Aristaless-related Homeobox Protein Research Articles

Identification and validation of the phosphorylation sites on Aristaless-related homeobox protein

The Aristaless-related homeobox protein (ARX) is a transcription factor expressed in the developing forebrain, skeletal muscle, pancreas, testis, and a variety of other tissues. It is known to have context-dependent transcriptional activator and repressor activity, although how it can achieve these opposing functions remains poorly understood. We hypothesized phosphorylation status might play a role in pivoting ARX between functioning as an activator or repressor. To gain further mechanistic insight as to how ARX functions, we identified multiple phosphorylation sites on ARX. We further established PKA as the kinase that phosphorylates ARX at least at Ser266 in mice. Two other kinases, CK2α and CDK4/cyclin D1, were also identified as kinases that phosphorylate ARX in vitro. Unexpectedly, phosphorylation status did not change either the nuclear localization or transcriptional function of ARX.

Splice variant in ARX leading to loss of C-terminal region in a boy with intellectual disability and infantile onset developmental and epileptic encephalopathy.

Pathogenic variants in the X-chromosome Aristaless-related homeobox (ARX) gene contribute to intellectual disability, epilepsy, and associated comorbidities in affected males. Here, we report a novel splice variant in ARX in a family with three affected individuals. The proband had early onset developmental and epileptic encephalopathy, his brother and mother had severe and mild intellectual disability, respectively. Massively parallel sequencing identified a novel c.1449-1G>C in intron 4 of the ARX gene, predicted to abolish the splice acceptor site, retaining intron 4 and leading to a premature termination codon immediately after exon 4. As exon 5 is the last exon of the ARX gene, the premature termination codon at position p.L484* would be predicted to escape nonsense-mediated mRNA decay, potentially producing at least some C-terminally truncated protein. Analysis of cDNA from patient lymphoblastoid cells confirmed retention of intron 4 and loss of detectable expression of ARX mRNA across exon 4 to exon 5. We review published cases of variants that lead to altered or early termination of the ARX protein, but not complete loss of function, and are associated with phenotypes of intellectual disability and infantile onset developmental and epileptic encephalopathies, including Ohtahara and West syndromes. Taken together, this novel splice variant retaining intron 4 is likely to be the cause of the early onset developmental and epileptic encephalopathy in the proband.

Regulating transcriptional activity by phosphorylation: A new mechanism for the ARX homeodomain transcription factor.

Aristaless-related homeobox (ARX) gene encodes a paired-type homeodomain transcription factor with critical roles in development. Here we identify that ARX protein is phosphorylated. Using mass spectrometry and in vitro kinase assays we identify phosphorylation at serines 37, 67 and 174. Through yeast-2-hybrid and CoIP we identified PICK1 (Protein interacting with C kinase 1) binding with the C-terminal region of ARX. PICK1 is a scaffold protein known to facilitate phosphorylation of protein partners by protein kinase C alpha (PRKCA). We confirm that ARX is phosphorylated by PRKCA and demonstrate phosphorylation at serine 174. We demonstrate that phosphorylation is required for correct transcriptional activity of the ARX protein using transcriptome-wide analysis of gene expression of phospho-null mutants (alanines replacing serines) compared to ARX wild-type (ARX-WT) overexpressed in pancreatic alpha TC cells. Compared to untransfected cells, ARX-WT overexpression significantly altered expression of 70 genes (Log2FC >+/-1.0, P-value <0.05). There were fewer genes with significantly altered expression compared to untransfected cells with the double phospho-null mutant Ser37Ala+Ser67Ala (26%) and Ser174Ala (39%), respectively. We demonstrate that the c-terminal region of ARX required to bind PICK1 causes a shift in PICK1 subcellular localisation to the nucleus to co-locate with the ARX protein, and truncation of this C-terminal region leads to the same loss of transcriptional activation as S174A mutant. In conclusion, we show that ARX is phosphorylated at several sites and that this modification affects its transcriptional activity.

