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
78 162 207
N-TermMBDID TRD* * * *GFP
DAPI MeCP2 GFPGFP (inset)Merge
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 weeks 7 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
Gapdh AfmSatellite L1 SstCrh
% IP / Input
Chromosome 5 Position ( x106 )
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.
(Mef2c and Grin2) in KO mice. We focused on the expression of
At 4 weeks of age, when KO mice appear largely asymptom-
atic, gene expression differences between WT and KO mice
were not significant (Figure 4C); this is consistent with a previous
report that found BDNF expression to be unchanged in
presymptomatic mice and to decrease after symptom onset
(Chang et al., 2006). Differences in gene expression between
the mutant and WT mice began to appear at 7 weeks, but there
were no significant differences among the KO, R270X, and
G273X mice, either at these early time points (Figure 4D) or
even by 9 weeks of age (Figure S4).
To determine if the differences in phenotypes are accompa-
nied by differential gene expression changes in other tissues,
campi of 4- and 9-week-old WT, KO, R270X, and G273X mice.
MeCP2 loss of function was associated with many subtle differ-
ences in gene expression (Table S1). Overall, the transcriptional
profiles of KO, R270X, and G273X mice appeared very similar at
both ages (Figures 4E and 4F). The total number of genes misre-
gulated in any mutant increased from 4 weeks of age (2,778
genes) to 9 weeks of age (3,082 genes). We searched for genes
that were misregulated in KO and R270X mice but rescued in
G273X mice. Interestingly, only 41 genes (38 upregulated and
3 downregulated) appeared similar to WT in G273X mice at
4 weeks of age (Figure 4E; Table S2), being reduced by 9 weeks
of age to only 8 genes (6 upregulated and 2 downregulated) (Fig-
ure 4F; Table S2). Of the 41 genes that were rescued in G273X
mice at 4 weeks, the majority (71%) was misregulated in
Bdnf SstTac1 Oprk1Mef2c
Rela?ve to WT
Rela?ve to WT
7 weeks old
4 weeks old
All Misregulated Genes
Genes Rescued in G273X
All Misregulated Genes
Genes Rescued in G273X
log2 fold change
Figure 4. MeCP2-R270X and MeCP2-G273X
(A) Schematic of constructed transcriptional assay.
The target reporter construct contains a 53 Gal
DNA-binding site immediately upstream of the
human b-actin promoter with endpoints relative to
TSSindicated. Theeffector constructconsistsofthe
CMV promoter driving expression of WT or mutant
MeCP2 fused at the N terminus to the Gal4 DNA-
binding domain (Gal4 DBD). A control reporter
lacking the53Gal sitewascotransfected tomonitor
(B) Average luciferase activity of N2a lysates trans-
fected with the target reporter construct and the
indicated effector construct. Data are normalized to
control reporter activity, and the percent difference
relative toempty effectorisindicated. ****p<0.0001,
***p < 0.001, *p < 0.05, Student’s t test. n = 3.
(C and D) Hypothalamic RNA levels were quantified
by reverse-transcription qPCR for four genes
downregulated in KO mice (Bdnf, Sst, Oprk1, and
Tac1) and two upregulated genes (Mef2c and
Grin2a). Transcript levels were normalized to Gapdh
and are represented as the fold expression relative
to WT. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p <
0.05. (C) Gene expression of 4-week-old animals
(n = 6, n = 7, n = 6, and n = 6 for WT, KO, R270X, and
G273X, respectively). (D) Gene expression of
7-week-old animals (n = 7, n = 6, n = 6, and n = 6 for
WT, KO, R270X, and G273X, respectively).
(E and F) Hippocampal RNA was quantified by
microarray, and the abundance of significantly
altered genes is displayed as the log2(fold change)
relative to WT. The intensity key indicates genes that
are upregulated in red and genes that are down-
regulated in green. (E) (Left) all genes that were
misregulated relative toWTinany mutant at4weeks
of age. (Right) genes that were misregulated in KO
and R270X mice but rescued in G273X mice at
all genes that were misregulated relative to WT in
any mutant at 9 weeks of age. (Right) genes that
were misregulated in KO and R270X mice but
rescued in G273X mice at 9 weeks of age (n = 4
animals per genotype).
Pooled data show mean ± SEM. See also Figure S4
and Tables S1 and S2.
