An etiologic regulatory mutation in IRF6 with loss- and gain-of-function effects

Abstract

DNA variation in Interferon Regulatory Factor 6 (IRF6) causes Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate (CLP). However, an etiologic variant in IRF6 has been found in only 70% of VWS families. To test whether DNA variants in regulatory elements cause VWS, we sequenced three conserved elements near IRF6 in 70 VWS families that lack an etiologic mutation within IRF6 exons. A rare mutation (350dupA) was found in a conserved IRF6 enhancer element (MCS9.7) in a Brazilian family. The 350dupA mutation abrogated the binding of p63 and E47 transcription factors to cis-overlapping motifs, and significantly disrupted enhancer activity in human cell cultures. Moreover, using a transgenic assay in mice, the 350dupA mutation disrupted the activation of MCS9.7 enhancer element and led to failure of lacZ expression in all head and neck pharyngeal arches. Interestingly, disruption of the p63 Motif1 and/or E47 binding sites by nucleotide substitution did not fully recapitulate the effect of the 350dupA mutation. Rather, we recognized that the 350dupA created a CAAAGT motif, a binding site for Lef1 protein. We showed that Lef1 binds to the mutated site and that overexpression of Lef1/β-Catenin chimeric protein repressed MCS9.7-350dupA enhancer activity. In conclusion, our data strongly suggest that 350dupA variant is an etiologic mutation in VWS patients and disrupts enhancer activity by a loss- and gain-of-function mechanism, and thus support the rationale for additional screening for regulatory mutations in patients with CLP.

Figure 1.

A Rare mutation mapped in IRF6 enhancer element. (A) A screen shot from UCSC Genome Browser that shows IRF6 coding and highly conserved regions. Exons 1–9 are connected rectangles from right to left. The track below is the multi-species conservation, and includes MCS9.7 enhancer element located 9.7 kb upstream of exon 1. The green tracks are the multi-species conservation in eleven vertebrates. (B) The chromatograph of the DNA sequence from unaffected and affected individuals from the Brazilian family. The red arrow indicates the position of the duplicated A nucleotide. (C) Pedigree of a Brazilian family with Van der Woude syndrome (VWS). DNA samples were collected from five individuals (II.2, II.4, III.1, III.3 and III.7). All carried the MCS9.7-350dupA variant (blue circle). (D) Digital photos of two affected patients showing pits (III.3) and bi-lateral pits (III.7) in lower lip and repaired cleft lip in both. (E) The nucleotide conservation of the cis-overlapping motifs of Ebox 3 and 4 and p63 Motif 1. (F) Diagram of MCS9.7 element shows the binding sites of AP2a (yellow box), p63 Motif 1 and Motif 2 (orange box) and Ebox motifs (grey box). The rest of the colored boxes represent putative binding sites of transcription factors that expressed or have a known role in orofacial clefting (Supplementary Material, Table S1). Below the diagram of MCS9.7 is the sequence of the cis-overlapping p63 Motif 1 and Ebox 3 and 4 Motifs. The position of the A duplication mutation is shown by the vertical red arrow. A Logo representation of p63 motif and Ebox motif (jaspar.genereg.net).

Figure 2.

The effect of the 350dupA mutation on MCS9.7 enhancer activity. (A) HEK293 cells were transfected with three different constructs, no enhancer (Control), MCS9.7 enhancer (MCS9.7) and MCS9.7 enhancer with 350dupA mutation (MCS9.7-350dupA). Luciferase activity was measured 24 h post-transfection and normalized to internal Renilla activity. (B) In vivo enhancer activity of MCS9.7 from E11.5 transgenic embryos and MCS9.7 with the 350dupA mutation (MCS-9.7-350dupA). Reproducible craniofacial staining was observed at the pharyngeal arches and the lambdoid structure (yellow arrow; fusion site of the lateral nasal (ln), medial nasal (mn) and maxillary (mx) prominences). (C) Annotation of MCS9.7 and MCS9.7-350dupA enhancer activity patterns. Embryos were classified into four categories of patterns: (i) no LacZ staining detected (white), (ii) non-craniofacial (ectopic) LacZ activity (gray), (iii) weak craniofacial LacZ activity as in MCS9.7 representative embryo (pink), (iv) strong craniofacial LacZ activity, pattern as MCS9.7 representative embryo (red). The data in each column are represented as the mean of five replicates ± SD.

Figure 3.