Detection of Aristaless-related homeobox protein in ovarian sex cord-stromal tumors

ObjectiveTo examine the potential of ARX as a novel biomarker of ovarian endometriosis and other ovarian pathologies. MethodsThe mRNA level of ARX in ovarian endometriosis and normal endometrium samples was determined by real-time PCR, while the protein level was determined by Western blotting and immunohistochemical staining. Immunohistochemical analysis was performed on nearly 200 tissue samples of different ovarian pathologies. GraphPad Prism was used for statistical analysis. ResultsThe expression of ARX was significantly increased in ovarian endometriosis samples as compared to normal endometrium. Also Western blotting data showed higher ARX levels in the ovarian endometriosis samples versus normal endometrium. Immunohistochemical analysis revealed that the protein is localized in the ovarian stroma and does not originate from endometriosis. Further immunohistochemical analysis performed on several different non-neoplastic and neoplastic ovarian tissue samples revealed that in the non-neoplastic ovary ARX protein is present only in the stromal cells and their derivates (luteinized stromal cells, theca and Leydig cells) and not in granulosa cells, oocites, surface epithelium or rete ovarii, while all stromal and sex cord tumors showed strong nuclear staining for ARX. All other primary or metastatic epithelial tumors of the ovary were ARX negative. ConclusionsARX is not associated with endometriosis and cannot be used as a biomarker for ovarian endometriosis. ARX is present in ovarian stroma and cells derived from ovarian stroma as well as in all types of sex cord-stromal tumors of the ovary and could thus be used as a marker for sex cord-stromal differentiation in ovarian tumors.

Embryonic forebrain transcriptome of mice with polyalanine expansion mutations in theARXhomeobox gene

The Aristaless-related homeobox (ARX) gene encodes a paired-type homeodomain transcription factor with critical roles in embryonic development. Mutations in ARX give rise to intellectual disability (ID), epilepsy and brain malformation syndromes. To capture the genetics and molecular disruptions that underpin the ARX-associated clinical phenotypes, we undertook a transcriptome wide RNASeq approach to analyse developing (12.5 dpc) telencephalon of mice modelling two recurrent polyalanine expansion mutations with different phenotypic severities in the ARX gene. Here we report 238 genes significantly deregulated (Log2FC > +/-1.1, P-value <0.05) when both mutations are compared to wild-type (WT) animals. When each mutation is considered separately, a greater number of genes were deregulated in the severe PA1 mice (825) than in the PA2 animals (78). Analysing genes deregulated in either or both mutant strains, we identified 12% as implicated in ID, epilepsy and autism (99/858), with ∼5% of them as putative or known direct targets of ARX transcriptional regulation. We propose a core pathway of transcription regulators, including Hdac4, involved in chromatin condensation and transcriptional repression, and one of its targets, the transcription factor Twist1, as potential drivers of the ID and infantile spasms in patients with ARX polyalanine expansion mutations. We predict that the subsequent disturbance to this pathway is a consequence of ARX protein reduction with a broader and more significant level of disruption in the PA1 in comparison to the PA2 mice. Identifying early triggers of ARX-associated phenotypes contributes to our understanding of particular clusters/pathways underpinning comorbid phenotypes that are shared by many neurodevelopmental disorders.

Nuclear import of aristaless-related homeobox protein via its NLS1 regulates its transcriptional function

Nucleocytoplasmic transport of transcription factors is essential in eukaryotes. We previously reported the presence of two functional NLSs in the homeodomain protein, aristaless-related homeobox (Arx) protein, which is a key transcriptional repressor of LMO1, SHOX2, and PAX4 during development. NLS2, that overlaps the homeodomain, is recognized directly by multiple importin βs, but not by importin αs. In this study, we found that the N-terminal NLS1 of Arx is targeted by multiple importin α proteins, including importin α3 and α5. Both in vivo and in vitro assays demonstrated that nuclear import of Arx via NLS1 is mediated by the importin α/β pathway. Mutagenesis analysis indicated that two basic amino acids, (84)K and (87)R, are essential to the function of NLS1, and that their mutation prevents interactions of Arx with importin αs. Interestingly, inhibition of nuclear import of Arx via NLS1 clearly attenuates its ability of transcriptional repression, suggesting that nuclear import of Arx via NLS1 contributes to its transcriptional function.

Aristaless-Related Homeobox Plays a Key Role in Hyperplasia of the Pancreas Islet α–Like Cells in Mice Deficient in Proglucagon-Derived Peptides