Cell 152, 984–996, February 28, 2013 ª2013 Elsevier Inc. 989
1 78 162 207 310 486
0 1.8 3.6 7.2 14 27 54 68 0 1.8 3.6 7.2 14 27 54 68
p = 8.1e-10
0 20 40 60
% Probe Bound
Protein Concentration (μM)
KD = 6.59µM
%IP / Input
M 0 0.5 1 2M 0 0.5 1 2 M 0 0.5 1 2
MeCP2 / Nucleosome Ratio
MeCP2-WT MeCP2-R270X MeCP2-G273X
M 0 2.5 0 2.5M 0 2.5 0 2.5
% NA Soluble
Figure 5. A Conserved AT-Hook Domain between R270 and G273 Enables MeCP2 to Alter Chromatin Structure
(A) Conserved MeCP2 residues shared by fish, frog, rat, mouse, and human. Sequences were aligned with ClustalW2, and blue lines indicate residues sharing
identity. Two blocks of conserved amino acids corresponding to AT-Hook 1 and AT-Hook 2 are shown. The asterisk (*) denotes identity within the aligned
(B) PROMALS3D alignment of the human MeCP2 C terminus (residues 163–486) and the human HMGA1 sequence. PairwiseStatSig method was used to
compute significance (Agrawal and Huang, 2011). p = 8.1 3 10-10after 200,000 iterations.
(C) (Top) a recombinant form of the MeCP2 AT-Hook 2 domain (amino acids 257–272) was used in an EMSA to test binding to a 64-mer double-stranded DNA
probe (66.7 nM). The AT-Hook 2 domain truncated at R270 (R270X) failed to bind DNA. The full AT-Hook 2 domain ending at G273 (G273X) readily forms the
(legend continued on next page)
990 Cell 152, 984–996, February 28, 2013 ª2013 Elsevier Inc.
G273X mice by 9 weeks. Thus, the transcriptional profiles of KO,
R270X, and G273X mice are similar but do reveal a delay in mis-
regulation for a small subset of genes in G273X mice.
A Conserved AT-Hook Domain Is Disrupted in MeCP2-
R270X but Not MeCP2-G273X
To understand the influence of the three amino acids positioned
between R270 and G273, we examined the MeCP2 protein
sequence surrounding this region. Sequence data indicate that
MECP2 arose soon after the appearance of vertebrates because
homologs are detectable in jawless fish such as Petromyzon
marinus but not in lower species (http://genome.ucsc.edu/).
There is a relatively low level of conservation between the
MeCP2 sequences from the single homologs found in zebrafish
to humans (49% identity and 62% similarity for zebrafish versus
human) (Figure 5A). One major block of conservation overlaps
with the MBD: the C terminus, including the TRD, is poorly
conserved and known to be highly disordered (Ghosh et al.,
2010). Nevertheless, we found highly basic clusters of amino
acids that are conserved from fish to humans within this
region (Figure 5A). One of these conserved basic clusters (amino
acids 185–194) is an AT-hook domain (AT-Hook 1) of unknown
significance (Klose et al., 2005; Lewis et al., 1992). A second
basic cluster (amino acids 265–272) contains another AT-hook
(AT-Hook 2) that was uncharacterized, although it had been
annotated by InterPro (http://www.ebi.ac.uk/interpro/). Given
the importance of AT-hooks to the nonhistone, chromatin-asso-
ciated proteins of the high-mobility group AT-hook (HMGA)
family, which contain two or three AT-hook domains connected
by a flexible polypeptide linker (Reeves, 2010), we explored the
possibility that MeCP2 and HMGA family members share
a common ancestry.
Both of MeCP2’s AT-hooks align well with the corresponding
AT-hook domains of HMGA1 (Figure 5B). There is an unexpect-
edlyhigh degree of sequence identity between the third AT-hook
of HMGA1 and a more C-terminal portion of MeCP2: this third
AT-hook-like domain within MeCP2 appears to have been dis-
rupted by the insertion of two serine-rich tracks. Further analysis
showed that this domain exists in multiple extant fish species
(e.g., Oryzias latipes and Takifugu rubripes), suggesting that it
arose before fish and mammals diverged (http://genome.ucsc.
edu/). To compare MeCP2 and HMGA1 using an unbiased
method relative to the human proteome, we computed the
percent identity for MeCP2 versus all proteins of similar length
to HMGA1 (Figures S5A and S5B). This analysis found that
sized proteins in the human proteome. We found an analogous
relationship when comparing HMGA1 to all human proteins of
similar length to MeCP2 (Figures S5C and S5D). These data
strongly suggest that MeCP2 shares a common ancestry with
the HMGA family of proteins.
