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 thyro id hormone receptor alpha (TR alpha) which has sustained mutations both in the ligand binding and DNA binding domains. The oncoprotein has therefore l ost its ability to bind ligand, and its heterodimerization with the retinoi d-X receptor (RXR) is impaired. Here, we have investigated the effects of t he mutations in the DNA binding domain. By applying a PCR-based screening a ssay we isolated DNA sequences to which P75(gag-v-erbA) binds as a heterodi mer with RXR, and characterized these with regard to their nucleotide seque nce 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 prefer ence 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 d ata were confirmed by gel retardation assays with synthetic oligonucleotide s as well as by transfection experiments using dominantly active VP16 fusio n proteins with p75(gag-v-erbA) and TR alpha. The comparison between RXR/P7 5(gag-v-erbA) and RXR/TR alpha heterodimers demonstrated that although thei r DNA binding properties are very similar, however, a relaxed specificity o f P75(gag-v-erbA) for the spacer length may allow it to interfere with more hormone signalling pathways than only that of thyroid hormone. (C) 1998 El sevier Science Ltd. All rights reserved.