and
increasing more slowly) than either KO or R270X lines at all
time points (Figure 2E). Because G273X mice lived much longer
than KO and R270X mice, we were able to analyze these mice
after 12 weeks of age. The severity scores of older G273X
mice continued to increase until the average reached a level
indistinguishable from the middle symptomatic period of either
KO or R270X mice. The KO, R270X, and G273X mice thus even-
tually developed the same phenotypes, but disease progression
was significantly delayed in G273X animals.
In severity of symptoms, patterns of body and brain weight
gain, and lifespan, then, the G273X mice show more moderate
disease withlateronset, whereasR270Xmiceexhibitthe severe,
Cell 152, 984–996, February 28, 2013 ª2013 Elsevier Inc. 985
N-TermMBDID TRD* * * *GFP
DAPI MeCP2 GFPGFP (inset)Merge
78162 207 1
Figure 1. Design and Characterization of WT;R270X and WT;G273X Transgenic Mice
(A) Schematic of the MECP2 locus and the corresponding WT protein product (top). Diagrams are not to scale, but positions along the primary sequence and
location of the canonical NLS are indicated. Schematic indicating the final modified loci containing a GFP tag inserted in place of the codon for R270 or G273 and
the corresponding mutant protein products (bottom). The asterisk (*) indicates a truncated TRD. N-Term, N-terminal; C-Term, C-terminal.
(B) Western blot analysis using whole-brain lysates for each transgenic line and their WT littermates and an antibody against the N terminus that recognizes WT
and both mutant forms of MeCP2. Mutant MeCP2 fused with GFP migrates below MeCP2-WT.
(C) Mutant MeCP2 localizes with MeCP2-WT in cortical tissue using double immunofluorescence. The C terminus MeCP2 antibody is specific for MeCP2-WT.
Scale bars represent 10 mm.
See also Figure S1.
986 Cell 152, 984–996, February 28, 2013 ª2013 Elsevier Inc.
early-onset disease course reminiscent of the more severely
affected male patients. These results confirm our hypothesis
that R270 and G273 mark a crucial region for MeCP2 function.
To exclude any potential effects of transgene insertion, we
generated two additional lines of WT;G273X mice (lines ‘‘B’’
and ‘‘C’’) and generated G273X mice from these independent
lines (Figures S2F and S2G). Like the original G273X line, these
mice lived significantly longer, had lower body weights, and
had better severity scores relative to KO and R270X mice
(Figures S2H–S2J). These data confirm that the loss of three
additional amino acids in R270X mice renders them much sicker
than G273X mice.
Both MeCP2-R270X and MeCP2-G273X Exhibit
Genome-wide DNA Binding
One possible explanation for the differences in phenotypes
between the R270X and G273X mutations is differential DNA
occupancy. To characterize the DNA-binding profiles of these
mutantproteins in vivo,weperformed chromatinimmunoprecip-
itation followed by high-throughput sequencing (ChIP-seq) from
mouse brain. We compared profiles of MeCP2-R270X and
MeCP2-G273X to the distribution of MeCP2-WT (Figure 3A),
which has been previously reported by Skene et al. (2010). All
three profiles show striking similarities across the mouse
genome (Figure 3B), including repetitive elements (Figure S3A).
The binding pattern of both mutants was reminiscent of
We next assayed specific sites where MeCP2-WT has been
reported to bind using ChIP followed by quantitative PCR
(ChIP-qPCR) (Chahrour et al., 2008; McGill et al., 2006). We
chose four gene promoters (Gapdh, Afm, Sst, and Crh) and
two repetitive elements (major satellite DNA and the L1 retro-
transposon). ChIP-qPCR revealed that MeCP2-R270X and
Both mutants seemed to be enriched at major satellite DNA,
consistent with MeCP2-WT (Lewis et al., 1992). These data
suggest that, like the WT protein, both MeCP2-R270X and
MeCP2-G273X bind widely throughout the genome.
We next assayed the ability of MeCP2-R270X and MeCP2-
G273X to bind to chromatin in whole-brain nuclei isolated from
R270X and G273X mice. We extracted purified nuclei in buffers
with increasing ionic strength (200 mM, 300 mM, 400 mM, and
1 M NaCl) and assayed the resulting supernatants for extracted
MeCP2. Nuclei from WT animals were included for comparison.
As expected, MeCP2-WT was increasingly extracted at higher
salt concentrations (Figures 3D and 3E). Both MeCP2-R270X
and MeCP2-G273X demonstrated similar profiles to MeCP2-
WT (Figures 3D and 3E). We also assayed the levels and extract-
either the total levels (Figures S3B and S3C) or H1 extractability
Severity Score (0-12)
357911 13 15 17
Body Weight (g)
WT (Mecp2+/y)KO (Mecp2-/y) R270X (Mecp2-/y; R270XTg) G273X (Mecp2-/y; G273XTg)
4 weeks7 weeks9 weeks 13.5 weeks
Brain Weight (mg)
Figure 2. Phenotypic Characterization of
R270X and G273X Mice
(A) Representative photographs of WT, KO,
R270X mice have disheveled fur and are over-
(B) Kaplan-Meier curves for four genotype classes.
