Repression of DR5-mediated transcription by TGIF. HepG2 cells were transfected with DR5-TATA-luc luciferase reporters, together with expression vectors encoding RXRα and TGIF, as indicated. +, present; −, absent. The DR5 reporters contained either 1, 2, or 4 tandem copies of the DR5 element as indicated. (A) AT-RA or 9C-RA was added 24 h prior to analysis, as indicated, to a final concentration of 10⁻⁷ M. The boxed inset in panel A shows the DR5-TATA-luc activity in the absence of added retinoic acid. (B) Cells were transfected with a control reporter lacking DR5 elements (TATA-luc) or with reporters containing 1 or 2 DR5 elements, together with RXRα and TGIF expression constructs, as indicated. 9C-RA was added as indicated 24 h prior to lysis. (C) Nuclear extracts from COS1 cells transfected with a TGIF or control vector were incubated with a radiolabeled probe containing a single consensus TGIF site (CTGTCAA). Unlabeled competitor oligonucleotides, either the consensus TGIF site or the DR5 RARE, were added as indicated. Triangles represent a titrations of a 5-, 20-, 80-, 320-, and 530-fold excesses of unlabeled competitor. The TGIF shifted band and free probe are indicated. (D) Cells were transfected with the DR1 or DR5 (two copy) reporters together with a control vector (pSUPER) or one expressing a TGIF-specific hairpin RNA (siTGIF) and treated with 9C-RA, as indicated, 24 h prior to analysis. Three days after transfection, luciferase activity was assayed. (E) Cells were transfected with a control vector, a TGIF expression plasmid, or a TGIF siRNA vector, together with a luciferase reporter in which transcription is activated by an estrogen response element (ERE). Estrogen (E2) was added, as indicated, 24 h prior to analysis.

Analysis of endogenous gene expression in Tgif null cells. (A) RNA was isolated from wild-type or mutant testes and subjected to real time RT-PCR analysis. Expression of the indicated genes was analyzed in triplicate together with actin, and results are presented as expression relative to actin for the wild type and mutant, with expression of each gene arbitrarily set equal to 1 for the wild-type samples. (B) Wild-type or Tgif null mouse embryo fibroblasts were treated with (+) or without (−) RA for 16 h, as indicated. Chromatin was cross-linked with formaldehyde and subjected to immunoprecipitation with a TGIF-specific antiserum (α-TGIF) or the preimmune serum (Pre-imm). Precipitates were analyzed by PCR for the presence of a region of the RARβ promoter which spans the RARE or part of the GAPDH promoter. In addition, the input chromatin samples were analyzed with the same oligonucleotide pairs.

Regulation of RXR-dependent transcription by TGIF. HepG2 cells were transfected with a DR1-TATA-luc luciferase reporter, together with expression vectors encoding RXRα and TGIF, as indicated (−, not used). 9C-RA was added 24 h prior to analysis, as indicated. In panel A, + indicates 10⁻⁷ M 9C-RA, and the titration of increasing 9C-RA was 10⁻⁹ M, 10⁻⁸ M, and 10⁻⁷ M. In panel B, increasing amounts of TGIF were cotransfected as indicated (in ng per duplicate). Luciferase activity was assayed 40 h after transfection and is presented, in arbitrary units, as the mean ± standard deviation of results from duplicate transfections.

TGIF interacts with RXRα. (A) COS1 cells were transfected with T7 epitope-tagged RARα or RXRα expression constructs, together with a Flag-tagged TGIF or control vector, and cells were treated with 9C-RA for 1 or 16 h (10⁻⁷ M) prior to lysis as indicated. Protein complexes were isolated on anti-Flag agarose and analyzed by Western blotting (WB) for the presence of coprecipitating RARα or RXRα. A portion of the lysates was subjected to analysis by direct Western blotting to monitor protein expression (below). +, present. (B) Cells were transfected as described for panel A, and 9C-RA was added for 1, 2, 4, or 6 h as indicated. Protein complexes were analyzed as described for panel A. (C) COS1 cells either treated for 16 h with 9C-RA (+) or left untreated (−) were lysed by sonication. Protein complexes were precipitated with a RXRα-specific monoclonal antibody and Western blotted with a TGIF-specific antiserum (above). The expression of TGIF and RXRα was monitored in the lysates by direct Western blotting (below). Arrowheads in panel C indicate specific bands; the bar indicates the heavy chain. The positions of molecular mass markers (in kDa) are shown to the left of each panel. IP, immunoprecipitation.

