Molecular Endocrinology, Vol 9, 86-95, Copyright © 1995 by Endocrine Society

ARTICLES

The dominant negative effect of thyroid hormone receptor splicing variant alpha 2 does not require binding to a thyroid response element

RT Liu, S Suzuki, T Miyamoto, T Takeda, M Ozata and LJ DeGroot
Department of Medicine, University of Chicago, Illinois 60637, USA.

The functionally inactive thyroid hormone receptor splicing variant- alpha 2 (TRv alpha 2) can inhibit transcriptional activation by TR alpha 1 or beta 1, demonstrating a dominant negative effect (DNE). We examine here the three commonly proposed mechanisms, namely, competition for binding to thyroid hormone response elements (TREs), formation of inactive heterodimers, and squelching. A mutation introduced into the DNA-binding domain (DBD) of the TRv alpha 2 was designed to prevent its binding to TREs. In transient cotransfection studies, the DBD mutant has nearly the same DNE as does TRv alpha 2 on three different TRE-containing reporter genes. The DNE of TRv alpha 2 is also not reversed by cotransfection with excess retinoid X receptor- alpha. Extracts of COS cells cotransfected with TR alpha 1 and either TRv alpha 2 or DBD mutant at different ratios were analyzed by gel shift assays. Neither TRv alpha 2 or the mutant altered binding of TR alpha 1 to four radiolabeled TREs. TRv alpha 2 itself can inhibit constitutive transactivation by a thymidine kinase promoter-driven reporter construct. Our results suggest that TRv alpha 2 can function in a dominant negative manner without binding to a TRE, at least for certain TREs. It is concluded that the DNE of TRv alpha 2 may occur through another unrecognized mechanism, perhaps by binding to basal transcription factors.

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