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. 2016 Mar 31;6:23944. doi: 10.1038/srep23944

Figure 3. The E-box-containing cis-element in intron 12 mediates HNF-4α-induced ChREBP-α transcription.

Figure 3

(a). Luciferase activity assay shows that HA-HNF-4α fails to increase transcriptional activity of the 4 kb ChREBP-α promoter compared with the HA-GFP-transfected sample. 24 hours after the 4 kb ChREBP-α promoter in pGL3-Basic plasmid and the HA-GFP, HA-LXR or HA-HNF-4α expression plasmid are transfected in 293T cells, luciferase activity is analyzed. *indicates p < 0.05 when compared with the HA-GFP-transfected sample. (b). Luciferase activity assay shows that HNF-4α enhances transcriptional activity of the pGL3-Promoter plasmid containing the 140 bp sequence in intron 12 of ChREBP-α (IV) compared with the control. I, II, III and IV are the pGL3-Promoter plasmids containing the 164 bp, 174 bp, 378 bp and 140 bp sequences located in intron 2, intron 6, intron 7 and intron 12 of ChREBP-α. 24 hours after I, II, III or IV and the HA-GFP or HA-HNF-4α expression plasmid are transfected in 293T cells, luciferase activity is analyzed. *indicates p < 0.05 when compared with the HA-GFP-transfected sample. (c). Luciferase activity assay shows that deletion of the E-box in IV reduced induction of the transcriptional activity of IV by HNF-4α compared with the control. 24 hours after the pGL3-Promoter plasmid, IV or IVΔE-box and the HA-GFP, HA-LXR or HA-HNF-4α expression plasmid are transfected in 293T cells, luciferase activity is analyzed. *indicates p < 0.05 when compared with the HA-GFP-transfected sample. Western blot analysis using the anti-HA antibody shows protein levels of ectopically expressed HA-HNF-4α and HA-LXR. (d). ChIP analysis for HepG2 cells using an anti-HNF-4α antibody, nonspecific IgG or anti-histone antibody shows that HNF-4α binds the 140 bp region in intron 12 of the ChREBP-α gene.