Defects in glucagon action can cause hyperplasia of islet α-cells, however, the underlying mechanisms remain largely to be elucidated. Mice homozygous for a glucagon-GFP knock-in allele (Gcggfp/gfp) completely lack proglucagon-derived peptides and exhibit hyperplasia of GFP-positive α-like cells. Expression of the transcription factor, aristaless-related homeobox (ARX), is also increased in the Gcggfp/gfp pancreas. Here, we sought to elucidate the role of ARX in the hyperplasia of α-like cells through analyses of two Arx mutant alleles (ArxP355L/Y and Arx [330insGCG]7/Y) that have different levels of impairment of their function. Expression of Gfp and Arx genes was higher and the size and number of islets increased in the Gcggfp/gfp pancreas compared to and Gcggfp/+ pancreas at 2 weeks of age. In male Gcggfp/gfp mice that are hemizygous for the ArxP355L/Y mutation that results in a protein with a P355L amino acid substitution, expression of Gfp mRNA in the pancreas was comparable to that in control Gcggfp/+Arx+/Y mice. The increases in islet size and number were also reduced in these mice. Immunohistochemical analysis showed that the number of GFP-positive cells was comparable in Gcggfp/gfp ArxP355L/Y and Gcggfp/+Arx+/Y mice. These results indicate that the hyperplasia is reduced by introduction of an Arx mutation. ArxP355L/Y mice appeared to be phenotypically normal; however, Arx [330insGCG]7/Y mice that have a mutant ARX protein with expansion of the polyalanine tract had a reduced body size and shortened life span. The number of GFP positive cells was further reduced in the Gcggfp/gfp Arx [330insGCG]7/Y mice. Taken together, our findings show that the function of ARX is one of the key modifiers for hyperplasia of islet α-like cells in the absence of proglucagon-derived peptides.

SnapShot: Genetics of Autism

6q23.3 (AHI1*) Jouberin; interacts with b-catenin in cilia Joubert syndrome. (Reduced brain and body size. Cerebellar, retinal, and kidney defects. Most die by P10. Neuron-specific loss leads to depressive-like phenotypes.)7q35-q36.1 (CNTNAP2*)Caspr2, a neurexin family member; clusters voltage-gated K+ channels Recessive EPI syndrome, ASD, ADHD, TS, OCD. (Neuronal migration defects. Reduced GABAergic neurons and decreased cortical synchrony. Seizures. Deficits in social, repetitive behaviors, and USV.) 9q34.13 (TSC1) Hamartin, a growth inhibitory protein that negatively regulates the mTOR pathwayTuberous Sclerosis type I. (Liver and neural tube defects. Die by E12. Abnormal kidney and liver growth in heterozygotes. Variable brain structure, function and behavior abnormalities in conditional mutants. Die at various postnatal ages. Neuron-specific loss causes abnormal spine morphology and cortical excitability. Loss of LTD.)10q23.31 (PTEN) Protein tyrosine phosphatase; negatively regulates the mTOR pathwayCowden disease. (Placenta and germ cell defects. Die by E9.5. Neuron-specific loss alters synaptic physiology. Heterozygotes have prostate, skin and colon defects, and spontaneous tumors. Macrocephaly, neuronal hypertrophy, abnormal social interaction, and increased survival in conditional mutants.)11q13.4 (DHCR7) Final enzyme in cholesterol biosynthetic pathwaySmith-Lemli-Opitz syndrome. (Craniofacial and lung abnormalities. Die by P1. Abnormal cholesterol regulation and enlarged bladders. Hypomorphic mutants are viable and fertile. Compound mutants have fused toes, enlarged ventricles, and 25% embryonic lethality.) 12p13.33 (CACNA1C)a-1 subunit of a voltage-dependent Ca2+ channelTimothy syndrome. (Die embryonically. Impaired pancreatic function. Motor defects and antidepressant-like behavior in heterozygotes; anxiety-like deficits in females. Neuron-specific loss, impaired cognition and LTP.) 15q11.2 (UBE3A) Ubiquitination ligase; targets protein degradation systemAngelman syndrome. (Small brain, seizure susceptibility, motor and learning deficits. Reduced spine density and impaired LTP. Impaired synapse maturation and plasticity.) 16p13.3 (TSC2) Tuberin; which negatively regulates the mTOR pathwayTuberous Sclerosis type II. (Heart, neural tube, and motor defects. Purkinje cell death. Die by E12. Various tumors and axon guidance defects in heterozygotes. Dominant-negative mutant has enhanced anxiety-like behaviors; motor, learning, social behavior deficits.) 17q11.2 (NF1) Neurofibromin; a GTPase activator and negative regulator of RAS signaling Neurofibromatosis. (Macrocephaly, small eyes, and heart defects. Delayed organ development. Embryonic lethal. Increased astrocytes and tumor susceptibility. LTP and learning and memory deficits in heterozygotes.) Xp21.2 (DMD) Dystrophin; cytoskeletal protein bridging ECMDuchenne muscular dystrophy. (Muscle and heart defects in hemizygous males and homozygous females. Reduced fertility. Abnormal retinal electrophysiology and synapse organization, density, and maturation.)Xp21.3 (ARX) Aristaless-related homeobox protein TFLIS, XLID, EPI, ASD. (Hemizygous males die perinatally. Decreased inhibitory synaptic transmission. Males hemizygous for point mutations or triple repeat expansions have seizures. Defects in behavior and GABAergic neuron generation and migration.)Xq27.3 (FMR1) Fragile X mental retardation protein; an RNA-binding protein that traffics mRNA Fragile X syndrome. (Seizures. Enlarged testes in males. Learning and social behavior defects. Dendritic spine abnormalities. Enhanced LTD and impaired LTP. Altered cortical drive and E/I neuronal cortical networks.) Xq28 (MECP2) MeCP2; involved in transcriptional regulation and chromatin organizationRett syndrome. (Brain, breathing, and motor defects in hemizygous males. Mild cognitive and anxiety-like phenotypes in heterozygous females. Various conditional loss and postnatal reduction mimic null phenotypes in adult hemizygous males. Impaired excitatory synapses and spine morphology. Increased neuronal connectivity.)

