High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

Marie-Lise Quillé,Caroline Benech,Daniel Baron,Gaëlle Friocourt,Edouard Hirchaud,Rémi Houlgatte,Claude Férec,Olivier Mignen,Morgane Placet,Sylvia Quéméner-Redon,Solenne Carat,Jeanne Guihot

doi:10.1371/journal.pone.0025181

Marie-Lise Quillé, Caroline Benech + Show 10 more

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https://doi.org/10.1371/journal.pone.0025181

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Abstract

Genetic investigations of X-linked intellectual disabilities have implicated the ARX (Aristaless-related homeobox) gene in a wide spectrum of disorders extending from phenotypes characterised by severe neuronal migration defects such as lissencephaly, to mild or moderate forms of mental retardation without apparent brain abnormalities but with associated features of dystonia and epilepsy. Analysis of Arx spatio-temporal localisation profile in mouse revealed expression in telencephalic structures, mainly restricted to populations of GABAergic neurons at all stages of development. Furthermore, studies of the effects of ARX loss of function in humans and animal models revealed varying defects, suggesting multiple roles of this gene during brain development. However, to date, little is known about how ARX functions as a transcription factor and the nature of its targets. To better understand its role, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified a total of 1006 gene promoters bound by Arx in transfected neuroblastoma (N2a) cells and in mouse embryonic brain. Approximately 24% of Arx-bound genes were found to show expression changes following Arx overexpression or knock-down. Several of the Arx target genes we identified are known to be important for a variety of functions in brain development and some of them suggest new functions for Arx. Overall, these results identified multiple new candidate targets for Arx and should help to better understand the pathophysiological mechanisms of intellectual disability and epilepsy associated with ARX mutations.

Highlights

Intellectual disability (ID) is usually defined as a heterogeneous group of disorders resulting from a variety of acquired and genetic causes, and is thought to affect approximately 1% of the population across the world [1]
Several other mutations have been described and implicated Aristaless-related homeobox (ARX) in a wide spectrum of disorders extending from phenotypes with severe neuronal migration defects such as Xlinked lissencephaly with abnormal genitalia (XLAG), to milder forms of mental retardation without apparent brain abnormalities but with associated features of dystonia and epilepsy [3,4,5,6,7,8,9,10]
Assessment of the specificity of the ChIP protocol To better understand the role of Arx and the effect of its mutations on brain development, we used chromatin immunoprecipitation on promoter arrays (ChIP-chip) to identify direct targets of this transcription factor

Summary

Introduction

Intellectual disability (ID) is usually defined as a heterogeneous group of disorders resulting from a variety of acquired and genetic causes, and is thought to affect approximately 1% of the population across the world [1]. More than 90 associated genes have been identified [2]. One of the most frequently mutated genes is the Aristaless-related homeobox (ARX) gene (Entrez Gene 170302) located on Xp22.13. It was first identified in 2002 as involved in non-syndromic X-linked mental retardation (OMIM 300382 and 300419) [3] and West syndrome (OMIM 308350) [4,5]. Several other mutations have been described and implicated ARX in a wide spectrum of disorders extending from phenotypes with severe neuronal migration defects such as Xlinked lissencephaly with abnormal genitalia (XLAG), to milder forms of mental retardation without apparent brain abnormalities but with associated features of dystonia and epilepsy [3,4,5,6,7,8,9,10]

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