Identification of DNA binding sites for the v-erbA oncoprotein, the viral homolog to thyroid hormone receptor alpha

Abstract

The v-erbA oncogene protein, P75 gag-v-erbA, is a mutant form of the thyroid hormone receptor α (TR α) which has sustained mutations both in the ligand binding and DNA binding domains. The oncoprotein has therefore lost its ability to bind ligand, and its heterodimerization with the retinoid-X receptor (RXR) is impaired. Here, we have investigated the effects of the mutations in the DNA binding domain. By applying a PCR-based screening assay we isolated DNA sequences to which P75 gag-v-erbA binds as a heterodimer with RXR, and characterized these with regard to their nucleotide sequence and ability to associate with RXR/P75 gag-v-erbA heterodimers in vitro and in vivo. In the PCR selection assay the heterodimer exhibited a preference for direct repeats with a 3′ half-site sequence AGGTCG and spacers of four or five nucleotides separating the two half-sites. These DNA binding data were confirmed by gel retardation assays with synthetic oligonucleotides as well as by transfection experiments using dominantly active VP16 fusion proteins with P75 gag-v-erbA and TR α. The comparison between RXR/P75 gag-v-erbA and RXR/TR α heterodimers demonstrated that although their DNA binding properties are very similar, however, a relaxed specificity of P75 gag-v-erbA for the spacer length may allow it to interfere with more hormone signalling pathways than only that of thyroid hormone.