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

BackgroundAristaless related homeobox (ARX) is a paired-type homeobox gene. ARX function is frequently affected by naturally occurring mutations. Nonsense mutations, polyalanine tract expansions and missense mutations in ARX cause a range of intellectual disability and epilepsy phenotypes with or without additional features including hand dystonia, lissencephaly, autism or dysarthria. Severe malformation phenotypes, such as X-linked lissencephaly with ambiguous genitalia (XLAG), are frequently observed in individuals with protein truncating or missense mutations clustered in the highly conserved paired-type homeodomain.ResultsWe have identified two novel point mutations in the R379 residue of the ARX homeodomain; c.1135C>A, p.R379S in a patient with infantile spasms and intellectual disability and c.1136G>T, p.R379L in a patient with XLAG. We investigated these and other missense mutations (R332P, R332H, R332C, T333N: associated with XLAG and Proud syndrome) predicted to affect the nuclear localisation sequences (NLS) flanking either end of the ARX homeodomain. The NLS regions are required for correct nuclear import facilitated by Importin 13 (IPO13). We demonstrate that missense mutations in either the N- or C-terminal NLS regions of the homeodomain cause significant disruption to nuclear localisation of the ARX protein in vitro. Surprisingly, none of these mutations abolished the binding of ARX to IPO13. This was confirmed by co-immunoprecipitation and immmuno fluorescence studies. Instead, tagged and endogenous IPO13 remained bound to the mutant ARX proteins, even in the RanGTP rich nuclear environment. We also identify the microtubule protein TUBA1A as a novel interacting protein for ARX and show cells expressing mutant ARX protein accumulate in mitosis, indicating normal cell division may be disrupted.ConclusionsWe show that the most likely, common pathogenic mechanism of the missense mutations in NLS regions of the ARX homeodomain is inadequate accumulation and distribution of the ARX transcription factor within the nucleus due to sequestration of ARX with IPO13.

Ohtahara syndrome in a family with an ARX protein truncation mutation (c.81C>G/p.Y27X).

Aristaless-related homeobox (ARX) gene mutations cause a diverse spectrum of disorders of the human brain, including lissencephaly, various forms of epilepsy and non-syndromic mental retardation. We have identified a novel mutation, c.81C>G (p.Y27X), within the ARX gene in a family with two affected male cousins. One of the boys was diagnosed with an early infantile epileptic encephalopathy also known as Ohtahara syndrome, whereas his cousin had been diagnosed with West syndrome (WS). Both patients have normal genitalia and neither have lissencephaly. The ARX mutation identified is predicted to yield a severely truncated protein of only 26 amino acids and can be considered as a null mutation. Somewhat surprisingly, however, it does not yield the X-linked lissencephaly with ambiguous genitalia (XLAG) syndrome. We proposed that the ARX mRNA translation re-initiated at the next AUG codon at position c.121-123 (aa 41) and, thus, partly rescued these patients from XLAG. Our in vitro studies show that this N-terminally truncated ARX protein (p.M41_C562) is detected by western immunoblot in lysates from cells transiently transfected with an ARX over-expression construct containing the c.81C>G mutation. Although these findings widen the spectrum of clinical phenotypes because of mutations in the ARX gene, they also emphasize the molecular pathogenetic effect of individual mutations as well as the effect of genetic background resulting in intrafamilial clinical heterogeneity for these mutations.

