ARX homeodomain mutations abolish DNA binding
and lead to a loss of transcriptional repression
Cheryl Shoubridge1,2,∗, May Huey Tan1,2,∗, Grace Seiboth1and Jozef Ge ´cz1,2
1Department of Genetics and Molecular Pathology, SA Pathology at the Women’s and Children’s Hospital,
North Adelaide, South Australia 5006, Australia and2Department of Paediatrics, University of Adelaide, Adelaide,
South Australia 5005, Australia
Received November 11, 2011; Revised and Accepted December 15, 2011
Mutations in the Aristaless-related homeobox (ARX) gene are one of the most frequent causes of X-linked
intellectual disability (ID). Several missense mutations, clustered in the paired-type homeodomain of ARX,
have been identified. These mutations lead to a range of phenotypes from X-linked lissencephaly with abnor-
mal genitalia to seizure disorders without brain malformations including X-linked infantile spasms with ID
(ISSX-ID) and X-linked myoclonic epilepsy with spasticity and ID (XMESID). The effect of these mutations
on the DNA-binding and transcriptional activity has been evaluated. Luciferase reporter assays showed
altered repression activity of ARX by all mutations, causing brain malformations and ISSX-ID phenotypes,
but not by the P353L mutation implicated in a milder phenotype of XMESID. Similarly, transient overexpres-
sion of wild-type ARX repressed endogenous expression of known ARX targets, LMO1 and SHOX2, when
measured by real-time quantitative polymerase chain reaction. Overall, the molecular consequence of mis-
sense mutations correlated well with the severity of the clinical phenotype. In all mutations tested, except
P353L, the DNA binding was abolished. Electrophoretic mobility shift assay results were validated using
chromatin immunoprecipitation following overexpression of normal and selected missense mutations.
Unlike wild-type ARX and clinically less severe mutations, the mutations leading to severe clinical pheno-
types were not able to specifically bind to DNA upstream of known, endogenous ARX-regulated genes,
LMO1 and SHOX2. In conclusion, the missense mutations in the ARX homeodomain represent loss-of-func-
tion mutations, which lead to a reduced or complete loss of DNA binding and as a consequence, a loss of
Intellectual disability (ID) is a large group of highly variable
disorders of the brain affecting approximately 1 in every 50
individuals across the world populations. Defects arising
from genes on the X-chromosome lead to clinically complex
and genetically heterogeneous X-linked ID (XLID), with
more than 100 genes currently known (1). One of the most fre-
quently mutated XLID genes is the Aristaless-related homeo-
box (ARX) gene. In excess of 100 mutations have been
identified in ARX, causing a range of phenotypes all with
ID, often with additional features including epilepsy, infantile
spasms, hand dystonia, lissencephaly, autism or dysarthria (2).
The overall burden of ARX mutations might still be underesti-
mated. This is because of the difficulty of the sequence capture
and massively parallel sequencing technologies to enrich and
sequence high-GC-rich ARX sequence (J.G., unpublished
data), broad clinical expressivity of ARX mutations as well
as the fact that many mutations identified by various diagnos-
tic services may go unreported.
ARX is a member of the paired-type homeobox transcription
factors, which are involved in the control of development and
differentiation, and all contain the conserved 60 amino acid
DNA-binding motif, referred to as the homeodomain. Muta-
tions (insertion, deletion, nonsense and splice) in the ARX
homeodomain that lead to loss or truncation of the mature
ARX protein cause severe brain malformation phenotypes
(2). Disease-causing single-nucleotide substitutions that lead
to missense changes are also found clustered in the ARX
homeodomain (3–9). Several of these missense mutations
∗To whom correspondence should be addressed. Tel: +61 881618105; Fax: +61 881617031; Email: firstname.lastname@example.org
# The Author 2011. Published by Oxford University Press. All rights reserved.
For Permissions, please email: email@example.com
Human Molecular Genetics, 2012, Vol. 21, No. 7
Advance Access published on December 21, 2011
by guest on December 30, 2015
are predicted to cause a complete loss-of-function as they lead
to severe phenotypes with brain malformations such as
X-linked lissencephaly with abnormal genitalia (XLAG)
(3,6,7,10,11) normally associated with protein truncation
mutations. In contrast, the remaining missense mutations in
this domain affect the same residues as mutated in patients
with XLAG but cause clinical presentations with seizures
but no gross malformation of the brain (9,12–14).