The conserved AT-Hook 2 domain terminates with G273, after
which the sequence diverges among species (Figure 5A). Trun-
cating the three amino acids between G273 and R270 would
disrupt the central RGR motif of this AT-hook and could disrupt
DNA binding. To test this possibility, we purified a recombinant
form of the AT-Hook 2 domain (amino acids 257–272) tagged
with GST. Electrophoretic mobility shift assay (EMSA) showed
that this domain binds double-stranded DNA with an apparent
KDof 6.59 mM (Figure 5C). We then tested whether the AT-
be predicted based on the loss of the central RGR motif, this
mutant form of AT-Hook 2 failed to bind DNA, even at very
high concentrations (Figure S5E). This conserved feature of
MeCP2 is therefore a DNA-binding domain that must retain
amino acids 270–272 in order to function.
Although MeCP2-R270X contains a truncated AT-Hook 2
domain, we failed to uncover any differences in the occupancy
of MeCP2-R270X and MeCP2-G273X using crosslinked ChIP-
qPCR (see Figure 3C). We considered that native ChIP-qPCR
might reveal subtle differences in DNA binding consistent with
the loss of the AT-Hook 2 domain because this assay requires
that a protein stably interact with chromatin in the absence of
crosslinking (Turner, 2001). Although native ChIP-qPCR binding
of both mutant proteins was similar across the Gapdh, Afm, Sst,
and Crh promoters and at the L1 retrotransposon, MeCP2-
R270X exhibited half the binding of MeCP2-G273X within major
satellite DNA (Figure 5D). Thus, truncation of the AT-Hook 2
domain impairs the ability of MeCP2-R270X to stably interact
with certain sequences in vivo. Given the similarity of MeCP2
to HMGA1, we next sought to determine whether loss of the
conserved AT-Hook 2 domain of MeCP2 has consequences
for chromatin structure that might help explain the differences
between R270X and G273X mice.
MeCP2-R270X and MeCP2-G273X Differ in Their
Activity Toward NAs
Prior work has shown MeCP2 to form higher-order structures
with nucleosomal DNA in vitro; the RTT-causing R168X mutation
impairs this ability (Georgel et al., 2003). We therefore sought to
characterize the interactions of MeCP2-R270X and MeCP2-
G273X with reconstituted NAs. Recombinant MeCP2-WT,
indicated complexes with the probe DNA. (Bottom) for AT-Hook 2-G273X, a hyperbolic model was fit to the data, and the apparent KDwas calculated to be
6.59 mM; quantification from three independent experiments.
(D) Native ChIP with anti-GFP on whole-brain chromatin from R270X and G273X mice. WT mice that lack GFP were used as a control for antibody specificity.
*p < 0.05. n = 3.
(E) EMSA reaction with methylated NAs and recombinant MeCP2. MeCP2-WT, MeCP2-R270X, and MeCP2-G273X were added at the indicated MeCP2/
nucleosome ratios. Each protein shifted the NAs at a similar concentration. MeCP2-WT induces the formation of higher-order complexes, evident as a long trail
above the shifted band. M, 1 kb plus DNA marker (n = 6).
by centrifugation. The NAs that remain in the soluble fraction were run on an agarose gel and visualized by ethidium bromide staining.
(G) Quantification of the experiment described in (F).
****p < 0.0001, ***p < 0.001, **p < 0.01, Student’s t test. n = 4. Pooled data show mean ± SEM. See also Figure S5.
Cell 152, 984–996, February 28, 2013 ª2013 Elsevier Inc. 991
MeCP2-R270X, and MeCP2-G273X were mixed with CpG-
methylated NAs and analyzed by EMSA. All three proteins
were able to shift the methylated NAs at a similar concentration
(Figure 5E). Consistent with previous reports, MeCP2-WT
formed higher-order structures with the NAs, evident as a broad
smear above the shifted band (Figure 5E). MeCP2-R270X and
MeCP2-G273X failed to exhibit this activity under identical
Adding magnesium to reconstituted NAs induces oligomeriza-
tion and is thought to model the formation of compacted
chromatin in vivo (Schwarz et al., 1996). NAs condensed in
this manner become insoluble and can be separated under
high-speed centrifugation. Interestingly, MeCP2 facilitates this
process (Nikitina et al., 2007b). Using this assay, we evaluated
the ability of MeCP2-R270X and MeCP2-G273X to oligomerize
methylated NAs (Figure 5F). With the addition of 2.5 mM
MgCl2, MeCP2-WT greatly facilitated the oligomerization of
NAs as evidenced by a reduction in the percentage remaining
soluble after centrifugation (Figures 5F and 5G). In contrast,
MeCP2-R270X failed to facilitate NA oligomerization and was
indistinguishable from NAs alone. MeCP2-G273X exhibited
intermediate activity and facilitated NA oligomerization, although
not to the same extent as MeCP2-WT. Across a broad range
of MgCl2 concentrations, MeCP2-R270X exhibited reduced
activity for oligomerizing NAs, whereas MeCP2-G273X showed
activity more similar to MeCP2-WT (Figures S5F and S5G).
