Roles of v-erbA homodimers and heterodimers in mediating dominant negative activity by v-erbA.
v-erbA, a viral oncogenic homolog of thyroid hormone receptor (TR), blocks the effect of T3 in TR-mediated transcription. The mechanism(s) for this dominant negative effect by v-erbA on TRs is unknown but may involve competition between v-erbA and TR-containing complexes for binding to thyroid hormone response elements (TREs) and/or protein-protein interactions between v-erbA and TR. To investigate these potential mechanisms, we used the electrophoretic mobility shift assay to compare in vitro translated v-erbA and TR alpha binding to two TREs-chick lysozyme TRE (F2) and direct repeat TRE (DR4). v-erbA bound as a homodimer to these TREs, whereas TR alpha bound as a homodimer and monomer. T3 decreased TR alpha homodimer binding to the TREs as we reported previously; however, surprisingly, high concentrations of T3 (10(-6) M) also decreased v-erbA homodimer binding to the TREs. Additionally, v-erbA formed heterodimers with nuclear proteins such as retinoid X receptor and T3 receptor auxiliary protein as well as with TR alpha. These dimers remained bound to DNA in the presence of T3. Finally, v-erbA could not mediate ligand-dependent transcriptional activation even at 10(-6) M T3 but could block ligand-dependent TR-mediated transactivation in co-transfection experiments. v-erbA also exhibited differential dominant negative activity on F2 and DR4 suggesting that half-site sequence and/or orientation may influence v-erbA-dominant negative activity. In sum, there are multiple v-erbA complexes that bind to TREs in the presence of T3, which all may contribute to v-erbA's dominant negative effect on TR-mediated transcription by competing with TR-containing complexes for binding to TREs.
Yen
P M
PM
Department of Medicine, Brigham and Women's Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115.
Ikeda
M
M
Brubaker
J H
JH
Forgione
M
M
Sugawara
A
A
Chin
W W
WW
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry; D005799:Genes, Dominant; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D018774:Oncogene Proteins v-erbA; D009857:Oncogenes; D011485:Protein Binding; D011988:Receptors, Thyroid Hormone / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins; D015682:Retroviridae Proteins, Oncogenic / Q000737:chemistry* / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D018774:Oncogene Proteins v-erbA; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D015682:Retroviridae Proteins, Oncogenic; D014284:Triiodothyronine