The Roles of Multiple Importins for Nuclear Import of Murine Aristaless-related Homeobox Protein

Nuclear import of proteins with nuclear localization signals (NLSs) is mediated by shuttling carriers, the importins. Some cargoes display more than a single NLS, and among these are homeodomain proteins such as Arx, which is critical for development of multiple tissues. Arx has two functional NLSs. The present studies show that several pathways can import Arx via its NLS2, which is within its DNA binding homeodomain. Using an in vitro nuclear import assay, we show that import of Arx via NLS2 can be mediated by importin beta1, importin 9, or importin 13, with binding being strongest to importin beta1. All binding is sensitive to RanGTP. Experiments based on precise domain deletions indicate that NLS2 binds impbeta1, imp9, and imp13 and includes both an importin binding subdomain and a regulatory subdomain with arginine residues being important for function. Moreover, Arx can be co-precipitated with these importins when NLS2 is present. Although nuclear import of Arx can be mediated by these three importin betas, importin beta1 seems to play the major role judging from in vivo small interfering RNA ablations and the in vitro import assay. This is the first evidence to show the role of importin beta1 in nuclear import of paired-type homeodomain proteins. We propose a novel and possibly quite general mechanism for nuclear import of paired-type homeodomain proteins which is critical for development.

Clinical study of two brothers with a novel 33 bp duplication in the ARX gene

Pathogenic variations of the ARX (aristaless-related homeobox) gene are associated with marked phenotypic pleiotropy. These phenotypes are X-linked neurological disorders that include brain and genital malformation and non-malformation syndromes. Typically, malformation phenotypes result from pathogenic variations that are predicted to truncate the ARX protein, or alter residues in the highly conserved homeodomain. While non-malformation phenotypes tend to be caused by pathogenic variations that are predicted to expand the first two polyalanine tracts of ARX, or alter residues outside of the homeodomain. The most common pathogenic variation of the ARX gene is a duplication of 24 bp, c.429_452 dup, which leads to an expansion of the second polyalanine tract of the ARX protein from 12 to 20 alanine residues. This pathogenic variation is associated with both sporadic and familial nonsyndromic mental retardation. Syndromic manifestations include mental retardation with hand dystonia (Partington syndrome), infantile spasms (West syndrome) and/or other epileptic seizures. Here, we report on a novel pathogenic variant of a tandem 33 bp duplication that is predicted to result in an expansion of polyalanine tract 2 in two brothers with mental retardation, epilepsy, dystonia, and the novel feature of intermittent hyperventilation. This pathogenic variation is predicted to result in a "non-homogeneous" polyalanine tract expansion that is longer than predicted expansion caused by the common 24 bp duplication. The location of the novel 33 bp duplication in the same region as the common 24 bp duplication supports this region as the ARX variation "hot spot."

Aristaless-related homeobox gene disruption leads to abnormal distribution of GABAergic interneurons in human neocortex: evidence based on a case of X-linked lissencephaly with abnormal genitalia (XLAG)

X-linked lissencephaly with abnormal genitalia (XLAG) is a rare disorder caused by mutations in the aristaless-related homeobox (ARX) gene, located on Xp22.13. Arx-null mice show loss of tangential migration of GABAergic interneurons, presumably being related to caudal ganglionic eminence tangential migration. In the present study, we investigated a subpopulation of GABAergic interneurons in the brain of an infant with XLAG, who had a novel nonsense mutation of the ARX gene, compared with those of age-matched normal controls and Miller-Dieker syndrome. We performed immunocytochemistry for interneuron and migration markers. We found that glutamic acid decarboxylase (GAD)- and calretinin (CR)-containing cells were significantly reduced in the neocortex and located in the white matter and neocortical subventricular zone, while neuropeptide Y- or cholecystokinin-containing cells were normally distributed. Moreover, in the neocortical subventricular region, the GAD- and CR-containing cells expressed the radial migration marker Mash-1 as well as nestin. Our findings suggest that ARX protein controls not only the tangential migration of GABAergic interneurons from the ganglionic eminence, but also may serve to induce radial migration from the neocortical subventricular zone.

X-linked mental retardation (XLMR)-associated mutations affect the activity of the aristaless-related homeobox protein, Arx

X-linked mental retardation (XLMR)-associated mutations affect the activity of the aristaless-related homeobox protein, Arx