FIG. 6.

PRMT1 is recruited to the Grp78 promoter in Tg-stressed cells and enhances activation mediated by YY1 and ATF6(373). (A) Cos-7 cells were stably transfected with F-YY1 and CMV-Bsd. Cells were either cultured under normal conditions or treated with Tg for 3 h and harvested. Whole-cell extracts were immunoprecipitated with either normal mouse IgG or anti-Flag M2 agarose. Immunoprecipitates were then run on an 8% polyacrylamide gel and probed with antibodies against either Flag or PRMT1. A31 cell extract was used as a positive control for the presence of PRMT1. *, nonspecific band. (B) HeLa cells were treated with Tg for 3 h, chromatin extracts were prepared as shown in Fig. 4, and immunoprecipitation was carried out with antibodies against YY1, PRMT1, and the methylated arginine 3 residue of histone H4, as well as normal IgG. Products of the PCR using primers for the 213-bp region of the Grp78 promoter are shown. In each transfection experiment, CV-1 cells were transfected with -169/Luc reporter vector and CMV-Renilla luciferase control vector; pBluescript was used as an empty vector. Cells were harvested 24 h after transfection and assayed for dual-luciferase activity. (C) Increasing amounts of PRMT1 expression plasmid (in nanograms) were cotransfected as indicated. (D) Increasing amounts of YY1 expression plasmid (in nanograms) were cotransfected to establish the synergistic induction of the Grp78 promoter by YY1 in the presence of ATF6(373). (E) Increasing amounts of PRMT1 (in nanograms) were cotransfected with YY1 and ATF6(373) expression plasmids. The luciferase activity of the -169/Luc reporter gene alone is set at 1. *, P value of <0.05.