Skip to main content
. 2008 Sep 25;105(39):15046–15051. doi: 10.1073/pnas.0801773105

Fig. 3.

Fig. 3.

AP4 directly represses p21 expression via E-box motifs. (A) Schematic presentation of putative AP4-binding sites (A1-A4) and their position in the p21 promoter region relative to the transcriptional start site (+1). Wild-type and mutant p21 promoter constructs used in transient reporter assays are depicted. Mutated AP4-binding sites are represented in bold and underlined. The initiator (Inr) element (TCAGTTCCT) localizes to position + 8 to + 16 relative to the transcription start site (“+1”) (13). luc: ORF encoding the firefly luciferase. (B) Determination of p21 reporter activity in H1299 cells. Cells were transfected in 12-well plates with wild type or the indicated mutant p21 reporter plasmids, pcDNA3-AP4-VSV plasmid or equimolar amounts of pcDNA3-VSV backbone. Shown are the median expression values and standard errors of two independent transfection experiments. p21 prom. wt, mA3 + 4, mA2–4, and mA1–4: reporter plasmids encoding for the p21 promoter sequence with wild-type or mutant AP4-binding sites (see Fig. 3A). (C) The CMV/p21 reporter (nucleotides −49 to +16 of p21), a mutant version (CMV/p21mut) harboring substitutions of two nucleotides within three potential Miz1-binding sequences, or the p21/CMV reporter (nucleotides −94/-50 of p21) containing one E2F and four Sp1/3-binding sites (13) were cotransfected with pcDNA3-AP4-VSV plasmid or equimolar amounts of pcDNA3-VSV backbone in H1299 cells. Shown are the median expression values and standard errors of two independent transfection experiments. (D) Schematic representation of AP4 mutants. B: Basic region, HLH: helix–loop–helix, LZ1/2: leuzine zipper motif 1 and 2, TIV: conserved motif of unknown function containing the amino acid sequence TIV. The amino acid sequence of the basic region (underlined) and flanking residues are indicated for the wild type and mutant AP4 versions. Altered residues are represented in bold. (E) Effect of AP4 variants on p21 reporter activity in H1299 cells. Cells were transfected in 12-well plates with wild-type p21 reporter plasmid, pcDNA3-AP4-VSV plasmids (wild type or mutant AP4 versions) or equimolar amounts of pcDNA3-VSV backbone. Shown are the median expression values and standard deviations of three independent experiments. (F) Effect of AP4 variants on endogenous p21 expression. Expression level of AP4-VSV, p21, and β-actin proteins was detected by immunoblot analysis 24 h after induction of conditional wild-type or mutant AP4 alleles in U-2OS cells.