Effect of 350dupA mutation on transcription factor binding to MCS9.7. (A) Chromatin immunoprecipitation from HaCaT cells followed by quantitative real-time PCR was used to detect binding of indicated transcription factors to MCS9.7 enhancer element, to the non-conserved sequence element (NCS10) that is 800 bp away from MCS9.7, and at the transcriptional start site for IRF6 (IRF6 TSS). (B) The effect of the 350dupA mutation on the MCS9.7 enhancer activity was tested before (−) and after (+) transactivation with ΔNp63 expression vector. Sosa2 cells were transfected with indicated luciferase construct in comparison with cotransfection of these plasmids with ΔNp63 expression vector. (C) EMSA assay was used to test the effect of 350dupA mutation on the binding of p63 protein to Motif 1. A unique shift band (arrow) in ascending concentrations of recombinant p63 protein was observed (arrow) compare with free probe alone and probe with only reticulocyte extract mixture (arrowhead). The 350dupA mutation completely disrupts p63 protein binding. The binding of p63 was confirmed by using monoclonal antibodies against p63 protein shown by the super shift band (double head arrow). (D) Binding of E47 to MCS9.7 probe using a dilution series of E47 protein. Recombinant E47 protein showed a unique shift band (arrow) when incubated with wild-type probe compare with free probe alone (FP). The binding of E47 to MSC9.7-350dupA was abolished in all three ascending concentrations. The specificity of E47 binding was confirmed using antibodies against E47 proteins shown by the super shift band (head arrow). The data in each column are represented as the mean of five replicates ± SD.

Figure 4.

Allelic Architecture of 350dupA Mutation within a cis-overlapping Motif. Substitution (lowercase, italic) and insertion (subscript) mutations were made in MCS9.7 to delineate effects on P63 motif1 (M1), Ebox 3 (E3) and Ebox 4 (E4) in HEK293 cells. The construct without MCS9.7 enhancer was used as base line control. The common-type MCS9.7 enhancer (M1/E3E4) increased luciferase activity 29-fold, whereas 350dupA mutation (A(m1/e3)E4) decreased the activity by 6-fold compared with the common sequence of MCS9.7. Disruption of Ebox 3 and 4 (M1/e3e4) did not change the luciferase activity, while abolishing only P63 motif1 (m1/E3E4) reduced the luciferase activity by 22%. Insertion of A nucleotide in the spacer between Ebox 3 and 4 (M1/E3AE4) slightly increased luciferase activity, while the insertion of C nucleotide (C(m1e3)E4) in the same position similar to 350dupA reduced the activity by 25%. Disruption of P63 Motif1 and insertion of T nucleotide (m1/E3E4T) reduced activity by 18%; however, disruption of P63 Motif 1, Ebox 3 and 4 and insertion of T nucleotide (m1/e3e4T) to create a CAAAG motif decreased the luciferase activity by 35%.

Figure 5.

350DupA mutation creates a novel binding site. (A) EMSA assay showed unique shift bands when MCS9.7 and MCS9.7-350dupA probes incubated with Lef1 recombinant protein. The binding specificity of Lef1 protein was confirmed using polyclonal antibodies against Lef1 as shown with the super shift (SS) bands when MCS9.7 and MCS9.7-350dupA probes were incubated with Lef1 protein and anti-Lef1 antibodies. The 350dupA mutation creates a novel Lef1 binding site. (B) Basic construct without MCS9.7 element showed a minimal luciferase activity, while MCS9.7 enhancer element increased luciferase activity 33-fold. The 350dupA mutation reduced the activity about 4-fold compared MCS9.7. Cotransfection with the Lef1-βCatenin expression vector did not change luciferase activity in basic construct but reduced the activity by 29% when driven by MCS9.7. Interestingly, luciferase activity driven by MCS9.7-350dupA was significantly reduced by 51% when cotransfected with Lef1-βCatenin vector. (C) A merge image of red and green fluorescent staining shows that both Irf6 and Lef1 are colocalized in the same epidermal cells. A pre-merged immuno-staining shows the expression of Irf6 in red fluorescent (D) and Lef1 in green fluorescent (E) in the epidermis of murine embryo at E14.5.

Figure 6.

A working model for the 350dupA mutation in patients with Van der Woude syndrome. (A) In normal situation, p63 protein binds to Motif1 (M1) and 2 (M2) which are 60 bp apart within MCS9.7 enhancer element and drives IRF6 expression. (B) The 350dupA mutation disrupts the binding of p63 to Motif1 and creates a repressive novel site for Lef1-βCat that interferes with the binding of p63 to the second Motif2 to significantly disrupt IRF6 expression.