An SNP in an ultraconserved regulatory element affects Dlx5/Dlx6 regulation in the forebrain

Abstract

Dlx homeobox genes play a crucial role in the migration and differentiation of the subpallial precursor cells that give rise to various subtypes of gamma-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.

Fig. 1.

The SNP found in I56i is located in a highly conserved sequence. Forty vertebrate genome sequences in the vicinity of the targeted variant (bold) were aligned. Non-consensus bases are underlined and inserted bases in tenrec and medaka are indicated as gaps in all other species. This region corresponds to bases 96,641,417-96,641,443 of chromosome 7 from the February 2009 human genome assembly (GRCh37).

Fig. 2.

The 182-SNP affects I56i enhancer activity in the developing forebrain. (A) Dlx5/Dlx6 bigene cluster. Exons are shown in black and white (coding), the enhancers I56i in red and I56ii in gray. The I56i enhancer is enlarged to show the position of the 182-SNP variant identified by Hamilton and collaborators (Hamilton et al., 2005). The sequence of the 182-SNP variant (vI56i) and wild-type I56i are shown beneath. The remainder of the mouse and human I56i sequences are virtually identical (Zerucha et al., 2000). (B) At E11.5, the mouse I56i enhancer drives lacZ reporter expression to the ventral telencephalon (VT), diencephalon (Di) and to the hyoid (Hyo) and mandibular (Md) arches. (C-J) Transverse sections through the telencephalon of I56i-lacZ (C,G,I) and vI56i-lacZ (D-F,H,J) mouse embryos at E11.5 (C-F) and E13.5 (G-J). (A-F) β-galactosidase assay staining. (G-J) Immunohistochemistry for β-galactosidase. (E,F) Sections from primary transgenic embryos. Black arrowheads (C-F) and white arrowheads (I,J) indicate reduced vI56i enhancer activity in the MGE at E11.5 and in the MGE, LGE and CGE at E13.5, respectively. The asymmetric staining observed in E,F could be attributed to tilted sectioning and is not the result of asymmetric expression of the lacZ transgene; it is similar to the results of transgenic experiments reported by Lettice and collaborators concerning a point mutation in the ZRS enhancer of the Shh gene (Lettice et al., 2008). Scale bars: 1 mm in B; 500 μm in C-J.

Fig. 3.

I56i enhancer activity is impaired by the 182-SNP in migrating neuronal precursors at E13.5. (A-H) Immunolocalization of β-galactosidase (red) in I56i-lacZ (A-D) and vI56i-lacZ (E-H) transgenic mouse brains at the level of the LGE/MGE (A,B,E,F) and CGE (C,D,G,H). The boxed regions in A,C,E,G are shown at higher magnification in B,D,F,H. Numerous lacZ-expressing neurons were observed migrating from the MGE and CGE towards the developing cortex in I56i-lacZ brains (A-D), whereas only a few scattered lacZ-expressing cells were detected in the migrating streams leaving both eminences from the vI56i-lacZ brains (E-H). Scale bars: 500 μm in A,C,E,G; 250 μm in B,D,F,H.

Fig. 4.

I56i enhancer activity is impaired by the 182-SNP in the adult cortex. Quantification of the percentage of lacZ-expressing cells that co-express various GABAergic interneuron subtypes (PV, parvalbumin; SOM, somatostatin; CB, calbindin; CR, calretinin) in the adult mouse somatosensory cortex at P35. The contribution of vI56i-lacZ-expressing precursors to adult cortical interneuron populations expressing PV, SOM, CB and CR was significantly reduced compared with that of I56i-lacZ-expressing cells. All data are presented as mean ± s.e.m. *, P<0.001.

Fig. 5.

Binding of a forebrain transcription factor to I56i is affected by the 182-SNP. (A) The 182-SNP is located within a nuclear protein binding site (FP3) in the I56i enhancer as shown by DNaseI footprinting analysis. A DNA fragment corresponding to the I56i enhancer (nucleotides 1-276) was incubated with a nuclear protein extract from E13.5 mouse forebrain (+ Extract). Four regions of the enhancer (FP1 to FP4) were bound by nuclear proteins. Protected areas (footprints) are represented by thick black bars. Protein-DNA interactions were mapped on the mouse I56i enhancer sequence using a Maxam-Gilbert guanine/adenine chemical sequencing reaction (GA) as a standard. The 182-SNP was pinpointed within the protected area FP3. The position on the I56i sequence is shown to the left (Hamilton et al., 2005) (see Fig. S1 in the supplementary material). (B) A competitive electrophoretic mobility shift assay (EMSA) on I56i FP3 was performed using E13.5 mouse embryonic forebrain nuclear extract. In the presence of nuclear extract, four protein-DNA complexes are observed (lanes 1 and 6, complexes E1 to E4). These four complexes were competed by increasing amounts of the unlabeled I56i FP3 oligonucleotide (lanes 2 to 5, I56i FP3). In competition using the SNP-containing vI56i FP3 oligonucleotide (see Fig. 1A for sequence), protein-DNA complexes E1 to E3 were competed, whereas the lower-mobility complex E4 was unaffected (lanes 7 to 10, vI56i FP3).

Fig. 6.

Transcriptional activation of I56i by Dlx and Gtf2i is impaired by the 182-SNP. (A) The molecular weight of the protein component(s) of the E4 complex is similar to that of Dlx2. EMSA using the I56i FP3 oligonucleotide with E13.5 mouse embryonic forebrain nuclear extract (Nuclear Extract) or increasing amounts of recombinant Flag-tagged Dlx2 (Flag-Dlx2). Preincubation of the recombinant Dlx2 with an anti-Flag antibody leads to the formation of a high molecular weight complex (asterisk, Flag-Ab lane). (B) Affinity of Dlx proteins for the FP3 site is affected by the 182-SNP. EMSA was performed using the I56i X3 oligonucleotide and a fixed amount of recombinant Dlx5. The resulting complexes (arrowhead) were competed more efficiently by the unlabeled wild-type I56i X3 (increasing amounts in lanes 3 to 5) than by the variant (vI56i X3, lanes 6 to 8) competitor. Competition (%) refers to the percentage of remaining complexes with respect to the control reaction (without competitor, 100%). (C) Replacement of I56i by the 182-SNP-containing variant (vI56i-pGL4.23) impaired transcriptional activation by Dlx2 or Dlx5. (D) Dlx2 and Dlx5 synergize with Gtf2i in activating the transcription of a luc2 reporter construct containing the I56i enhancer. Values shown in C and D represent the mean of relative luciferase activity obtained from five (C) or six (D) independent experiments ± s.e.m. Relative luciferase activity was normalized to Dlx2 plus mI56i-pGL4 (100%). *, P<0.05.