Journal: Scientific Reports

Article Title: Evidence for NR2F2 /COUP-TFII involvement in human testis development

doi: 10.1038/s41598-024-68860-3

Figure Lengend Snippet: Identification and modeling of the COUP-TFII p.Arg246His variant. ( A ) The pedigree of the family. Squares denote male and circles denote female family members. Solid square represents the patient. ( B ) Location of the Arg 246 residue. Chromatogram shows the de novo heterozygous change in NR2F2 (NM_021005: c.G737A > p.Arg246His). Schematic representation of the functional domains of the COUP-TFII protein is shown with an arrow indicating the position of the p.Arg246His mutation. The N-terminus Activation-Function 1 (AF-1), the DNA-Binding domain (DBD), the Ligand-Binding domain (LBD) and the C-terminus Activation-Function 2 (AF-2) domains are indicated. The mutated residue is located in the LBD. ( C ) Structure of COUP-TFII LBD showing the location and polar interactions of the WT and mutant residues. The LBD structure is drawn as cartoon; wild-type (blue, left), mutated (red, middle) or superimposed (right) amino acids are shown as sticks. The wild-type Arg 246 residue is involved in two polar interactions. One involves the neighboring Val 242 located on the same alpha-helix, the other one, the residue Glu 393 located on an opposite alpha-helix, stabilizing AF-2 domain. The mutant His 246 residue has lost the ability to interact with Glu 393. ( D ) Evolutionary conservation of Arg 246 residue. Sequence alignment of the partial LBD of human COUP-TFII protein with other species (left) and the LBD of several nuclear receptors (right) shows a high degree of amino acid conservation. The position of the mutation is highlighted by the box.

Article Snippet: Subsequently, overnight incubation at 4 °C with both mouse anti-NR5A1 (1:200, #sc-393592, Santa-Cruz Biotechnologies) and rabbit anti-COUP-TFII (1:200, #ab42672, Abcam) primary antibodies was performed.

Techniques: Variant Assay, Residue, Functional Assay, Mutagenesis, Activation Assay, Binding Assay, Ligand Binding Assay, Sequencing

Journal: Scientific Reports

Article Title: Evidence for NR2F2 /COUP-TFII involvement in human testis development

doi: 10.1038/s41598-024-68860-3

Figure Lengend Snippet: The mutation does not impact the protein stability, subcellular localization or interaction with NR5A1. ( A ) Protein stability. Left: Whole cell protein was extracted from the HEK293-T cells transfected with either WT or p.Arg246His COUP-TFII-encoding plasmids. Western blot analysis showed a single band around 46 kDa for both COUP-TFII-WT and COUP-TFII-p.Arg246His. The image is cropped from the original full-length blot shown in Supplementary Figure and is representative of three independent experiments. Right: the band intensity was normalized to that of β-Actin and plotted as mean ± SEM of COUP-TFII/β-Actin ratios obtained from three independent experiments. There was no significant difference between the COUP-TFII-WT and COUP-TFII-p.Arg246His protein production/stability (Student t-test, P = 0.84). ( B ) Nuclear localization of COUP-TFII. HEK293-T cells were transfected with WT or mutant COUP-TFII expression vectors. Immunocytochemistry showed strong nuclear localization for both WT and mutant proteins (green). Images were taken at 40X magnification (Scale bar: 20 µm). ( C ) The mutation does not affect the interaction between COUP-TFII and NR5A1. Left: NR5A1 and WT or p.Arg246His COUP-TFII-encoding plasmids were transiently expressed for 48 h in HEK293-T cells. Protein–protein interaction was assessed by the Duolink proximity ligation assay (PLA). Nuclei are stained with DAPI (blue) while each signal/dot (green) represents a COUP-TFII/NR5A1 interaction event. Right: quantification of PLA. For each condition, the number of interactions of at least 50 individual cells was counted and the median (interquartile range) was analyzed. Statistical analysis revealed that the binding of NR5A1 to COUP-TFII-p.Arg246His was comparable to that of COUP-TFII-WT (Mann–Whitney test, P = 0.22). Images were taken at 63X magnification (Scale bar: 20 µm).

