The region of residues -145 to -119 (CD/L) of the cathepsin D gene promoter contains a GC-rich motif that binds Sp1 protein and an adjacent pentanucleotide (CACGC) that corresponds to the core sequence of a dioxin responsive element (DRE) and binds the aryl hydrocarbon receptor (AhR)-AhR nuclear translocator (Arnt) complex. This Spl (N)4DRE(core) motif has been identified in promoters of several genes in which Sp1 plays an important role in basal gene expression. In transient transfection assays with MCF-7 human breast cancer cells using wild-type pCD/L and constructs mutated in the core DRE (pCD/L(m1)) and Sp1 (pCD/L(m2)) sites, it was shown that both motifs were required for maximal basal activity. The requirements for AhR-Arnt interactions with Sp1 protein for maximal activity of pCD/L were confirmed in wild-type MCF-7 and Hepa 1c1c7 cells and Arnt- deficient Hepa 1c1c7 cells using antisense Arnt and Arnt expression plasmids. The functional interactions of Sp1 with AhR- Arnt were paralleled by physical interactions showing that AhR- Arnt and Sp1 proteins were co-immunoprecipitated and AhR-Arnt enhanced Sp1-[32P]CD/L binding in electrophoretic mobility shift assays. The physical and functional interactions of Sp1 with AhR-Arnt proteins bound to the Sp1(N)4DRE(core) motif were also dependent on the proximity of these sites, and both the activity and the extent of Sp1-DNA binding decreased as the number of intervening nucleotides increased from 4 to 20. These studies show that regulation of basal expression of some genes by Spl may also require interactions with AhR-Arnt.
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