The dominant negative effect of thyroid hormone receptor splicing variant alpha 2 does not require binding to a thyroid response element.
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.
Liu
R T
RT
Department of Medicine, University of Chicago, Illinois 60637, USA.
Suzuki
S
S
Miyamoto
T
T
Takeda
T
T
Ozata
M
M
DeGroot
L J
LJ
D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics / Q000378:metabolism; D003864:Depression, Chemical; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012326:RNA Splicing; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012045:Regulatory Sequences, Nucleic Acid; D015533:Transcriptional Activation / Q000187:drug effects*; D014407:Tumor Cells, Cultured
D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D004247:DNA