Figure 8.
HNF4α determines the chromatin structure of the SULT1E1 promoter. (A) A schematic presentation of ‘plasmid’ 3 C assay of the SULT1E1 gene. Arrows indicate positions of PCR primers. (B) Huh7 cells were reverse transfected with pGL3-BasicVec3C or pGL3/5.3 kb-hSULT1E1Vec3C for 24 h and then treated with DMSO in FBS-free MEM for another 2 h. After cross-linking by CH2O treatment, nuclei were prepared and subjected to ‘plasmid’ 3 C assays as described in ‘Experimental Procedures' section. From purified DNAs, formation of 3 C ligated product was detected by PCR amplification using VecTP1 and VecTP2 primers. Control product was also amplified using VecCP1 and VecCP2 primers to verify the transfection efficiency and quality of the DNAs. (C) Huh7 cells were reverse transfected with pGL3/5.3 kb-hSULT1E1Vec3C, pGL3/5.3 kb-hSULT1E1DRbVec3C or pGL3/5.3 kb-hSULT1E1FHa/bVec3C. The relative looping formation in the purified DNAs was determined by real-time PCR. Values were normalized by amplification of control product and expressed by taking those in cells transfected with pGL3/5.3 kb-hSULT1E1Vec3C as one. Bars represent the mean ± SD. *P < 0.05 (5.3 kb + ligation versus DRb + ligation).