Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Phosphorylation regulates FOXC2-mediated transcription in primary LECs. (A) Immunofluorescent staining for Myc (green), lymphatic marker PROX1 (red), and DNA (blue) of LECs transduced with adenoviruses expressing wild-type Myc-FOXC2, phosphorylation-deficient mutant Myc-pmFOXC2, or control bacterial β-galactosidase (LacZ). Note that wild-type and mutant FOXC2 have similar expression levels and subcellular localization. Bars, 20 μm. (B) Phosphorylation regulates FOXC2 transcriptional activity. Gene expression profiling was performed on the adenovirus-transduced LECs shown in panel A. Genes whose expression changed >2-fold in response to the loss of FOXC2 phosphorylation (FDR < 0.01) are shown in orange (upregulated) and purple (downregulated) in the Volcano plot of significance against the fold change in gene expression. Vertical dotted lines mark the 2-fold change limits. (C) RT-PCR validation of the gene expression profiling results. Genes upregulated or downregulated >2-fold in response to the loss of FOXC2 phosphorylation are shown in orange and purple, respectively; genes affected <2-fold are shown in gray. No change in FOXC2 expression reflects equally efficient cell transduction with Ad-Myc-FOXC2 and Ad-Myc-pmFOXC2. The data are presented as log2-transformed fold change in gene expression normalized to a housekeeping gene (GAPDH). Horizontal dotted lines mark the 2-fold change limits. Shown are the means and standard deviations of triplicate determinations in a single experiment representative of two independent experiments. (D) Heat map representation of the differences in gene expression in response to the loss of FOXC2 phosphorylation. The left heat map shows expression levels of 57 of 59 genes downregulated >2-fold (FDR < 0.01) in Ad-Myc-pmFOXC2-transduced LECs compared to Ad-Myc-FOXC2-transduced LECs. The right heat map shows expression levels of 57 out of 88 genes upregulated >2-fold (FDR < 0.01) in the same cells. Three biological replicates are shown for each condition. The color key at the lower left corresponds to the mean-centered, arctan-transformed log2-fold change in gene expression falling within the range from −π/2 to π/2. Blue denotes genes with relative decreased expression; red denotes genes with relative increased expression.

Article Snippet: For immunofluorescence staining, we used mouse anti-Myc (clone 9E10; Santa Cruz Biotechnology), rabbit anti-Myc-Tag (clone 71D10; Cell Signaling Technology, used for transgene detection in vivo ), rat anti-mouse PECAM-1 (BD Pharmingen), rat anti-mouse FOXC2 ( 19 ), sheep anti-human FOXC2 (R&D Systems), and goat anti-human PROX1 (R&D Systems).

Techniques: Staining, Marker, Transduction, Expressing, Mutagenesis, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Transformation Assay

Journal: Nucleic Acids Research

Article Title: Prospero-related homeobox 1 (Prox1) functions as a novel modulator of retinoic acid-related orphan receptors α- and γ-mediated transactivation

doi: 10.1093/nar/gkt447

Figure Lengend Snippet: Prox1 interacts with the AF2 domain of RORα and RORγ. HEK293 cells were transfected with pCMV-Myc-Prox1 and pCMV10-3xFlag-RORγ( A ) or -RORα ( B ) or the respective AF2 deletion mutant. Cell lysates were prepared and used for co-immunoprecipitation analysis with an anti-Myc antibody. Immunoprecipitated proteins were separated by PAGE and examined by western blot analysis with an anti-Flag antibody. ( C and D ) Prox1 enhances RORγ protein stability. HEK293 cells were transfected with pCMV-Myc-Prox1 and pCMV10-3xFlag-RORγ and 36 h later treated with 10 µg/ml cycloheximide for the times indicated. RORγ and Prox1 protein levels were normalized against the Gapdh loading and their level at 0 h (100%). Flag-RORγ ( C ) and Myc-Prox1 ( D ) were examined by western blot analysis, and the intensity of the bands was determined and plotted. ( E ) Prox1 interaction with RORγ was increased by RORγ antagonist treatment. HEK293 cells were co-transfected with pCMV-Myc-Prox1 and pCMV10-3xFlag-RORγ and then incubated with each ROR antagonists, T0901317 or ursolic acid, or dimethyl sulfoxide (DMSO) at 1 or 10 μM for the last 24 h. The amount of RORγ in complex with Prox1 was examined by co-immunoprecipitation/western blot analysis as described earlier in the text. The level of total Flag-RORγ and Myc-Prox1 (input) was also analyzed.

