Journal: Nucleic Acids Research
Article Title: Prospero-related homeobox 1 (Prox1) functions as a novel modulator of retinoic acid-related orphan receptors ?- and ?-mediated transactivation
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
Article Snippet: Prox1 and Prox1 mutants were generated by polymerase chain reaction (PCR) amplification and cDNA fragments inserted into the EcoRI and XhoI sites of pCMV-Myc and pEGFP-C1 (Clontech, Palo Alto, CA, USA), or the EcoRI and SalI sites of pMAL-c2X (New England BioLabs, Inc., Ipswich, MA, USA).
Techniques: Expressing, Genome Wide, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Mouse Assay, Amplification, Transfection, Plasmid Preparation, Quantitative RT-PCR, Isolation