ATRX Mislocalization Distinguishes G273X Mice
The loss of MeCP2 causes mislocalization of the chromatin-
remodeling protein a-thalassemia/mental retardation syndrome
X linked (ATRX) in the hippocampus and cortex of symptomatic
KO mice (Nanetal.,2007).Wedecided to investigate the nuclear
localization of ATRX in the hippocampi of our mutant mice over
the course of disease.
WT mice show ATRX foci colocalizing with pericentric hetero-
chromatin (PCH) at 4, 7, and 9 weeks of age (Figures 6Ai–6Aiii
and S6A). At 4 weeks of age, when mutant mice are largely
asymptomatic, the average number of ATRX foci within PCH
was similar among KO, R270X, G273X, and WT mice, although
these foci appeared less intense in the mutant mice (Figures
6Ai, 6B, and 6C). By 7 weeks of age, the average number of
ATRX foci in KO and R270X animals was significantly less than
WT (p < 0.0001) (Figures 6Aii and 6B). At 9 weeks of age, only
a few ATRX foci were detected in KO and R270X mice (Fig-
ure 6Aiii), consistent with the previous report on null mice (Nan
et al., 2007).
The average number of ATRX foci observed within neurons of
G273X mice at 7 weeks of age was indistinguishable from WT:
more than six times that of either KO or R270X mice (p <
0.0001 and p < 0.001 for KO and R270X, respectively) (Figures
6Aiiand 6B). By9weeks,significantly fewer fociwere detectable
in G273X mice than in WT (p < 0.001) (Figures 6Aiii and 6B), but
there were still more foci than in either KO or R270X mice (p <
0.05 and p < 0.01 for KO and R270X, respectively). ATRX target-
ing to PCH within the hippocampus is therefore disrupted in
Mecp2 mutant lines but much more gradually in G273X mice.
To understand the cause of ATRX loss from PCH, we studied
this phenomenon in other Mecp2 mutants. The brains of symp-
tomatic Mecp2+/?heterozygous female mice (5–6 months old)
present a mixture of MeCP2-positive and MeCP2-negative
neurons because Mecp2 is subject to X chromosome inactiva-
tion. Within the hippocampus, only MeCP2-negative neurons
showed loss of ATRX from PCH (Figures 6D and 6E). We found
the same dichotomous pattern in the cortex (data not shown)
and hippocampus of younger Mecp2+/?female mice (9 weeks
old) (Figure S6B). The effects of MeCP2 loss on ATRX localiza-
tion are therefore cell autonomous. Interestingly, we found the
opposite phenomenon within hippocampi of female MECP2-
TG3 (TG3) mice, which overexpress MeCP2 from a transgene
integrated on the X chromosome (Collins et al., 2004). In TG3-
positive neurons, ATRX foci remained localized to PCH (Figures
S6C and S6D), but these foci were often much brighter than in
adjacent WT neurons (Figures S6C and S6E). Overexpression
of MeCP2 thus leads to greater accumulation of ATRX within
ATRX localization in mouse neurons was previously attributed
to MeCP2 directly recruiting ATRX to PCH (Nan et al., 2007). The
ATRX-interacting domain of MeCP2 overlaps with the MBD
and remains intact in both MeCP2-R270X and MeCP2-G273X
(amino acids 108–169). Consistent with the presence of this
domain, both mutant proteins interact with ATRX to an equal
extent as MeCP2-WT by coimmunoprecipitation (Figure S7A).