These missense mutations have each been reported in a
single family, often with multiple affected individuals. The
molecular mechanism(s) underlying disease in these cases
are not known. Missense mutations in the ARX homeodomain
occur in the residues predicted to be important for DNA
binding and the specificity of DNA binding (15) or nuclear
import (9,16,17). The nuclear localization sequences (NLSs)
that flank the homeodomain contain the residues required for
nuclear import of this transcription factor through interaction
with importin proteins, including Importin 13 (IPO13) (17)
and Importin b1 (16). In the N-terminal NLS (NLS2) of
ARX, there are several point mutations that have been identi-
fied in individuals with neurological disease: p.R332P (6),
p.R332H (3) p.R332C (5) and p.R332L (7), which cause
XLAG, and a residue adjacent to this arginine, p.T333N (6),
which causes Proud syndrome (agenesis of the corpus callo-
sum with ambiguous genitalia) (ACC/AG; MIM 30004). In
the C-terminal NLS (NLS3), there are two naturally occurring
missense mutations in the same residue, p.R379L (9), which
causes XLAG, and p.R379S (8,9), which causes infantile
spasms and ID. Our recent study modeling the missense muta-
tions in the NLS2 and NLS3 regions of ARX homeodomain
has shown that the mutant ARX protein remains in complex
with Importin 13 (IPO13) instead of uncoupling in the
RanGTP-rich nuclear environment, leading to inadequate ac-
cumulation and distribution of the ARX transcription factor
within the nucleus (9).
The remaining missense mutations in the ARX homeodo-
main include p.L343Q (3) and p.P353R (6)—both lead to
severe brain malformation phenotype of XLAG, whereas mu-
tation in the same residue p.P353L causes X-linked myoclonic
epilepsy with spasticity and ID (XMESID) (4). The two most
recently reported mutations in p.R358S (13) and p.R358W
(14) cause similar clinical outcomes. When p.R358 is
mutated to a serine, the patient presented with ACC, ID, in-
fantile spasms and intractable epilepsy and abnormal genitalia
(13), whereas substitution to tryptophan caused ACC, severe
ID, infantile spasms and subsequent intractable epilepsy but
also spastic/dyskinetic quadriparesis, severe limb contractures
and scoliosis (14). As these mutations do not occur in the NLS
regions of the homeodomain, altered interaction with IPO13 is
unlikely and alternate molecular mechanisms explaining their
pathogenesis need to be considered.