Article Snippet: After a wash in phosphate-buffered saline, an aliquot of the cross-linked chromatin was sonicated and incubated overnight with an antibody against Prox1 (51043-1-AP; Proteintech Group Inc., Chicago, IL, USA), RORα or RORγ as described previously ( ).

Techniques: Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Incubation

Journal: Nucleic Acids Research

Article Title: Prospero-related homeobox 1 (Prox1) functions as a novel modulator of retinoic acid-related orphan receptors α- and γ-mediated transactivation

doi: 10.1093/nar/gkt447

Figure Lengend Snippet: Both the N- and C-terminus of Prox1 are able to interact with RORγ. ( A ) MBP pull-down assays were performed using radiolabeled 35 S-RORγ (full-length), 35 S-RORγΔAF2 lacking the AF2 domain, 35 S-RORγΔLBD lacking the LBD and a series of MBP–Prox1 fragments, N(1-106), N(1-106)m, M(107-340), M(341-573), C(574-737) and C(574-737)m, as shown in the schematic. After incubation with amylose resin, MBP–Prox1 complexes were analyzed by PAGE, and radiolabeled RORγ was detected by autoradiography. Five percent of the input of each radiolabeled RORγ was loaded in the first lane. MBP was used as negative control. ( B ) MBP pull-down assays were performed using radiolabeled full-length RORγ ( 35 S-RORγ) and several N- and C-terminal fragments of Prox1, N(1-106), N(1-66), N(1-28), C(574-737), C(574-635) and C(636-737) as shown in the schematic. Samples were processed as described under A. ( C ) Loss of the N- and C-terminus of Prox1 diminishes its stabilizing effect on RORγ protein. HEK293 cells were transfected with pCMV10-3xFlag-RORγ and the pCMV-Myc-Prox1 or the pCMV-Myc-Prox1 mutant indicated and the level of RORγ and Myc-Prox1 protein examined by western blot analysis. Co-transfection with a β-Gal reporter indicated no significant difference in transfection efficiency between cells transfected with different Prox1 mutants.

Article Snippet: After a wash in phosphate-buffered saline, an aliquot of the cross-linked chromatin was sonicated and incubated overnight with an antibody against Prox1 (51043-1-AP; Proteintech Group Inc., Chicago, IL, USA), RORα or RORγ as described previously ( ).

Techniques: Incubation, Autoradiography, Negative Control, Transfection, Mutagenesis, Western Blot, Cotransfection

Journal: Nucleic Acids Research

Article Title: Prospero-related homeobox 1 (Prox1) functions as a novel modulator of retinoic acid-related orphan receptors α- and γ-mediated transactivation

doi: 10.1093/nar/gkt447

Figure Lengend Snippet: RORγ promotes translocation of Prox1 into the nucleus through the N- and C-terminus of Prox1. ( A ) Co-localization of Prox1 and RORγ or RORα. COS-1 cells were transfected with pEGFP-Prox1 and pCMV10-3xFlag-RORγ or -RORα as indicated. After immunohistochemical staining with anti-Flag M2 antibody and 4,6-diamidino-2-phenylindole (DAPI), immunofluorescence was examined by confocal microscopy. ( B ) Schematic presentation of a series of N- and C-terminal Prox1 deletion mutants. ( C–I ) COS-1 cells were transfected with pLVX-RORγ-mCherry-N1 and pCMV-Myc expression plasmids containing Prox1 or the N- or C-terminal mutants, PΔN106, PΔC636, PΔN106ΔC636, PΔN106ΔC716, PΔN106ΔC729 or PΔN106ΔC722. Subsequently, their subcellular localization was examined as described under A. The percentage of cells in which Prox1 was predominantly localized in the nucleus (N) or in the cytoplasm (C) or distributed equally between nucleus and cytoplasm (N + C) was calculated. In cells co-transfected with both RORγ and Prox1, only cells ( n > 100) in which both RORγ and Prox1 were co-expressed were counted.

Article Snippet: After a wash in phosphate-buffered saline, an aliquot of the cross-linked chromatin was sonicated and incubated overnight with an antibody against Prox1 (51043-1-AP; Proteintech Group Inc., Chicago, IL, USA), RORα or RORγ as described previously ( ).

