FIGURE 5.

HNF1α binds to the promoter of Xbp1 and is required for basal Xbp1 transcription. A, alignment of sequences of HNF1α binding sites with the consensus sequence. The numbers in parentheses indicate the position of the site relative to the transcription start site of Xbp1. B, PCR products from ChIP with anti-HNF1α antibody and Xbp1-specific or insulin 1-specific primers (representative image of three experiments). C, quantitative PCR measurement of Xbp1 and insulin promoter binding relative to negative control promoter and antibody. Values are the mean ± S.D. (error bars) of two independent experiments (separate from those of Fig. 5B), each analyzed in duplicate. D, EMSA to demonstrate binding of an oligonucleotide containing the HNF1α binding site in the rat Xbp1 promoter to HNF1α in vitro (representative image of three experiments). The band corresponding to unique binding of HNF1α to the probe is marked with an arrow. E, luciferase assay of DN Hnf1α cells co-transfected with the Xbp1 promoter-luciferase and the β-galactosidase reporter plasmids (n = 4). F, luciferase assay of WT Hnf1α cells co-transfected with the Xbp1 promoter-luciferase and the β-galactosidase reporter plasmids (n = 4). G and H, BiP and Xbp1 mRNA after 24 h with or without Dox, followed by 1 h with ActD or vehicle (DMSO) (n = 3).