Journal:

Article Title: The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development

doi:

Figure Lengend Snippet: Targeted disruption of the COUP-TFII locus. (A) Targeting vector construction. (Shaded boxes I, II, and III) COUP-TFII-coding regions; (open box) 5′-untranslated region; (arrows in boxes) direction of transcription. (TK) Thymidine kinase gene; (neor) neomycin-resistance gene. (R) EcoRI; (St) StuI; (Sl) SalI; (X) XbaI; (H) HindIII; (Sp) SpeI; (Xh) XhoI. (B) Southern blot analysis. (WT) Wild-type allele; (KO) recombined allele. Of the 680 colonies screened, 17 positive clones were obtained for a targeting efficiency of 2.5%. To ensure proper recombination had occurred at both the 5′ and 3′ ends of the neor cassette, Southern analysis was performed with probes 5′ and 3′ of the targeting vector. The 5′ probe was a 0.8-kb EcoRI fragment with XbaI as the diagnostic enzyme as the neor cassette (see A) introduced a new XbaI site to the locus. The expected 10-kb wild-type and 8-kb mutant fragments were generated (left). The 3′ probe was a 1-kb EcoRV–NsiI fragment downstream of the 3′ homologous sequence and SpeI was the diagnostic enzyme that produced a 12-kb wild-type and a 6-kb mutant band (right). Reprobing the blots with the neor gene revealed no other insertions in the genome (data not shown). Therefore, correct recombination was obtained at the COUP-TFII locus to inactivate the gene. (C) PCR genotyping showing wild-type (+/+), heterozygote (+/−), and mutant (−/−) embryos from the two independent (E5 and H7) COUP-TFII targeted lines. (D) RT–PCR analysis of transcripts from E9.5 embryos. There are no products in the absence of reverse transcriptase (RT) indicating that the bands are products of reverse transcription, and there are no wild-type COUP-TFII transcripts in the mutants (−/−). (KO) An amplified product from the 5′ end of the locus and the neor gene.

Article Snippet: Whole-mount and section in situ hybridization Whole-mount or section in situ hybridization was performed with a 1-kb ( Nsi I– Eco RV) COUP-TFII 3′ UTR cDNA fragment or an Ang1 cDNA fragment (from G.D. Yancopoulos, Regeneron, Tarrytown, NY) as templates for transcription of digoxigenin-labeled (whole mount) or [ 35 S]UTP-labeled (section) antisense riboprobes.

Techniques: Plasmid Preparation, Southern Blot, Clone Assay, Diagnostic Assay, Mutagenesis, Generated, Sequencing, Produced, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: PLoS Biology

Article Title: Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid–Activated Receptor

doi: 10.1371/journal.pbio.0060227

Figure Lengend Snippet: (A) Front and side views of the COUP-TFII LBD monomer with its AF2 helix colored in red. (B) Organization of the COUP-TFII LBD dimer, showing that its dimer interface is formed predominantly by helix α10 (cyan). (C) The packing of the ligand-binding pocket within the bottom half of the COUP-TFII LBD. (D) Space-filling diagram shows two small cavities in COUP-TFII colored with magenta (18 Å 3 ) and white (12 Å 3 ). (E and F) Overlay of the COUP-TFII LBD structure with the SRC-1 LXXLL motif (green in E) from the RXR structure or with the SMRT corepressor motif (magenta in F) from the antagonist bound PPARα structure. (G) Hydrogen bonds (yellow dashed lines) and hydrophobic interactions of the COUP-TFII AF2 helix (green) within the cofactor binding site.

Article Snippet: Cells were transiently transfected with 50 ng COUP-TFII expression vector, 150 ng of reporter plasmid and 0.5 ng of phRL-CMV (Promega).

Techniques: Ligand Binding Assay, Binding Assay

Journal: PLoS Biology

Article Title: Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid–Activated Receptor

doi: 10.1371/journal.pbio.0060227

Figure Lengend Snippet: (A) Activation of the NGFI-A promoter reporter construct with increasing concentrations of COUP-TFII (0ng, 50ng, 100ng, 150ng, and 200 ng of the expression vector, respectively, for each cell line). (B) Domain structure of COUP-TFII. (C) Effects of the COUP-TFII deletion mutants on activation of the NGFI-A promoter-driven reporter. The AF1-DBD construct activates ∼2-fold above empty vector control. (D) Activation by the GAL4-DBD-COUP-TFII-LBD. The fold activation is the relative fly luciferase activity of the NGFI-A promoter induced by COUP-TFII versus the control vector without COUP-TFII. All data are normalized to the activity of Renilla luciferase that was used as transfection control. For statistical analysis, the fold induction was compared with full-length COUP-TFII or GAL4-DBD in (C) and (D), respectively.