Techniques: Translocation Assay, Transfection, Immunohistochemical staining, Staining, Immunofluorescence, Confocal Microscopy, Expressing

Journal: Nucleic Acids Research

Article Title: Prospero-related homeobox 1 (Prox1) functions as a novel modulator of retinoic acid-related orphan receptors α- and γ-mediated transactivation

doi: 10.1093/nar/gkt447

Figure Lengend Snippet: Prox1 represses RORγ- and RORα-mediated transcriptional activation. ( A ) Huh-7 cells were co-transfected with the pGL4.27-(RORE) 5 or pGL4.10- Npas2 (-1534/+81) reporter plasmid, pCMV-β-Gal, pCMV10-3xFlag-RORγ or -RORα, and increasing amounts of pCMV-Myc-Prox1 expression plasmid (ROR:Prox1 = 1:0.2, 1:0.5, 1:1). Luciferase and β-galactosidase activities were measured 24 h later. ( B ) Huh-7 cells were co-transfected with pGL4.10 reporter plasmid containing Npas2 promoter region (−1534/+81) and expression vectors as described earlier in the text. ( C ) Mammalian monohybrid analysis. CHO cells were transfected with pGL4.27-(UAS) 5 reporter plasmid containing UAS, pCMV-β-Gal, pM-RORγLBD and increasing amounts of pCMV-Myc-Prox1 expression plasmid (ROR:Prox1 ratios are 1:0.1, 1:0.2, 1:0.5 and 1:1). ( D ) Mammalian two-hybrid analysis. CHO cells were transfected with pGL4.27-(UAS) 5 , pCMV-β-Gal, pM-GAL4-LXXLL(EBIP96), VP16-RORγ(LBD) or -RORα(LBD) and increasing amounts of pCMV-Myc-Prox1 (ROR:Prox1 = 1:0.2, 1:0.5, 1:1). Luciferase and β-galactosidase activities were measured 24 h later. ( E ) Schematic presentation of several N- or C-terminal Prox1 deletion constructs and mutants containing mutations in the LXXLL motifs or the homeodomain. ( F ) Huh-7 cells were co-transfected with pGL4.10- Npas2 (-1534/+81), pCMV-β-Gal, pCMV10-3xFlag-RORγ and pCMV-Myc expression vector containing Prox1 or the Prox1 mutants P(LXXLL)m, PΔN106, P(HD)m, PΔN106(HD)m, PΔC573, PΔC636 and PΔN106ΔC636) as indicated. ( G ) Mammalian two-hybrid analysis. CHO cells were transfected with pGL4.27-(UAS) 5 , pCMV-β-Gal, pM-GAL4-LXXLL(EBIP96), VP16-RORγ(LBD) and pCMV-Myc expression vector containing Prox1, PΔN106 or PΔC636. ( H ) The region of Prox1 between amino acids 723 and 729 is required for its repression of RORγ-mediated transactivation of Npas2 (−1534/+81). Huh-7 cells were transfected with pGL4.10- Npas2 (-1534/+81), pCMV-β-Gal, pCMV10-3xFlag-RORγ and pCMV-Myc expression vector containing Prox1, PΔN106, PΔN106ΔC729, PΔN106ΔC722, PΔN106ΔC716, PΔN106ΔC686 or PΔN106ΔC636, as indicated. Luciferase and β-galactosidase activities were measured 24 h later. All the experiments were performed in triplicate. Data represent mean ± SEM; * P < 0.05 by ANOVA.

Article Snippet: After a wash in phosphate-buffered saline, an aliquot of the cross-linked chromatin was sonicated and incubated overnight with an antibody against Prox1 (51043-1-AP; Proteintech Group Inc., Chicago, IL, USA), RORα or RORγ as described previously ( ).

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

Journal: Nucleic Acids Research

Article Title: Prospero-related homeobox 1 (Prox1) functions as a novel modulator of retinoic acid-related orphan receptors α- and γ-mediated transactivation

doi: 10.1093/nar/gkt447

Figure Lengend Snippet: Prox1 is recruited to the RORE sites of ROR-target clock genes in vivo . ( A ) ChIP–QPCR was performed using an anti-Prox1 antibody and chromatin prepared from liver collected at ZT22 from WT and RORγ − / − mice ( n = 4). Fold enrichment as percentage of input DNA was calculated. The Pepck promoter, which is not a RORγ target, was used as a positive control for Prox1 recruitment. Amplification of Gapdh served as a negative control. ( B ) ChIP–QPCR was performed using anti-Prox1 antibody and chromatin prepared from the livers ( n = 4) collected from WT mice at ZT8 and ZT20. The recruitment of Prox1 to the ROREs of Bmal1 , Npas2 and Cry1 was analyzed. ( C ) Re-ChIP analysis was performed with chromatin prepared from WT and ROR -deficient livers ( n = 4) collected at ZT20. The chromatin was immunoprecipitated with anti-Prox1 antibody first, then the extract was further immunoprecipitated with either anti-RORα or anti-RORγ antibody. Data represent mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 by ANOVA.