Mutations in the homeodomain of ARX are likely to affect
DNA binding and hence, transcriptional activity. Ablation of
Arx expression in the subpallium and ganglionic eminences
of the mouse brain have identified direct targets of ARX tran-
scriptional repression (18,19). The genes repressed via Arx
binding to a specific (TAATTA) transcription factor-binding
site (TFBS) include Lmo1, Ebf3 and Shox2 (19). Ectopic ex-
pression of Ebf3 in ventral telencephalon prevented tangential
migration of neurons, and silencing of Ebf3 expression in Arx
mutant mice partially restored this neuronal migration (18). To
investigate the mechanism(s) underlying the pathogenesis of
ARX homeodomain mutations, we evaluated the effect of mis-
sense mutations on the DNA-binding capacity and transcrip-
tional activity of ARX. Our findings demonstrate altered
transcriptional repression due to a loss of DNA binding as
the molecular mechanism of most but not all ARX mutations
Missense mutations in the ARX homeodomain abolish
the repression activity of ARX
Our first aim was to measure the functional impact of single-
nucleotide substitution mutations in the homeodomain of
ARX on its transcriptional activity. We generated a luciferase
reporter construct by cloning three copies of the orthologous
region identified as an Arx-binding site in the enhancer
Co-transfection of a renilla expression vector was used to
normalize the expression of luciferase. Reporter constructs
were transiently transfected into HEK293T cells along with
(Fig. 1A) or an empty expression (Myc-empty) vector. The
ratio of luciferase to renilla expression for the empty Myc-
vector is set to 100%. Compared with this value, the
ARX-Wt repressed the expression of luciferase by 41%
(Fig. 1B). This is in agreement with previous studies in
which this Arx-binding site was identified in the mouse
(19). The repression activity of ARX in our assay indicates
that ARX is able to bind and repress transcriptional activity
in the orthologous sequence upstream of LMO1 in the
human. When the ARX protein contains a mutation within
the homeodomain, the repression activity on luciferase ex-
pression was abolished. Indeed, all mutations tested gave
higher levels of luciferase expression than the Myc-empty
control (Fig. 1B). The only exception to this was the
p.P353L mutation, which displayed similar (32%), if slightly
less, repression activity than ARX-Wt.
Homeodomain mutations derepress LMO1 and SHOX2,
known target genes of ARX
To complement our luciferase reporter assay results, we mea-
sured the endogenous expression of LMO1 and SHOX2,
known ARX targets, following transient overexpression of
wild-type and mutant ARX. When compared with the en-
dogenous expression in untransfected HEK293T cells, the
levels of both genes were repressed following overexpression
of ARX-Wt (Fig. 2). In comparison, overexpression of ARX
missense mutations generally resulted in higher levels of
LMO1 and SHOX2 expression, with several displaying levels
similar to those of untransfected cells (Fig. 2). In agreement
with the luciferase assay data, the LMO1 and SHOX2 gene ex-
pression mirrored the severity of the clinical phenotype caused
by the mutations tested, with the p.P353L mutation still be
able to repress both target genes to levels achieved with
1640Human Molecular Genetics, 2012, Vol. 21, No. 7
by guest on December 30, 2015
This work was supported by grants from the National Health
and Medical Research Council of Australia (Project Grant
1002732 to C.S.; Principal Research Fellowship 508043 to
J.G.) and MS McLeod Foundation Fellowship to C.S.
1. Gecz, J., Shoubridge, C. and Corbett, M. (2009) The genetic landscape of
intellectual disability arising from chromosome X. Trends. Genet., 25,
2. Shoubridge, C., Fullston, T. and Gecz, J. (2010) ARX spectrum disorders:
making inroads into the molecular pathology. Hum. Mutat., 31, 889–900.
3. Kitamura, K., Yanazawa, M., Sugiyama, N., Miura, H., Iizuka-Kogo, A.,
Kusaka, M., Omichi, K., Suzuki, R., Kato-Fukui, Y., Kamiirisa, K. et al.
(2002) Mutation of ARX causes abnormal development of forebrain and
testes in mice and X-linked lissencephaly with abnormal genitalia in
humans. Nat. Genet., 32, 359–369.
4. Stromme, P., Mangelsdorf, M.E., Shaw, M.A., Lower, K.M., Lewis, S.M.,
Bruyere, H., Lutcherath, V., Gedeon, A.K., Wallace, R.H., Scheffer, I.E.
et al. (2002) Mutations in the human ortholog of Aristaless cause X-linked
mental retardation and epilepsy. Nat. Genet., 30, 441–445.
5. Uyanik, G., Aigner, L., Martin, P., Gross, C., Neumann, D.,
Marschner-Schafer, H., Hehr, U. and Winkler, J. (2003) ARX mutations in
X-linked lissencephaly with abnormal genitalia. Neurology, 61, 232–235.
