hnf4α mutations  (MedChemExpress)

Journal:

Article Title: Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4? dimerisation and transcriptional activity

doi: 10.1093/nar/gkg850

Figure Lengend Snippet: Structure-based amino acid sequence alignment of H7 of nuclear receptor LBDs (adapted from references 27–29). Usual names of nuclear receptors are indicated on the left side whereas names proposed by the Nuclear Receptor Nomenclature Committee are indicated on the right side. The arrow and boxed R indicate the glutamate and arginyl residues specifically found in members of the NR2 subfamily. Positions of the functional domains, of the I-box and of activation function 2 (AF-2) are shown. *, position in isoform 2 of HNF4α (HNF4α2), which was used in this study.

Article Snippet: Wild-type and mutated HNF4α were in vitro synthesised in reticulocyte lysates (Promega).

Techniques: Sequencing, Functional Assay, Activation Assay

Journal:

Article Title: Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4? dimerisation and transcriptional activity

doi: 10.1093/nar/gkg850

Figure Lengend Snippet: E262 and R258 are involved in HNF4α2 dimerisation in solution. (A and B) Analyses by co-immunoprecipitation assays and GST pull-down assays, respectively, of dimerisation between immobilised wild-type HNF4α2 fused to c-myc or GST (c-myc-HNF4α2 WT or GST-HNF4α2 WT) and wild-type or mutated [35S]methionine-labelled HNF4α2. Graphs in (A) and (B) indicate means ± SE of HNF4α2 mutant binding relative to that of the wild-type protein from three independent experiments. Inputs were taken into account for binding quantifications. (C) Dimerisation, analysed by co-immunoprecipitation assays of HNF4α2 WT, -ΔE262 or -E262A. For each assay, [35S]methionine-labelled HNF4α2 was incubated with the same protein fused to the c-myc tag. Control of synthesis of c-myc-HNF4α2 WT and mutated proteins is shown in the insert. (D) Dimerisation, analysed by GST pull-down assays of HNF4α2 WT, -ΔE262 and -E262A. For each assay, [35S]methionine-labelled HNF4α2 was incubated with the same protein fused to GST. Pull-down assays were performed in the indicated ionic strength conditions. The graph indicates means ± SE of HNF4α binding at 300 or 600 mM KCl relative to binding at 100 mM KCl (set to 100%) from three independent experiments. (E) Dimerisation, analysed by co-immunoprecipitation assays, between immobilised c-myc-HNF4α2 WT and [35S]methionine-labelled HNF4α2 WT or -R258M. The graph indicates mean ± SE of HNF4α2-R258M binding relative to that of the wild-type protein from four independent experiments. Inputs were taken into account for binding quantifications.

Article Snippet: Wild-type and mutated HNF4α were in vitro synthesised in reticulocyte lysates (Promega).

Techniques: Immunoprecipitation, Mutagenesis, Binding Assay, Incubation

Journal:

Article Title: Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4? dimerisation and transcriptional activity

doi: 10.1093/nar/gkg850

Figure Lengend Snippet: Mutations of E262 and R258 residues do not impair HNF4α2 DNA binding. (A) DNA binding of HNF4α2 mutants to the 32P-labelled HNF4α response element of the HNF1α promoter (HNF1 site). Control of in vitro synthesis of wild-type and mutated HNF4α2, used in EMSA, is shown in the insert (values on the right end indicate molecular size markers). The graph indicates means ± SE of mutated HNF4α2 DNA binding relative to that of the wild-type protein from three independent experiments. (B) Specificity of binding. Unprogrammed reticulocyte lysate (mock) yielded no shifted band. Supershifting was performed in the presence of the specific α455 HNF4α antiserum. (C) EMSA performed with a constant amount of HNF4α2 WT or HNF4α2-E262A and increasing amounts of labelled HNF1 probe. (D) HNF4α2-E262A did not bind as a monomer to the half-site of the HNF4α response element (HNF1 mt). (E) Competition experiments with COUP-TFII ΔAB. EMSA were performed on the HNF4 response element of the apoCIII promoter (CIIIB site) using in vitro synthesised HNF4α2 WT or HNF4α2-E262A and increasing amounts of the competitor COUP-TFII ΔAB. The amount of reticulocyte lysate in each lane was held constant by the appropriate addition of unprogrammed lysate. The positions of HNF4α2 and COUP-TFII ΔAB homodimers bound to DNA are indicated.

Article Snippet: Wild-type and mutated HNF4α were in vitro synthesised in reticulocyte lysates (Promega).