To determine whether progressive loss of ATRX from PCH
of MeCP2 from these sites, we compared MeCP2-R270X and
MeCP2-G273X localization at two time points. Immunofluores-
cence staining detected both mutant forms of MeCP2 at PCH
in the hippocampi of 4- and 9-week-old animals (Figure S7B).
The staining pattern remained unchanged regardless of age.
These truncated forms of MeCP2 appear to stably associate
with PCH in vivo, and ATRX loss from these sites is independent
of MeCP2 presence.
To rule out fixation artifacts as the cause of ATRX mislocaliza-
tion, we purified fresh whole-brain nuclei from both WT and KO
mice at 9 weeks of age and stained them for ATRX. In WT brain
nuclei, ATRX appeared within dense foci colocalized with
Hoechst-bright PCH. In nuclei purified from KO brains, ATRX
was depleted specifically from PCH (Figures S7C–S7E). We
then used this assay to study the localization of ATRX in periph-
eral tissues. The number of nuclei with ATRX foci colocalizing
with PCH was fewer in liver, heart, lung, and kidney than in the
brain (Figures 7A–7E). There was no difference between WT
and KO mice in the number of nuclei from peripheral tissues
with ATRX foci (Figure 7E). The pathological processes leading
to ATRX mislocalization are therefore brain specific.
Severalmodels forMeCP2functioninthebrain havebeenbased
on abnormalities observed in Mecp2KO mice, but KO mice have
not shed light on the postnatal regression so characteristic of
RTT. In this study, we generated two mouse models of MeCP2
dysfunction that develop phenotypes at distinctly different rates,
providing a parallel to the human male patients. We exploited
these differences and the general features of G273X mice to
gain insight into domains critical for MeCP2 function.
992 Cell 152, 984–996, February 28, 2013 ª2013 Elsevier Inc.
Many lines of evidence support a role for MeCP2 in transcrip-
tional repression. Principal among these is the presence of
a well-defined TRD mapped to amino acids 207–310 (Nan
et al., 1997). When the TRD is shortened by even 10 amino acids
from the C terminus, terminating at amino acid 300, repression
activity is completely lost (Nan et al., 1997). Neither MeCP2-
R270X nor MeCP2-G273X displayed transcriptional repressor
activity, consistent with the requirement for a fully intact TRD
for these functions. Despite this shared loss, however, G273X
mice live much longer and remain healthier for a significantly
greater period of time than KO mice. MeCP2 clearly performs
important functions besides classical transcriptional repression.
HMGA family members contain two or three AT-hooks con-
nected by a flexible linker sequence (Reeves, 2010) and require
multiple contacts with DNA in order to alter its conformation (Li
et al., 2000). These features are highly reminiscent of the
MeCP2 C terminus. In contrast to the MBD, the C terminus
including the TRD region is highly disordered, and multiple
nonoverlapping fragments within the C terminus are capable of
binding DNA in vitro (Ghosh et al., 2010). One of these fragments
(amino acids 261–330) contains the conserved AT-Hook 2
domain located between amino acids 265 and 272. In addition
to binding naked DNA, this fragment can bind and compact
NAs, a function that is enhanced by fusion with adjacent DNA-
binding fragments of the C terminus (Ghosh et al., 2010). Like
MeCP2, HMGA proteins have a special relationship with histone
H1. For example, HMGA proteins compete with H1 for binding
sites (Catez et al., 2006), and overexpression of HMGA1 leads
to reduced levels of certain H1 isoforms (Brocher et al., 2010).
The homology of MeCP2 to HMGA1 could explain many of
MeCP2’s effects on chromatin structure.
The most likely explanation for the functional differences
between MeCP2-R270X and MeCP2-G273X is the loss of func-
tion of the AT-Hook 2 domain. We propose a model (Figure 7F)
DAPIATRX MergeDAPI ATRX MergeDAPIATRX Merge
4 weeks7 weeks9 weeks
(Mecp2-/y ; G273XTg)
Foci / Cell
(% of WT)
Foci / Cell
WT NeuronsKO Neurons
Figure 6. ATRX Mislocalization in Various
(A) Immunofluorescence for ATRX in the hippo-
campus of WT, KO, R270X, and G273X mice. PCH
appears as DAPI-bright foci within neurons and
colocalizes with ATRX staining in WT animals at all
ages. (i) At 4 weeks of age, ATRX localization is
similar in WT and mutant mice. (ii) At 7 weeks of
age, KO and R270X mice show a noticeable
reduction in the number of bright ATRX foci,
whereas G273X mice remain similar to WT mice.
