title: ARX homeodomain mutations abolish DNA binding and lead to a loss of transcriptional repression.
abstract: 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 abnormal 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 overexpression 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 missense 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 phenotypes 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-function mutations, which lead to a reduced or complete loss of DNA binding and as a consequence, a loss of transcriptional repression.
authors.affiliation: Department of Genetics and Molecular Pathology, SA Pathology at the Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia. cheryl.shoubridge@adelaide.edu.au
authors.forename: Cheryl
authors.initials: C
authors.lastname: Shoubridge
authors.forename: May Huey
authors.initials: MH
authors.lastname: Tan
authors.forename: Grace
authors.initials: G
authors.lastname: Seiboth
authors.forename: Jozef
authors.initials: J
authors.lastname: Gécz
mesh_terms: D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D060588:LIM Domain Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: C469264:ARX protein, human; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D060588:LIM Domain Proteins; C065072:LMO1 protein, human; D012097:Repressor Proteins; C484664:SHOX2 protein, human; D014157:Transcription Factors; D004247:DNA