TGIF interacts with the RXRα ligand binding domain. (A) COS1 cells were transfected with expression constructs encoding T7 epitope-tagged RXRα deletion constructs, together with a Flag-tagged TGIF or control vector. Protein complexes were isolated on anti-Flag agarose and analyzed by Western blotting (WB) for the presence of coprecipitating T7-tagged RXRα constructs. (B) COS1 cells were transfected with T7 epitope-tagged ΔAF1 RXRα, together with a series of Flag-tagged TGIF expression constructs. Protein complexes were isolated on anti-Flag agarose and analyzed by Western blotting for the presence of coprecipitating RXRα. A portion of the lysates was subjected to analysis by direct Western blotting to monitor protein expression (below). (C) The RXRα deletion constructs used in panel A are shown schematically, together with the amino acids present in each construct. TGIF interaction is indicated to the right (+, interaction; −, none). AF1, activation function 1; DBD, DNA binding domain; LBD, ligand binding domain; AF2, activation function 2. (D) The TGIF deletion constructs are shown schematically, together with the amino acids in each and their interaction with RXRα. The two major conserved domains of vertebrate TGIFs are indicated. HD + 20, homeodomain plus 20 amino acid extension; RD2, repression domain 2; IP, immunoprecipitation.

TGIF and RXR cocomplexes. (A) COS1 cells were transfected with T7-tagged ΔAF1 RXRα, β, and γ constructs with or without Flag-TGIF. Complexes were isolated on Flag agarose and analyzed for coprecipitating T7-tagged RXRs. (B and C) COS1 cells were transfected with expression plasmids encoding T7-tagged RARα (B) or PPARγ (C), together with T7-RXRα ΔAF1 and Flag-TGIF, as indicated. Complexes were collected on Flag agarose and analyzed for the presence of coprecipitating T7-tagged proteins. A portion of the lysates was analyzed by direct Western blotting (below). IP, immunoprecipitation; WB, Western blotting; +, present.

TGIF recruits CtBP to RXRα. (A) COS1 cells were transfected with expression plasmids encoding T7-tagged RXRα ΔAF1 and TGIF (amino acids 1 to 116) and Flag-tagged CtBP, as indicated. Proteins were precipitated on Flag agarose and analyzed for the presence of coprecipitating T7-RXRα ΔAF1. A portion of the lysates was analyzed by direct Western blotting (below). (B) HepG2 cells were transfected with a DR1-TATA-luc luciferase reporter, and expression vectors encoding RXRα and TGIF or a mutant form of TGIF (S28C), as indicated. 9C-RA was added for the indicated times prior to analysis. Luciferase activity is presented, in arbitrary units, as the mean ± standard deviation of the results from duplicate transfections. (C) COS1 cells were transfected with T7-RXRα ΔAF1 and a control vector or one encoding Flag-TGIF or the S28C mutant form of TGIF. Flag immunoprecipitates were analyzed for the presence of T7-RXRα ΔAF1, and expression of transfected proteins in the lysates is shown below. IP, immunoprecipitation; WB, Western blotting; +, present.

A Tgif null mutation in mice. (A) The targeting vector, Tgif locus, and targeted allele are shown, with restriction enzyme sites and predicted sizes of restriction fragments. Coding exons are shown in black, and noncoding exons are shown in gray. The arrows indicate the positions of PCR primers used for genotype analyses. The hatched gray bar indicates the position of the probe used for Southern blotting. (B) Genomic DNA from mice of the indicated genotypes was subjected to restriction enzyme digestion and Southern blotting. (C) RNA from mice of the indicated genotypes was subjected to Northern analysis with probes for Tgif, GFP, Tgif2, and Gapdh. (D) Heterozygous Tgif mutants were intercrossed, and the genotypes of the offspring at 21 days after birth were determined by PCR from genomic DNA. The positions of the PCR primers are indicated by arrows in panel A. The numbers of mice of each genotype and the percentage of the total are shown, together with the number of litters and average litter size.

Retinoic acid teratogenesis in Tgif mutant embryos. Pregnant females, from heterozygous intercrosses, were treated with retinoic acid at gestational day 7.5. Embryos were examined for developmental defects at E10.5. (A) Embryos representative of each defect are shown. A normal wild type embryo and a homozygous mutant with exencephaly (severe open neural tube) are shown above. Below are three heterozygous mutants, one with a reduced fore and hind brain (left) and normal and arrested littermates (right). (B) The percentage of defective embryos (all defects) of each Tgif genotype is shown, together with the percentage with exencephaly. The total number of embryos of each genotype analyzed is shown below. (C) The numbers of embryos with each defect and the numbers of normal embryos are shown for each Tgif genotype. Defects: fb + hb, reduced fore and hind brain; arrest, severe developmental arrest or delay; exenc, exencephaly.