FIGURE 9.

The transcriptional activity of E93 in the Atg1 promoter region is both dependent and independent of 20E-EcR-USP. A, HEK293 cells were co-transfected with the E93 (GFP as a control) expression construct, the pGL3 basic plasmids containing the indicated promoter regions of Atg1, the hsp70 basal promoter regulating expression of firefly luciferase (Fluc) and a reference reporter plasmid carrying Renilla luciferase (Rluc). After 48 h of transfection, the dual luciferase assays were performed. Luciferase activity fold-change is defined as the relative luciferase activity induced by E93/GFP. B, HEK293 cells were co-transfected with the E93, EcR, and USP (E93 and GFP as a control) expression constructs, Fluc with the indicated promoter regions of Atg1, and Rluc. Luciferase activity fold-change is defined as the relative luciferase activity induced by E93 + EcR-USP/E93 + GFP. The others are the same as in A. C, HEK293 cells were co-transfected with the wild-type E93 or E93 mutant expression constructs (E93^ΔLLQHLL and E93^ΔPLDLSAK), Fluc with the 1-kb Atg1 promoter, and Rluc. The others are the same as in A. D, the mutated 1-kb Atg1 promoter regions, but not different Atg1 promoter regions, were used here, whereas the others are the same as in A. E, a ChIP assay of FLAG-E93 binding to the 1.82-kb region of ATG1 promoter. Bm-N cells were transfected with FLAG-E93 expression plasmid for 48 h, and cells were immunoprecipitated with IgG or antibodies against FLAG. Results of qPCR analyses are presented as FLAG-E93/IgG. F, HEK293 cells were co-transfected with the wild-type E93 or E93 mutant expression constructs (E93^ΔHTH1, E93^ΔHTH2, and E93^ΔHTH1ΔHTH2), Fluc with the 1-kb Atg1 promoter, and Rluc. The others are the same as in A.