6. Kato, M., Das, S., Petras, K., Kitamura, K., Morohashi, K., Abuelo, D.N.,
Barr, M., Bonneau, D., Brady, A.F., Carpenter, N.J. et al. (2004)
Mutations of ARX are associated with striking pleiotropy and consistent
genotype-phenotype correlation. Hum. Mutat., 23, 147–159.
7. Halac, I., Habiby, R., Curran, J. and Zimmerman, D. (2006) Central and
gonadal hypogonadism in X-linked lissencephaly. J. Pediatr. Endocrinol.
Metab., 19, 955–957.
8. Marsh, E., Fulp, C., Gomez, E., Nasrallah, I., Minarcik, J., Sudi, J.,
Christian, S.L., Mancini, G., Labosky, P., Dobyns, W. et al. (2009)
Targeted loss of Arx results in a developmental epilepsy mouse model and
recapitulates the human phenotype in heterozygous females. Brain, 132,
9. Shoubridge, C., Tan, M.H., Fullston, T., Cloosterman, D., Coman, D.,
McGillivray, G., Mancini, G.M., Kleefstra, T. and Gecz, J. (2010)
Mutations in the nuclear localization sequence of the Aristaless related
homeobox; sequestration of mutant ARX with IPO13 disrupts normal
subcellular distribution of the transcription factor and retards cell division.
Pathogenetics, 3, 1–15.
10. Proud, V.K., Levine, C. and Carpenter, N.J. (1992) New X-linked
syndrome with seizures, acquired micrencephaly, and agenesis of the
corpus callosum. Am. J. Med. Genet., 43, 458–466.
11. Dobyns, W.B., Berry-Kravis, E., Havernick, N.J., Holden, K.R. and
Viskochil, D. (1999) X-linked lissencephaly with absent corpus callosum
and ambiguous genitalia. Am. J. Med. Genet., 86, 331–337.
12. Scheffer, I.E., Wallace, R.H., Phillips, F.L., Hewson, P., Reardon, K.,
Parasivam, G., Stromme, P., Berkovic, S.F., Gecz, J. and Mulley, J.C.
(2002) X-linked myoclonic epilepsy with spasticity and intellectual
disability: mutation in the homeobox gene ARX. Neurology, 59,
13. Fullston, T., Finnis, M., Hackett, A., Hodgson, B., Brueton, L., Baynam,
G., Norman, A., Reish, O., Shoubridge, C. and Gecz, J. (2011) Screening
and cell-based assessment of mutations in the Aristaless-related
homeobox (ARX) gene. Clin. Genet., 80, 510–522.
14. Conti, V., Marini, C., Gana, S., Sudi, J., Dobyns, W.B. and Guerrini, R.
(2011) Corpus callosum agenesis, severe mental retardation, epilepsy, and
dyskinetic quadriparesis due to a novel mutation in the homeodomain of
ARX. Am. J. Med. Genet. A, 155, 892–897.
15. Gecz, J., Cloosterman, D. and Partington, M. (2006) ARX: a gene for all
seasons. Curr. Opin. Genet. Dev., 16, 308–316.
16. Lin, W., Ye, W., Cai, L., Meng, X., Ke, G., Huang, C., Peng, Z., Yu, Y.,
Golden, J.A., Tartakoff, A.M. et al. (2009) The roles of multiple importins
for nuclear import of murine aristaless-related homeobox protein. J. Biol.
Chem., 284, 20428–20439.
17. Shoubridge, C., Cloosterman, D., Parkinson-Lawerence, E., Brooks, D.
and Gecz, J. (2007) Molecular pathology of expanded polyalanine tract
mutations in the Aristaless-related homeobox gene. Genomics, 90, 59–71.
