Fig. 2.
Glucocorticoid activates MAO B 2-kb (−2099/−99) promoter via GRE4. A, a schematic diagram of MAO B 2-kb promoter showing four consensus GREs and two clusters of overlapping Sp1 sites. The GRE4 is 577 bp upstream of the distal cluster of Sp1 sites. The sequence and location of each GRE is shown. The nucleotide identity shared with canonical GRE of each consensus GRE is underlined. B, wild-type (2 kb) or deleted MAO B promoter-luc was cotransfected with GR expression construct into 1242-MG cells. Cells were then treated with 100 nM dexamethasone for 24 h followed by luciferase activity determination. Ethanol was used as a vehicle. Activity of MAO B promoter-luc cotransfected with GR expression construct under no treatment was set as 1. C, the radioactive probe harboring GRE4 derived from MAO B promoter was incubated with nuclear extracts of 1242-MG cells treated either with or without dexamethasone (100 nM, 48 h). Ethanol was used as a vehicle. Anti-GR antibody was added into DNA-protein binding reaction when required. Arrows indicate GR-DNA complex and shifted GR-DNA complex conjugated with anti-GR antibody. A representative gel is shown. D, a schematic diagram of MAO B 2-kb promoter showing the location of GRE4 (−832/−817) and 5′-irrelevant locus (−1940/−1780) used in ChIP assay. 1242-MG cells treated with or without dexamethasone (100 nM, 24 h, ethanol used as a vehicle) were subjected to ChIP assay using anti-GR antibody followed by real-time PCR with primers specific for the region encompassing GRE4. PCR with primers targeting 5′-irrelevant locus was used as a negative control. GR occupancy at specific locus was quantitated using the 2−ΔΔCT method and presented as the percentage of input. All data were presented as the mean ± S.D. from at least three independent experiments.*, p < 0.05; **, p < 0.01.