Article Snippet: Subsequently, overnight incubation at 4 °C with both mouse anti-NR5A1 (1:200, #sc-393592, Santa-Cruz Biotechnologies) and rabbit anti-COUP-TFII (1:200, #ab42672, Abcam) primary antibodies was performed.

Techniques: Mutagenesis, Transfection, Western Blot, Expressing, Immunocytochemistry, Proximity Ligation Assay, Staining, Binding Assay, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Evidence for NR2F2 /COUP-TFII involvement in human testis development

doi: 10.1038/s41598-024-68860-3

Figure Lengend Snippet: Mutant COUP-TFII shows loss of inhibitory effect on NR5A1-dependent activation of target promoters. The transcriptional activities of COUP-TFII-WT and COUP-TFII-p.Arg246His were studied using the human ( A ) LHB and ( B ) INSL3 promoters as reporters, following transfection in HEK293-T cells. The data shown represent the dot plot of results with mean ± SEM of a minimum of three independent experiments, each with twelve replicates. The reporter constructs were transfected into HEK293-T cells with either the COUP-TFII-WT and COUP-TFII-p.Arg246His expression vector with or without NR5A1-WT expression vector. All data were standardized for Renilla activity. The results are expressed as relative percentage of NR5A1-WT activity (100%). COUP-TFII-WT exerts an inhibitory effect on NR5A1-mediated activation of both ( A ) LHB and ( B ) INSL3 . However, there is a loss of inhibition by COUP-TFII-p.Arg246His. The statistical significances are marked by the horizontal lines (dash line *, P < 0.05; bold line ***, P < 0.001).

Article Snippet: Subsequently, overnight incubation at 4 °C with both mouse anti-NR5A1 (1:200, #sc-393592, Santa-Cruz Biotechnologies) and rabbit anti-COUP-TFII (1:200, #ab42672, Abcam) primary antibodies was performed.

Techniques: Mutagenesis, Activation Assay, Transfection, Construct, Expressing, Plasmid Preparation, Activity Assay, Inhibition

Journal: Scientific Reports

Article Title: Evidence for NR2F2 /COUP-TFII involvement in human testis development

doi: 10.1038/s41598-024-68860-3

Figure Lengend Snippet: Identification and modeling of the COUP-TFII p.Arg246His variant. ( A ) The pedigree of the family. Squares denote male and circles denote female family members. Solid square represents the patient. ( B ) Location of the Arg 246 residue. Chromatogram shows the de novo heterozygous change in NR2F2 (NM_021005: c.G737A > p.Arg246His). Schematic representation of the functional domains of the COUP-TFII protein is shown with an arrow indicating the position of the p.Arg246His mutation. The N-terminus Activation-Function 1 (AF-1), the DNA-Binding domain (DBD), the Ligand-Binding domain (LBD) and the C-terminus Activation-Function 2 (AF-2) domains are indicated. The mutated residue is located in the LBD. ( C ) Structure of COUP-TFII LBD showing the location and polar interactions of the WT and mutant residues. The LBD structure is drawn as cartoon; wild-type (blue, left), mutated (red, middle) or superimposed (right) amino acids are shown as sticks. The wild-type Arg 246 residue is involved in two polar interactions. One involves the neighboring Val 242 located on the same alpha-helix, the other one, the residue Glu 393 located on an opposite alpha-helix, stabilizing AF-2 domain. The mutant His 246 residue has lost the ability to interact with Glu 393. ( D ) Evolutionary conservation of Arg 246 residue. Sequence alignment of the partial LBD of human COUP-TFII protein with other species (left) and the LBD of several nuclear receptors (right) shows a high degree of amino acid conservation. The position of the mutation is highlighted by the box.

Article Snippet: Cells were incubated overnight with rabbit anti-COUP-TFII antibody (1:200, #ab42672, Abcam) diluted in PBS-3% BSA at 4 °C.

