FIG. 3.

ATF6α is required for full induction of Grp78 and its nuclear form co-localizes with YY1. (A) Primary MEFs (wild type) or with a βgeo gene trap mutation in the ATF6α gene were cultured and treated with Tg for 16 h. Western blotting of protein extracts from control cells and cells with Tg was performed with antibody against ATF6 (C1.12) (24). The bands corresponding to the wild-type ATF6 (p90) and its nuclear form ATF6(N), as well as the ATF6/βgeo fusion protein (p250), are shown. (B) RNA was isolated from the wild-type and mutant mouse ES cells treated with Tg for 16 h. Northern blotting was performed to probe for Grp78 transcript level with GAPDH as a control. The results were quantitated on a phosphorimager and are shown in graph format. (C) Cos-7 cells were grown to 50% confluence and transfected with either full-length HA-ATF6 or HA-ATF6(373) plasmids. After a 24-h incubation, the cells were fixed and stained with anti-HA monoclonal antibody (red) and anti-YY1 (green) polyclonal antibody. The cells were visualized on a Zeiss LSM510 confocal microscope (magnification, ×380). The merged image shows colocalization between HA-ATF6(373) and YY1 in the transfected cells (yellow). (D) 293T cells were transfected with either HA-ATF6(273) or HA-ATF6(373), and equal amounts of whole-cell extracts were subjected to sequential Western blotting to determine HA-ATF6 expression levels, with the GAPDH level serving as a loading control. (E) The transfected cells from the results shown in panel D were concurrently cross-linked with formaldehyde, and chromatin preparations were subjected to the ChIP assay with antibody against YY1 performed in duplicate and normal IgG as control. PCR primers for the 21-bp region of the human Grp78 promoter encompassing the three ERSEs were used, and the products are shown. Cells transfected with HA-ATF6(273) (top) and cells transfected with HA-ATF6(373) (bottom) are shown.