Censored animals are indicated with a black tick
mark (n = 54, n = 41, n = 28, and n = 21 for WT, KO,
R270X, and G273X, respectively).
(C) Average body weight plotted versus age. *p <
0.05 compared to WT. The maximum number of
animals analyzed was 54, 41, 28, and 21 for WT,
KO, R270X, and G273X, respectively.
(D) Average brain weights for four genotype
classes shown at 4, 7, and 9 weeks. WT and
G273X brains were also weighed at 13.5 weeks.
****p < 0.0001 and *p < 0.05. n.s., not significant.
The number of animals analyzed was six, seven,
eight, and three for WT, and six, six, six, and three
for G273X at 4, 7, 9, and 13.5 weeks, respectively.
The number of animals analyzed was eight, seven,
and eight for KO, and six, six, and five for R270X at
4, 7, and 9 weeks, respectively.
(E) Average cumulative severity scores plotted
against age. ***p < 0.001, Mann-Whitney U test.
The number of observations analyzed was 25, 20,
15, and 22 for WT, and 12, 10, 10, and 8 for G273X
at 4–6, 7–9, 9–12, and >12 weeks, respectively.
The number of observations analyzed was 17, 15,
and 11 for KO, and 12, 6, and 6 for R270X at 4–6,
7–9, and 9–12 weeks, respectively.
All error bars show SEM. See also Figure S2.
Cell 152, 984–996, February 28, 2013 ª2013 Elsevier Inc. 987
(Figure S3D). MeCP2-R270X and MeCP2-G273X thus show
Both R270X and G273X Disrupt the TRD of MeCP2
Wenext comparedthe effect ofthe R270Xand G273Xmutations
on the functions of the TRD in a heterologous repression assay
(Figure 4A). The initial report characterizing the MeCP2 TRD
found that an intact MBD limits proper localization of the Gal4-
MeCP2 fusion protein to the reporter construct (Nan et al.,
1997), so we used both MeCP2-WT and a mutant form with
a point mutation in the MBD, MeCP2-R111G, which abolishes
methyl-CpG binding without disrupting MBD folding (Free
et al., 2001). Both MeCP2-WT and MeCP2-R111G showed
repressor activity (Figure 4B). Consistent with the idea that an
intact MBD restricts the ability of the MeCP2 fusion product to
localize to the Gal4-binding site, MeCP2-R111G showed
stronger repressor activity.
We then generated MeCP2-R270X and MeCP2-G273X both
with and without the R111G mutation. Neither mutant showed
repressor activity, either in the presence or the absence of the
R111G mutation (Figure 4B). Curiously, we noted occasional
transcriptional activation using Gal4-MeCP2 containing these
mutated TRDs, a phenomenon previously observed with a trun-
cated TRD (Nan et al., 1997). This assay indicates that both the
R270X and G273X mutations disrupt the TRD, suggesting that
both R270X and G273X mice express mutant proteins that are
defective in transcriptional repression.
Transcriptional Dysregulation Is Similar in R270X
and G273X Mice
We have identified a number of genes whose expression varies
reliably with MeCP2 function in the hypothalamus (Chahrour
et al., 2008). We therefore measured, in the hypothalami of
R270X and G273X mice, the RNA levels of particular genes that
are downregulated (Bdnf, Sst, Tak1, and Oprk1) or upregulated
GapdhAfmSatellite L1 SstCrh
% IP / Input
Chromosome 5 Position ( x106 )
5060 7080 90
ρ = 0.995
ρ = 0.978
ρ = 0.973
Figure 3. MeCP2-R270X and MeCP2-G273X
Bind DNA Globally
(A) GFP antibodies were used to immunoprecipi-
tate MeCP2-R270X and MeCP2-G273X from
or G273X mice, respectively. Recovered DNA was
subjected to deep sequencing. ChIP-seq counts
MeCP2-R270X and MeCP2-G273X are compared
and the number of counts per bin is displayed over
the indicated chromosome position.
(B) Correlation plots between the number of ChIP-
seq reads per 100 kb bin across the genome
compared for WT versus R270X (top), WT versus
(bottom). r denotes Spearman’s correlation.
(C) Isolated DNA as in (A) was subjected to qPCR
analysis using primers designed for the promoters
of the indicated genes or repetitive elements.
Satellite refers to mouse major satellite DNA; L1
to R270X and G273X mice, ChIP-qPCR was also
performed on WT mice that lack GFP as a control
for antibody specificity and is plotted to the left of
R270X and G273X). IP, immunoprecipitate.
(D) Whole-brain nuclei were purified from WT,
R270X, or G273Xmiceand resuspended inbuffers
comprised of 200 mM, 300 mM, 400 mM, or 1 M
NaCl. An antibody for the MeCP2 N terminus
shows the amount of MeCP2 extracted under
each condition (n = 3).
(E) The average extracted fractions from the
experiment described in (D) are plotted for each
genotype above the corresponding NaCl concen-
tration. Data are normalized for the amount of
MeCP2 extracted in 1 M NaCl (n = 3 mice per
Pooled data show mean ± SEM. See also Fig-
988 Cell 152, 984–996, February 28, 2013 ª2013 Elsevier Inc.