Techniques: Variant Assay, Residue, Functional Assay, Mutagenesis, Activation Assay, Binding Assay, Ligand Binding Assay, Sequencing

Journal: Scientific Reports

Article Title: Evidence for NR2F2 /COUP-TFII involvement in human testis development

doi: 10.1038/s41598-024-68860-3

Figure Lengend Snippet: The mutation does not impact the protein stability, subcellular localization or interaction with NR5A1. ( A ) Protein stability. Left: Whole cell protein was extracted from the HEK293-T cells transfected with either WT or p.Arg246His COUP-TFII-encoding plasmids. Western blot analysis showed a single band around 46 kDa for both COUP-TFII-WT and COUP-TFII-p.Arg246His. The image is cropped from the original full-length blot shown in Supplementary Figure and is representative of three independent experiments. Right: the band intensity was normalized to that of β-Actin and plotted as mean ± SEM of COUP-TFII/β-Actin ratios obtained from three independent experiments. There was no significant difference between the COUP-TFII-WT and COUP-TFII-p.Arg246His protein production/stability (Student t-test, P = 0.84). ( B ) Nuclear localization of COUP-TFII. HEK293-T cells were transfected with WT or mutant COUP-TFII expression vectors. Immunocytochemistry showed strong nuclear localization for both WT and mutant proteins (green). Images were taken at 40X magnification (Scale bar: 20 µm). ( C ) The mutation does not affect the interaction between COUP-TFII and NR5A1. Left: NR5A1 and WT or p.Arg246His COUP-TFII-encoding plasmids were transiently expressed for 48 h in HEK293-T cells. Protein–protein interaction was assessed by the Duolink proximity ligation assay (PLA). Nuclei are stained with DAPI (blue) while each signal/dot (green) represents a COUP-TFII/NR5A1 interaction event. Right: quantification of PLA. For each condition, the number of interactions of at least 50 individual cells was counted and the median (interquartile range) was analyzed. Statistical analysis revealed that the binding of NR5A1 to COUP-TFII-p.Arg246His was comparable to that of COUP-TFII-WT (Mann–Whitney test, P = 0.22). Images were taken at 63X magnification (Scale bar: 20 µm).

Article Snippet: Cells were incubated overnight with rabbit anti-COUP-TFII antibody (1:200, #ab42672, Abcam) diluted in PBS-3% BSA at 4 °C.

Techniques: Mutagenesis, Transfection, Western Blot, Expressing, Immunocytochemistry, Proximity Ligation Assay, Staining, Binding Assay, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Evidence for NR2F2 /COUP-TFII involvement in human testis development

doi: 10.1038/s41598-024-68860-3

Figure Lengend Snippet: Mutant COUP-TFII shows loss of inhibitory effect on NR5A1-dependent activation of target promoters. The transcriptional activities of COUP-TFII-WT and COUP-TFII-p.Arg246His were studied using the human ( A ) LHB and ( B ) INSL3 promoters as reporters, following transfection in HEK293-T cells. The data shown represent the dot plot of results with mean ± SEM of a minimum of three independent experiments, each with twelve replicates. The reporter constructs were transfected into HEK293-T cells with either the COUP-TFII-WT and COUP-TFII-p.Arg246His expression vector with or without NR5A1-WT expression vector. All data were standardized for Renilla activity. The results are expressed as relative percentage of NR5A1-WT activity (100%). COUP-TFII-WT exerts an inhibitory effect on NR5A1-mediated activation of both ( A ) LHB and ( B ) INSL3 . However, there is a loss of inhibition by COUP-TFII-p.Arg246His. The statistical significances are marked by the horizontal lines (dash line *, P < 0.05; bold line ***, P < 0.001).

Article Snippet: Cells were incubated overnight with rabbit anti-COUP-TFII antibody (1:200, #ab42672, Abcam) diluted in PBS-3% BSA at 4 °C.

Techniques: Mutagenesis, Activation Assay, Transfection, Construct, Expressing, Plasmid Preparation, Activity Assay, Inhibition