FIG. 1.

YY1 selectively activates the Grp78 promoter in Tg-stressed cells and is required for its full stress induction. (A) Summary of ER stress-induced changes in the DMS methylation pattern of the Grp78 promoter as revealed by in vivo footprinting (27). The sequence of the highly conserved human and mouse Grp78 promoter containing ERSE#3 where most of the ER stress-induced site occupancy changes occur is aligned. The solid arrow indicates constitutive protection from DMS methylation of the G residue. The open arrows and star indicate ER stress-inducible DMS methylation protection and hypersensitivity, respectively. The binding sites for the transcription factors NF-Y, TFII-I, and ATF6/YY1 are underlined. (B) CV-1 cells were transfected with the -169/Luc reporter plasmid and with either CMV-driven YY1 full-length expression plasmid or empty vector (V). Twenty-four hours after transfection, the cells were treated with 300 nM Tg for 16 h, harvested, and assayed for luciferase activity. (C) 293T cells were transfected with the EGFP vector and either U6 siControl or U6 siYY1. After 96 h, the cells were treated with Tg for 4 h and subjected to cell sorting by fluorescence. RNA was isolated from each sample, and equal amounts were used as template in RT-PCRs with oligo(dT) primers for cDNA synthesis and gene-specific primers as indicated. PCRs were carried out with 30, 26, 22, and 18 amplification cycles for determination of linear ranges. Data shown were subjected to 22 cycles of PCR. (D) Quantitation of Grp78 PCR products in panel C is shown, corrected for GAPDH levels.