FIG. 4.

NRF1 activates transcription from the 21-bp X gene minimal promoter, and NRF1 activation is inhibited by dominant-negative mutant NRF1 or NRF1 siRNAs. (A) HepG2 was transfected with the X minimal promoter-CAT construct pXMP2CAT plus an empty plasmid (lane 1) or a wild-type (wt) NRF1 expression plasmid (lanes 2 and 3) with (lane 2) or without (lane 3) a dominant-negative (d.n.) NRF1 mutant expression plasmid. Two days after transfection, cells were harvested and the CAT activity of the cell lysate was assayed. Relative CAT activity was calculated on the basis of the conversion rate of ¹⁴C-chloramphenicol into the acetylated forms. (B) HepG2 was transfected with the X minimal promoter-CAT construct pXMP2CAT plus an empty plasmid (lanes 1 and 2) or a wild-type NRF1 expression plasmid (lanes 3 to 10) with (lanes 5 to 10) or without (lanes 3 and 4) three different NRF1 siRNA expression DNAs (numbered 1 to 3) that target different parts of the X mRNA. NRF1 siRNA expression DNAs (0.5 μg/6-cm-diameter dish [lanes 5, 7, and 9] or 3.5 μg/6-cm dish [lanes 6, 8, and 10]) were transfected. CAT assays were performed as described above. (C) Analysis of the transcripts expressed from pXMP2CAT. HepG2 cells were transfected with pXMP2CAT plus the NRF1 expression plasmid (lane 6) or pXMP2CAT plus an empty plasmid (lane 5). Two days after transfection, poly(A)⁺ RNAs were purified and the transcripts from pXMP2CAT were detected by primer extension with a ³²P-labeled CAT primer.