Article Snippet: After a wash in phosphate-buffered saline, an aliquot of the cross-linked chromatin was sonicated and incubated overnight with an antibody against Prox1 (51043-1-AP; Proteintech Group Inc., Chicago, IL, USA), RORα or RORγ as described previously ( ).

Techniques: In Vivo, ChIP-qPCR, Positive Control, Amplification, Negative Control, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Prospero-related homeobox 1 (Prox1) functions as a novel modulator of retinoic acid-related orphan receptors α- and γ-mediated transactivation

doi: 10.1093/nar/gkt447

Figure Lengend Snippet: Prox1 represses transcription of ROR target genes. ( A ) Confirmation of the downregulation of Prox1 mRNA and protein levels in Huh-7 cells transfected with either control siRNA or Prox1 siRNA for 3 days. Gapdh mRNA and protein was used as an internal control. ( B ) Effect of the downregulation of Prox1 expression on the expression of RORγ , Bmal1 , Npas2 , Cry1 , Avpr1a , Pepck and Elovl3 . Gene expression levels in Huh-7 cells treated with either siRNA-control or siRNA-Prox1 ( n = 3) were analyzed by QRT–PCR. Data represent mean ± SD. ( C ) ChIP–QPCR was performed using anti-Prox1 antibody and chromatin prepared from Huh-7 treated with either siRNA-control or siRNA-Prox1 ( n = 3). The recruitment of Prox1 to the conserved ROREs of human Bmal1 , Npas2 and Cry1 was analyzed. Using non-specific IgG antibody and amplification of Gapdh served as a negative control. Data represent mean ± SEM, ** P < 0.01, *** P < 0.001 by ANOVA. ( D ) Effect of the downregulation of Prox1 expression on the activation of the RORE-containing regulatory regions, Npas2 (-1534/+81), Bmal1 (−650/+105) and Cry1 (+22976/+23214). Huh-7 cells treated with either siRNA-control or siRNA-Prox1 were transfected with a pGL4 reporter vector under control of the indicated RORE-containing region. Relative reporter activity was analyzed 24 h later. ( E ) Increased association of H3K9Ace on the ROREs of Bmal1 , Npas2 and Cry1 genes in Huh-7 cells in which Prox1 is downregulated. ChIP–QPCR analysis was performed with chromatin from Huh-7 cells treated with either siRNA-control or siRNA-Prox1 and an anti-H3K9Ace antibody. An IgG antibody and the amplification of Gapdh gene were used as negative controls. ChIP–QPCR data are represented as fold relative enrichment as percentage of input DNA. ( F ) Chromatin accessibility on the ROREs of Bmal1 , Npas2 and Cry1 genes was assessed by FAIRE–QPCR analysis using chromatin samples prepared from Huh-7 cells treated with either siRNA-control or siRNA-Prox1. FAIRE signal is represented as fold relative enrichment as percentage of input DNA. ( G ) H3K9 acetylation and chromatin accessibility was analyzed on the proximal promoter of Elovl3 gene as described earlier in the text. Data represent mean ± SEM; * P < 0.05 by ANOVA.

Article Snippet: After a wash in phosphate-buffered saline, an aliquot of the cross-linked chromatin was sonicated and incubated overnight with an antibody against Prox1 (51043-1-AP; Proteintech Group Inc., Chicago, IL, USA), RORα or RORγ as described previously ( ).