(iii) At 9 weeks, KO, R270X, and G273X mice show
a decrease in the number of bright ATRX foci
relative to WT. Scale bars represent 2 mm. Images
are of individual neuronal nuclei; for lower-power
images of more neurons, see Figure S6A.
(B) Quantification of the average number of ATRX
foci detected per neuron in each genotype at 4, 7,
and 9 weeks of age. ***p < 0.001 and *p < 0.05 for
G273X compared to both KO and R270X. n = 6
high-power fields from two mice per genotype
(C) Quantification of the average foci intensities for
each genotype at 4 weeks of age. ****p < 0.0001.
n = 651 WT foci, n = 513 KO foci, n = 344 R270X
foci, and n = 735 G273X foci from two mice per
(D) Double immunofluorescence for MeCP2 and
ATRX in hippocampus of symptomatic Mecp2+/?
heterozygous female mice. (Top) MeCP2-ex-
pressing neurons (solid circle) and neurons not
expressing MeCP2 (dashed circle). ATRX foci are
brighter in MeCP2-positive neurons compared to
adjacent MeCP2-negative neurons. Scale bars
represent 10 mm (n = 2 mice). (Bottom) higher-
magnification image of the WT and KO neuron
circled in the upper panel.
(E) Quantification of the average number of
ATRX foci detected per neuron in symptomatic
Mecp2+/?heterozygous female mice (n = 4 high-
power fields from two mice). *p < 0.05.
Pooled data show mean ± SEM. See also Fig-
Cell 152, 984–996, February 28, 2013 ª2013 Elsevier Inc. 993
wherein MeCP2 binds with high affinity to methyl-CpG sites
located throughout the genome and at concentrated domains
of heterochromatin using its MBD. Once there, MeCP2 manipu-
lates the nearby chromatin structure in a manner similar to other
AT-hook-containing proteins, e.g., by altering local DNA confor-
mation or adjusting nucleosome positioning. Additional residues
C terminal to G273 might perform separate roles or enhance the
activity of the N-terminal amino acids such that without the full C
terminus, MeCP2 function is reduced. Loss of the three amino
acids between G273 and R270 largely abolishes the already
impaired ability of MeCP2 to maintain chromatin order,
hastening the loss of ATRX from PCH. The presence of disorga-
nized chromatin might well prevent neurons from responding
appropriately to stimuli important for normal synaptic function.
Ultimately, impaired synaptic function causes the clinical symp-
toms observed in RTT. At the opposite end of the spectrum,
overabundance of MeCP2 leads to a progressive restructuring
with ATRX Foci
Conserved Basic Clusters
Figure 7. ATRX Mislocalization Is Specific
to the Brain
(A–D) Immunofluorescence for ATRX in fresh
nuclei prepared from WT and KO mice at 9 weeks
of age. Nuclei are counterstained with Hoechst
from liver (A), heart (B), lung (C), and kidney (D).
Scale bars represent 10 mm. n = three mice per
(E) Quantification of the experiment described in
(A)–(D and Figure S7C. The percentage of ATRX-
positive nuclei with foci localizing to PCH from WT
and KO animals is plotted for each tissue. Error
bars represent 95% confidence intervals. The
number of nuclei analyzed for brain was 226 and
183; for liver, 192 and 195; for heart, 112 and 121;
for lung, 151 and 195; and for kidney, 184 and 204
from WT and KO mice, respectively.
(F) Proposed model for MeCP2 in chromatin
homeostasis. MeCP2 contains highly conserved
C-terminal region. MeCP2 first binds DNA through
its MBD, but the presence of multiple DNA-binding
conformation,leading tohomeostasis asindicated
by the recruitment of ATRX to PCH. When the
C terminus becomes truncated (e.g., G273X), this
activity is reduced. Further truncation beyond the
AT-Hook 2 domain (e.g., R270X) severely impairs
the function of MeCP2, chromatin is no longer
maintained in a physiological conformation, and
ATRX is lost from PCH.
See also Figure S7.
of chromatin that would ordinarily be
MeCP2 and other factors. The excessive
such as ATRX to domains such as PCH
might induce hyperresponsiveness to
stimuli and excessive synaptic function.
This would lead to progressive but less-
severe symptoms, as observed in mice
and humans that overexpress MeCP2.
