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

The v-erbA oncogene protein, p75(gag-v-erbA), is a mutant form of the thyroid hormone receptor alpha (TR alpha) 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 alpha. The comparison between RXR/P75(gag-v-erbA) and RXR/TR alpha 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.