18. Colasante, G., Collombat, P., Raimondi, V., Bonanomi, D., Ferrai, C.,
Maira, M., Yoshikawa, K., Mansouri, A., Valtorta, F., Rubenstein, J.L.
et al. (2008) Arx is a direct target of Dlx2 and thereby contributes to the
tangential migration of GABAergic interneurons. J. Neurosci., 28,
19. Fulp, C.T., Cho, G., Marsh, E.D., Nasrallah, I.M., Labosky, P.A. and
Golden, J.A. (2008) Identification of Arx transcriptional targets in the
developing basal forebrain. Hum. Mol. Genet., 17, 3740–3760.
20. McKenzie, O., Ponte, I., Mangelsdorf, M., Finnis, M., Colasante, G.,
Shoubridge, C., Stifani, S., Gecz, J. and Broccoli, V. (2007)
Aristaless-related homeobox gene, the gene responsible for West
syndrome and related disorders, is a Groucho/transducin-like enhancer of
split dependent transcriptional repressor. Neuroscience, 146, 236–247.
21. Collombat, P., Mansouri, A., Hecksher-Sorensen, J., Serup, P., Krull, J.,
Gradwohl, G. and Gruss, P. (2003) Opposing actions of Arx and Pax4 in
endocrine pancreas development. Genes. Dev., 17, 2591–2603.
22. Collombat, P., Hecksher-Sorensen, J., Broccoli, V., Krull, J., Ponte, I.,
Mundiger, T., Smith, J., Gruss, P., Serup, P. and Mansouri, A. (2005) The
simultaneous loss of Arx and Pax4 genes promotes a
somatostatin-producing cell fate specification at the expense of the alpha-
and beta-cell lineages in the mouse endocrine pancreas. Development,
23. Esposito, G., Fogolari, F., Damante, G., Formisano, S., Tell, G., Leonardi,
A., Di Lauro, R. and Viglino, P. (1996) Analysis of the solution structure
of the homeodomain of rat thyroid transcription factor 1 by 1H-NMR
spectroscopy and restrained molecular mechanics. Eur. J. Biochem., 241,
24. Qian, Y.Q., Billeter, M., Otting, G., Muller, M., Gehring, W.J. and
Wuthrich, K. (1989) The structure of the Antennapedia homeodomain
determined by NMR spectroscopy in solution: comparison with
prokaryotic repressors. Cell, 59, 573–580.
25. Tsao, D.H., Gruschus, J.M., Wang, L.H., Nirenberg, M. and Ferretti, J.A.
(1995) The three-dimensional solution structure of the NK-2
homeodomain from Drosophila. J. Mol. Biol., 251, 297–307.
26. D’Elia, A.V., Tell, G., Paron, I., Pellizzari, L., Lonigro, R. and Damante,
G. (2001) Missense mutations of human homeoboxes: a review. Hum.
Mutat., 18, 361–374.
27. Chi, Y.I. (2005) Homeodomain revisited: a lesson from disease-causing
mutations. Hum. Genet., 116, 433–444.
28. Stromme, P., Mangelsdorf, M.E., Scheffer, I.E. and Ge ´cz, J. (2002)
Infantile spasms, dystonia, and other X-linked phenotypes caused by
mutations in Aristaless related homeobox gene, ARX. Brain. Dev., 24,
29. Kitamura, K., Itou, Y., Yanazawa, M., Ohsawa, M., Suzuki-Migishima,
R., Umeki, Y., Hohjoh, H., Yanagawa, Y., Shinba, T., Itoh, M. et al.
(2009) Three human ARX mutations cause the lissencephaly-like and
mental retardation with epilepsy-like pleiotropic phenotypes in mice.
Hum. Mol. Genet., 18, 3708–3724.
30. Kumar, R., Selth, L.A., Schulz, R.B., Tay, B.S., Neilsen, P.M. and Callen,
D.F. (2011) Genome-wide mapping of ZNF652 promoter binding sites in
breast cancer cells. J. Cell. Biochem., 112, 2742–2747.
Human Molecular Genetics, 2012, Vol. 21, No. 71647
by guest on December 30, 2015