Functional analysis of mutations in TGIF associated with holoprosencephaly

Abstract

Holoprosencephaly (HPE) is the most common structural malformation of the forebrain and face in humans. Our current understanding of the pathogenesis of HPE attempts to integrate genetic susceptibility, evidenced by mutations in the known HPE genes, with the epigenetic influence of environmental factors. Mutations or deletions of the human TGIF gene have been associated with HPE in multiple population cohorts. Here we examine the functional effects of all previously reported mutations, and describe four additional variants. Of the eleven sequence variations in TGIF, all but four can be demonstrated to be functionally abnormal. In contrast, no potentially pathogenic sequence alterations were detected in the related gene TGIF2. These results provide further evidence of a role for TGIF in HPE and demonstrate the importance of functional analysis of putative disease-associated alleles.

Fig. 1

Facial photographs of the first proband (A and B) with the pE45X mutatiom (C). Patient two (D) carries two different variations the S46fs (E) and H76Q (F). Red arrows indicate the missense changes and the deleted bases are boxed. The expected chromatogram from the normal allele is above and the frameshifted allele is below. A representative dHPLC chromatogram from patient three (G) shows heterozygosity for the normal allele (blue) and the variant allele (red). In panel H, the deleted base is boxed and the expected sequence from the normal allele is above and the frameshifted allele is below the chromatogram.

Fig. 2

(A) Summary of the positions of the studied mutations within the TGIF protein. Note that the P63R mutation [11] was incorrect; instead, the observed sequence change predicts P63A. (B) HPE mutations in the homeodomain of TGIF affect DNA binding ability. COS1 cells were transfected with a Flag-tagged TGIF wildtype or HPE mutant construct as indicated. 36 hours after transfection, cell lysates were incubated with biotinylated double-stranded oligonucleotide containing either mutated (m) or consensus TGIF binding site (CTGTCA). TGIF protein bound to DNA was isolated on Streptavidin agarose and analyzed by western blot for the presence of TGIF. These experiments were performed repeatedly to assure consistency of the results. Portions of each lysate and pellet sample were subjected to direct western blot analysis to monitor protein expression and solubility (below).

Fig. 3

TGIF HPE mutants interact with corepressors. (A and B) COS1 cells were transfected with His6-CtBP and Flag-TGIF wildtype or HPE mutant, as indicated. Protein complexes were isolated on anti-Flag agarose and analyzed by western blot for the presence of co-precipitating CtBP. A portion of the lysates was subjected to direct western blot analysis to monitor protein expression (below). (C) COS1 cells were transfected with Myc-epitope tagged Sin3 expression construct, together with the indicated Flag-tagged TGIF constructs or control vector. Protein complexes were isolated on anti-Flag agarose and analyzed by western blot for the presence of co-precipitating Sin3. A portion of the lysates was subjected to direct western blot analysis to monitor protein expression (below).

Fig. 4

TGIF HPE mutants interact with Smad3 and affect repression of TGFβ-activated transcription. (A) COS1 cells were transfected with untagged Smad3 and Flag-TGIF wildtype or HPE mutants or control vector, as indicated. Protein complexes were isolated on anti-Flag agarose and analyzed by western blot for the presence of coprecipitating Smad3. A portion of the lysates was subjected to direct western blot analysis to monitor protein expression (below). Due to the low expression level of the K259fs construct, a longer exposure of the lysate western blot is shown below. (B, C and D) HepG2 cells were transfected with a TGFβ-responsive luciferase reporter, 3TP-lux, and increasing amounts of TGIF wildtype or HPE mutant expression constructs. All cells were treated with 100 pM TGFβ prior to analysis. Firefly luciferase activity was normalized to Renilla luciferase and is represented in arbitrary units, as the mean +/− s.d. of duplicate transfections. (B) HepG2 cells were transfected with 10 ng and 50, or 10, 25 and 50 ng of TGIF expression construct (the height of the grey steps indicate the amount of input TGIF DNA: 10, 25 or 50 ng). The black bar indicate the activity of the reporter alone, without TGIF (C) 10 or 100 ng of TGIF constructs were transfected (grey steps). (D) Dose-dependent repression of luciferase activity by TGIF is shown as a graph of luciferase activity in arbitrary units as the mean +/− s.d. of duplicate transfections as a function of the amount (in ng) of TGIF expression vector transfected.

Fig. 5

TGIF HPE mutants interact with RXRα and affect repression of retinoic acid regulated transcription. (A) COS1 cells were transfected with T7-RXRα and Flag-TGIF wildtype or HPE mutant construct, as indicated. Protein complexes in lysates were isolated on anti-Flag agarose and analyzed by western blot for the presence of co-precipitating RXRα. Coprecipitating RXR is indicated with an arrow, the Ig heavy chain with a bar. A portion of the lysates was subjected to direct western blot analysis to monitor protein expression (below). (B and C) HepG2 cells were transfected with a DR1-TATA-luc luciferase reporter and RXRα (control and experimental), together with the indicated TGIF wildtype or HPE mutant expression constructs in the experimental lanes. Cells were either left untreated (black bars) or treated with 9-cis-retinoic acid (9C-RA) for 24 hours prior to analysis (striped bars). Reporter luciferase activity (normalized to Renilla luciferase activity) was assayed 40 hours after transfection and is presented, in arbitrary units, as the mean +/− s.d., of duplicate transfections.

Fig. 6

Analysis of Shh/Tgif double mutant mice. (A) Genotyping data at postnatal day 21 (P21) of offspring obtained from Shh/Tgif double heterozygote intercrosses in a mixed strain background. Numbers of mice with each genotype, the percentage of the total number of mice with each genotype observed, and the expected percentage of mice with each genotype, based on Mendelian ratios, are shown. (B) Genotyping data of embryos at 10.5 dpc obtained from Tgif heterozygotes crossed with Shh/Tgif double heterozygotes in a C57BL6/J strain background. The number of embryos with each genotype observed and the expected frequency of each genotype are shown, as in A. (C) The number of embryos of each genotype from the breeding in B with each type of observed defect is shown. (D–G) Examples of defective embryos. (D) Wildtype (left) 10.5 dpc embryo and a littermate with growth delay (right). (E) Growth arrested embryo. (F). Embryo with open neural tube defect. (G) Embryo with brain reduction.