Fig. 6.

Atf4 binds to the Ihh promoter to activate transcription. (A) Northern blot analysis showing that Atf4 and Runx2 stimulate endogenous Ihh mRNA expression in TMC23 cells. Gapdh serves as a loading control. (B) Atf4 activates Ihh promoter reporters in COS1 cells. Co-transfection assay showing that Atf4 activates Ihh promoter reporters (pIhh-1.3kbp and pIhh-742bp, as shown to left) but not the construct containing a mutated A9 (arrowhead), the Atf4 binding site. (C) Sequences of nine putative Atf4 binding sites (A1-A9) in the proximate 742 bp promoter region of the mouse Ihh gene. Core sequences are indicated in bold, being identical to those within the Atf4 consensus sequence [i.e. TTACATCA, OSE1 in osteocalcin (OG2) and T(T/G)(A/G)C(A/G)T(C/G)A in other Atf4 target genes]. (D) Atf4 binds to A9 in the Ihh promoter. EMSA using the nine ³²P-labeled putative Atf4 binding sites, A1-A9, as probes with purified His-tagged Atf4 recombinant protein. OSE1 serves as a positive control. Arrow, Aft4-probe complex. (E) A9 mediates Atf4 transactivation of Ihh. p5XA9-Luc and p5XmA9-Luc contain five copies of WT and mutant A9 [which binds Atf4 only weakly (see F,G)], respectively, linked to a TATA-box vector. (F) EMSA using ³²P-labeled probes at equal counts per minute of OSE1, A9 and A9 mutant (A9mut). Note the large amount of unbound A9mut probe at the bottom of the gel. (G) Competition EMSAs. Fold molar excess of unlabeled double-stranded DNA competitor over labeled probe is indicated. (H) Endogenous Atf4 binds to A9. The Ap1 probe, the cJun/cFos binding site, was used as a control for nuclear extract quality. (I) Supershift EMSAs showing that an antibody against Atf4 inhibits the binding of endogenous Atf4 to A9 (lane +). N, unrelated antibody.