SUMMARYDlx homeobox genes play a crucial role in the migration and differentiation of the subpallial precursor cells that give rise to various subtypes of -aminobutyric acid (GABA)-expressing neurons of the forebrain, including local-circuit cortical interneurons. Aberrant development of GABAergic interneurons has been linked to several neurodevelopmental disorders, including epilepsy, schizophrenia, Rett syndrome and autism. Here, we report in mice that a single-nucleotide polymorphism (SNP) found in an autistic proband falls within a functional protein binding site in an ultraconserved cis-regulatory element. This element, I56i, is involved in regulating Dlx5/Dlx6 homeobox gene expression in the developing forebrain. We show that the SNP results in reduced I56i activity, predominantly in the medial and caudal ganglionic eminences and in streams of neurons tangentially migrating to the cortex. Reduced activity is also observed in GABAergic interneurons of the adult somatosensory cortex. The SNP affects the affinity of Dlx proteins for their binding site in vitro and reduces the transcriptional activation of the enhancer by Dlx proteins. Affinity purification using I56i sequences led to the identification of a novel regulator of Dlx gene expression, general transcription factor 2 I (Gtf2i), which is among the genes most often deleted in Williams-Beuren syndrome, a neurodevelopmental disorder. This study illustrates the clear functional consequences of a single nucleotide variation in an ultraconserved non-coding sequence in the context of developmental abnormalities associated with disease.

The Dlx homeodomain transcription factors play important roles in the differentiation and migration of GABAergic interneuron precursors. The mouse and human genomes each have six Dlx genes organized into three convergently transcribed bigene clusters (Dlx1/2, Dlx3/4, and Dlx5/6) with cis-regulatory elements (CREs) located in the intergenic region of each cluster. Amongst these, the I56i and I12b enhancers from the Dlx1/2 and Dlx5/6 locus, respectively, are active in the developing forebrain. I56i is also a binding site for GTF2I, a transcription factor whose function is associated with increased sociability and Williams–Beuren syndrome. In determining the regulatory roles of these CREs on forebrain development, we have generated mutant mouse-lines where Dlx forebrain intergenic enhancers have been deleted (I56i(–/–), I12b(–/–)). Loss of Dlx intergenic enhancers impairs expression of Dlx genes as well as some of their downstream targets or associated genes including Gad2 and Evf2. The loss of the I56i enhancer resulted in a transient decrease in GABA+ cells in the developing forebrain. The intergenic enhancer mutants also demonstrate increased sociability and learning deficits in a fear conditioning test. Characterizing mice with mutated Dlx intergenic enhancers will help us to further enhance our understanding of the role of these Dlx genes in forebrain development.

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