Regulation of constitutive gene expression through interactions of Sp1 protein with the nuclear aryl hydrocarbon receptor complex

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.