Article Snippet: Cells were transiently transfected with 50 ng COUP-TFII expression vector, 150 ng of reporter plasmid and 0.5 ng of phRL-CMV (Promega).

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

Journal: PLoS Biology

Article Title: Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid–Activated Receptor

doi: 10.1371/journal.pbio.0060227

Figure Lengend Snippet: (A) Top view of the COUP-TFII dimer showing the close packing of L364 (gray) and L365 (green) from helices α10 (cyan) in the dimer interface. (B) Effects of the L364A/L365A double mutant and the AF2 deletion mutant on COUP-TFII activation of the NGFI-A promoter. For easy comparison, the relative fold of activation by the wild-type receptor is set to 1. The statistical analysis for the fold induction of the mutants was compared with wild type COUP-TFII.

Article Snippet: Cells were transiently transfected with 50 ng COUP-TFII expression vector, 150 ng of reporter plasmid and 0.5 ng of phRL-CMV (Promega).

Techniques: Mutagenesis, Activation Assay

Journal: PLoS Biology

Article Title: Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid–Activated Receptor

doi: 10.1371/journal.pbio.0060227

Figure Lengend Snippet: (A) Comparison of the RXRα charge clamp (K284 and E434 in the left) with that of COUP-TFII (right). The SRC-1 LXXLL motif is shown in green. (B) Effects of the charge clamp mutations on COUP-TFII activation. The relative fold of activation by the wild-type receptor is set to 1 in (B), (C), and (D). The statistical analysis for the fold induction of the mutants was compared with wild-type COUP-TFII. (C) Effects of coactivators on COUP-TFII activation. The statistical analysis for the fold induction by coactivators was compared with the wild-type COUP-TFII in the absence of additional coactivators. (D) Effects of mutations in the three conserved SRC-3 LXXLL coactivator binding motifs (M1-M3) to LXXAA on the SRC-3-mediated enhancement of COUP-TFII induction. For statistical analysis, the fold induction was compared with COUP-TFII in (B) and (C) and COUP-TFII and SRC-3 cotransfection in (D). The statistical analysis for the fold induction by mutated coactivators was compared with that of wild type SRC-3.

Article Snippet: Cells were transiently transfected with 50 ng COUP-TFII expression vector, 150 ng of reporter plasmid and 0.5 ng of phRL-CMV (Promega).

Techniques: Activation Assay, Binding Assay, Cotransfection

Journal: PLoS Biology

Article Title: Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid–Activated Receptor

doi: 10.1371/journal.pbio.0060227

Figure Lengend Snippet: (A) An active model of COUP-TFII. This model is based on the agonist-bound RXR structure with its helix α10 (cyan) extended and AF2 (red) in the active conformation. One cavity (magenta) with size of 659 Å 3 is found in this conformation. (B) The potential ligand binding pocket (magenta mesh) in the active model of the COUP-TFII LBD and its surrounding residues. (C) Effects of pocket residue mutations on COUP-TFII activation. The relative fold of activation by the wild-type receptor is set to 1. The statistical analysis for the fold induction of the mutants was compared with wild-type COUP-TFII.

Article Snippet: Cells were transiently transfected with 50 ng COUP-TFII expression vector, 150 ng of reporter plasmid and 0.5 ng of phRL-CMV (Promega).

Techniques: Ligand Binding Assay, Activation Assay

Journal: PLoS Biology

Article Title: Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid–Activated Receptor

doi: 10.1371/journal.pbio.0060227

Figure Lengend Snippet: (A) Effects of charcoal-treated FBS on COUP-TFII activation in the presence or absence of SRC-3 coactivator. The basal activity of the NGFI-A reporter construct in the presence of FBS and absence of COUP-TFII and SRC-3 is set as 1. (B) Addition of 50 μM of 9cRA or ATRA to promote COUP-TFII binding to SRC-3–1 coactivator motif where the addition of steroids (50 μM) has little effect. (C) Concentration-response curves of 9cRA and ATRA, which show the binding affinity (EC50) of 9cRA and ATRA is 17 μM and 26 μM, respectively. (D) Effects of ATRA and 9cRA on COUP-TFII activation of the NGFI-A promoter in COS-7 cells.

Article Snippet: Cells were transiently transfected with 50 ng COUP-TFII expression vector, 150 ng of reporter plasmid and 0.5 ng of phRL-CMV (Promega).

Techniques: Activation Assay, Activity Assay, Construct, Binding Assay, Concentration Assay