Techniques: Transfection, Control, Expressing, Gene Expression, Quantitative RT-PCR, ChIP-qPCR, Amplification, Negative Control, Activation Assay, Plasmid Preparation, Activity Assay

Journal: Nucleic Acids Research

Article Title: Prospero-related homeobox 1 (Prox1) functions as a novel modulator of retinoic acid-related orphan receptors α- and γ-mediated transactivation

doi: 10.1093/nar/gkt447

Figure Lengend Snippet: RORγ regulates the rhythmic expression of Prox1. ( A ) Genome-wide mapping of ROR-binding sites by ChIP-Seq analysis showed a strong association of both RORγ and RORα with several sites within the Prox1 gene in mouse liver. Arrows indicate the peaks corresponding to ROR recruitment. Gene tracks were taken from the UCSC Genome Browser using the mouse mm9 reference genome. A, B and C indicate peaks common to RORα and RORγ ChIP-Seq analysis. ( B ) ChIP–QPCR was performed using either anti-RORγ or -RORα antibody and chromatin prepared from the livers ( n = 4) collected from WT mice at ZT22. The putative ROR-binding sites A, B and C were amplified by ChIP–QPCR analysis. Amplification of Gapdh and a non-specific IgG antibody served as negative controls. Data represent mean ± SEM. ( C ) ROR enhanced the transactivation of the Luc reporter driven by the A and B sites. Huh-7 cells were co-transfected with pCMV-β-Gal, pCMV10-3× Flag-RORγ or -RORα and pGL4.27 reporter plasmid under the control of either the ROR-binding site A, B or C. Data represent mean ± SEM. ( D ) RORγ regulates the rhythmic expression of Prox1 . Circadian expression of Prox1 was analyzed by QRT–PCR in liver tissue isolated from WT, RORγ − / − or RORα sg/sg mice ( n = 4) every 4 h over a period of 24 h. The 24 h expression pattern was double-plotted. ( E ) Exogenous expression of RORγ in mouse primary hepatocyte ( n = 3) increased Prox1 transcription. Data represent mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001 by ANOVA.

Article Snippet: After a wash in phosphate-buffered saline, an aliquot of the cross-linked chromatin was sonicated and incubated overnight with an antibody against Prox1 (51043-1-AP; Proteintech Group Inc., Chicago, IL, USA), RORα or RORγ as described previously ( ).

Techniques: Expressing, Genome Wide, Binding Assay, ChIP-sequencing, ChIP-qPCR, Amplification, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, Isolation

Journal: Nucleic Acids Research

Article Title: Prospero-related homeobox 1 (Prox1) functions as a novel modulator of retinoic acid-related orphan receptors α- and γ-mediated transactivation

doi: 10.1093/nar/gkt447

Figure Lengend Snippet: Schematic model of the interrelationship between RORs, Prox1 and the circadian and metabolic networks. Prox1 interacts directly with RORα and RORγ. The AF2 domain of RORs and the N-terminal 28 amino acids and the prospero-like domain of Prox1, particularly the 723 EIFKSPN 729 region (black box), are mediating this interaction. Prox1 is able to repress the activation and expression of RORγ target genes. The homeo/prospero-like domain of Prox1 is essential for this repression. Prox1 is recruited by RORs to ROREs in the RORE-containing regulatory regions of ROR target genes, including several circadian clock and metabolic genes, suggesting that it functions as a negative regulator of RORγ-mediated transcription. Prox1 also functions as a repressor of RORγ transcription as indicated by data showing that Prox1 knockdown significantly enhanced RORγ mRNA expression and by the recruitment of Prox1 to the RORγ promoter . Inversely, RORγ functions as a positive regulator of Prox1 transcription . This is supported by the repression of the Prox1 expression in RORγ − / − mice, the recruitment of RORs to the Prox1 gene and the increased expression of Prox1 by exogenously expressed RORγ. Our observations suggest that Prox1 and RORγ are part of a feedback loop in which RORγ positively regulates Prox1 and Prox1 negatively regulates RORγ. Modulation of the rhythmic expression of Prox1 by RORγ and their regulation of several clock and metabolic genes supports a role for both RORγ and Prox1 in the control of circadian rhythm and metabolism. Prox1 is increasing when RORγ-target gene expression (e.g. Bmal1 , Npas2 , Avpr1a and Elovl3 ) peak at ZT0. Prox1 reaches optimum expression at ZT4 when these genes are downregulated. We propose that by inhibiting RORγ transcriptional activity and expression, increased expression of Prox1 might contribute to the downregulation of RORγ target genes, including several clock and metabolic genes. N, HD and PD refer to the N-terminus, homeodomain and prospero-like domain of Prox1, respectively.

Article Snippet: After a wash in phosphate-buffered saline, an aliquot of the cross-linked chromatin was sonicated and incubated overnight with an antibody against Prox1 (51043-1-AP; Proteintech Group Inc., Chicago, IL, USA), RORα or RORγ as described previously ( ).

Techniques: Activation Assay, Expressing, Knockdown, Control, Targeted Gene Expression, Activity Assay