Techniques: Binding Assay, In Vitro

Journal:

Article Title: Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4? dimerisation and transcriptional activity

doi: 10.1093/nar/gkg850

Figure Lengend Snippet: Differential effects on dimerisation in solution and DNA binding of mutations in H7 and H10 of the HNF4α LBD. (A) Dimerisation, analysed by co-immunoprecipitation assays, between immobilised c-myc-HNF4α2 WT and [35S]methionine-labelled HNF4α2 WT or -E327M. The graph indicates mean ± SE of HNF4α2-E327M binding relative to that of the wild-type protein from three independent experiments. Inputs were taken into account for binding quantifications. (B) Dimerisation, analysed by GST pull-down assays, between immobilised GST-HNF4α2 WT and [35S]methionine-labelled HNF4α2 WT, -R258M and -E327M. Pull-down assays were performed in various ionic strength conditions as in Figure ​Figure2D.2D. The graph indicates means ± SE of mutant binding relative to that of the wild-type HNF4α from three independent experiments. Inputs were taken into account for binding quantifications. (C) Dimerisation, analysed by co-immunoprecipitation assays using the α455 antiserum, between immobilised HNF4α2 WT, -R258M, -E262A or -E327M and [35S]methionine-labelled HNF4α3, which is not recognised by the α455 antiserum. Control of HNF4α2 protein synthesis is shown in the insert. The graph indicates means ± SE of HNF4α3 retention by HNF4α2 mutants relative to HNF4α3 retention by HNF4α2 WT from four independent experiments. Control of protein synthesis was taken into account for binding quantifications. (D) DNA binding of HNF4α2-E327M on the HNF1 site, analysed by EMSA performed as in Figure ​Figure3A.3A. Supershifts were obtained in the presence of the α455 HNF4α antiserum as indicated. Control of protein synthesis is shown in the insert. The graph indicates mean ± SE of HNF4α2-E327M DNA binding relative to that of the wild-type protein from three independent experiments.

Article Snippet: Wild-type and mutated HNF4α were in vitro synthesised in reticulocyte lysates (Promega).

Techniques: Binding Assay, Immunoprecipitation, Mutagenesis

Journal:

Article Title: Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4? dimerisation and transcriptional activity

doi: 10.1093/nar/gkg850

Figure Lengend Snippet: Deletion of E262 strongly affects HNF4α2 transcriptional activity. HeLa (A), HEK 293 (B) and COS-1 cells (C) were transiently transfected with 12.5 ng of expression vector for wild-type or mutated HNF4α2 or the corresponding empty vector (–) together with 250 ng of HNF1α promoter construct. Fold induction refers to the activity with no HNF4α2 derivative (–), which was set to 1. Results are means ± SE of three independent experiments performed in triplicate. **, P = 0.0015, 0.0060 and 0.0018 for the ΔE262 mutant in (A–C), respectively; ***, P < 0.0001 for the ΔD261 mutant in (A). (D) Western blotting of HeLa cell extracts. (E) HNF4α-ΔE262 does not exhibit a dominant-negative activity on wild-type HNF4α. COS-1 cells were transfected as in (C), except that equal amounts of wild-type HNF4α and HNF4α-ΔE262 or control vector (–) were co-transfected. (F and G) Effects of substitution mutations on the dominant-negative activity of HNF4α-ΔAF-2. COS-1 cells were transfected as in (C), except that in (F) plasmids expressing wild-type, E262A or E262K HNF4α were co-transfected with an equal amount of vector expressing HNF4α-ΔAF-2 or the control vector (–), whereas in (G) pcDNA3 HNF4α2 WT was co-transfected with an equal amount of vectors expressing HNF4α-ΔAF-2 or HNF4α-ΔAF-2-E262A or the control vector (–). Activation of the HNF1α promoter is expressed relative to that obtained when only full-length proteins were expressed. Results are means ± SE of three independent experiments performed in triplicate. **, P = 0.0040 in (F); ***, P < 0.0001 in (F); *, P = 0.0278 in (G).

Article Snippet: Wild-type and mutated HNF4α were in vitro synthesised in reticulocyte lysates (Promega).

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Construct, Mutagenesis, Western Blot, Dominant Negative Mutation, Activation Assay

Journal:

Article Title: Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4? dimerisation and transcriptional activity

doi: 10.1093/nar/gkg850

Figure Lengend Snippet: Deletion of E262 markedly decreases recruitment of transcriptional partners. (A) GST pull-down assays were performed using GST-SRC-1a (570–780), GST-p300 (340–528), GST-PGC-1 (36–797) or GST-COUP-TFII and [35S]methionine-labelled WT or mutated HNF4α2. Inputs correspond to 5 or 2% (for the experiment with GST-SRC-1a) of amounts of labelled proteins used in the assays. (B and C) Effects of mutations of the E262 residue on the enhancement of HNF4α2 transcriptional activity by p300 and COUP-TFII, respectively. HeLa cells were transiently transfected with 12.5 ng of wild-type or mutated HNF4α2 expression vector, 250 ng of HNF1α promoter construct and 250 ng of empty control vector (white bars) or expression vectors (black bars) for p300 or COUP-TFII. Shown are per cent enhancements of wild-type and mutated HNF4α activities. Results are means ± SE of three independent experiments performed in triplicate. *, P = 0.0109; **, P = 0.0037.

Article Snippet: Wild-type and mutated HNF4α were in vitro synthesised in reticulocyte lysates (Promega).

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Construct