In this study, we have used the loss of ATRX from PCH as
a tool to gain insight into the effects of MeCP2 on neuronal chro-
matin. However, the impaired binding of ATRX to PCH has itself
been associated with mutations that cause the a-thalassemia
X-linked intellectual disability (ATRX) syndrome (Iwase et al.,
2011). In Mecp2 mutant mice, it will be important to investigate
the consequences of ATRX loss from PCH. Knockdown of
ATRX leads to genomic instability in cultured cells and is associ-
ated with impairments in metaphase chromatin condensation
(Ritchie et al., 2008). ATRX interacts with the H3.3 chaperone
DAXX (Drane ´ et al., 2010), and loss of ATRX leads to depletion
of H3.3 from telomeric DNA (Goldberg et al., 2010). In addition,
ATRX binds to non-B form DNA structures (i.e., G quadruplex)
that are associated with repetitive sequences (Law et al.,
2010). ATRX is highly related to the SNF2 family members
Rad54 and ARIP4, and it has been suggested that ATRX may
resolve non-B forms of DNA to regular forms (Law et al., 2010).
994 Cell 152, 984–996, February 28, 2013 ª2013 Elsevier Inc.
ATRX syndrome shows a high degree of overlap with RTT (and
other MECP2-related disorders) (Gibbons and Higgs, 2000).
Cognitive deficits are one common feature of both diseases.
Microcephaly is another. Males are more sensitive to identical
patients with RTT and those affected by ATRX syndrome is that
the latter never show a period of normal development. Like
MeCP2, ATRX is widely expressed in many tissues, and both
proteins seem important for the proper functioning of mature
neurons. If progressive ATRX dysfunction occurs downstream
of MeCP2 loss only in neurons, this observation could account
for the absence of hematopoietic, craniofacial, and urological
abnormalities in RTT. The clinical course of males with MECP2
mutations is often more severe than male patients with disrup-
tions in ATRX (premature lethality is not common in ATRX
syndrome), but this could reflect the spectrum of mutations in
ATRX that causes disease (many of which are hypomorphic
alleles) (Gibbons and Higgs, 2000). Indeed, male Atrx mutant
mice die in utero (Garrick et al., 2006). Given the severity
of phenotypes in male patients with MECP2 mutations, it is
possible that other proteins in addition to ATRX are also disrup-
ted in neurons lacking MeCP2. The underlying processes that
lead to improper ATRX targeting may also affect these other as
yet unidentified proteins, with consequences for chromatin
Animals and Phenotypic Analysis
Generation, breeding, and characterization of phenotypic severity scores are
detailed in Extended Experimental Procedures. All mouse studies were
approved by the Institutional Animal Care and Use Committee for Baylor
College of Medicine. Mice were evaluated for six phenotypic categories as
previously described by Guy et al. (2007).
Statistical analysis was performed using GraphPad Prism 5 software, Micro-
soft Excel, or R statistical software with the exception of the ChIP-seq analysis
dures). One-way ANOVA followed by Bonferroni-Holm post hoc analysis were
used to test for significance unless otherwise specified.
Modification of the human PAC PAC671D9 to insert the EGFP tag along with
a poly-serine linker in MECP2 in place of the codons for amino acids R270
and G273 was performed as previously described by Chao et al. (2010).
Gene Expression Analysis
RNA extraction, processing, and reverse-transcription qPCR analysis were
performed as previously described by Chao et al. (2010). Hippocampal RNA
waspolyA selected and measured usingGeneChip Mouse Gene 1.0 ST Arrays
(Affymetrix) by the Genomic and RNA Profiling Core at Baylor College of
Plasmid Cloning and Primer Sequences
Standard cloning procedures were followed for the generation of plasmids
used in this study. For specific details, please see Extended Experimental
Procedures and Table S3.
ChIP-seq and microarray data are deposited under GEO records GSE36536
and GSE42987, respectively.
figures, and three tables and can be found with this article online at http://dx.
We thank Christopher Woodcock for the pUC19-601x12 vector, Chris
McGraw, Hsiao-Tuan Chao, Laura Lombardi, and Jeffrey Neul for critical
reading of this manuscript, and Gabriele Schuster for pronuclear injections.
This work was supported by the Howard Hughes Medical Institute, NINDS
grant NS053862(toH.Y.Z.), thecoresoftheBaylorCollegeofMedicine IDDRC
(HD024064), and NINDS F30NS066527 (to S.A.B.).
Received: February 27, 2012
Revised: December 3, 2012
Accepted: January 22, 2013
Published: February 28, 2013
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