Active repression by thyroid hormone receptor splicing variant alpha2 requires specific regulatory elements in the context of native triiodothyronine-regulated gene promoters.
Structural requirements for the inhibitory action of thyroid hormone receptor splicing variant alpha2 (TR alpha2) on T3/TRbeta1-mediated transactivation were investigated in native promoters of two T3-regulated genes: the brain-specific myelin basic protein (MBP) and the housekeeping malic enzyme (ME). T3/TRbeta1 transactivation of MBP256-chloramphenicol acetyl transferase (CAT) and ME315-CAT constructs was inhibited and unaffected by TR alpha2, respectively. In electrophoretic mobility shift assays, TR alpha2 bound MBP-thyroid response element (TRE) as a monomer but failed to interact with ME-TRE. Mutations of ME-TRE allowed TR alpha2 binding but not inhibition of T3/TRbeta1-mediated transactivation. In the context of the MBP promoter, replacement of MBP-TRE with ME-TRE or exchange of MBP TATA-like box with the ME GC-rich region spanning the transcription start site abolished TR alpha2 dominant negative action. Simultaneous introduction of both MBP-TRE and MBP TATA-like box in the context of ME promoter, however, triggered TR alpha2 inhibition of T3/TRbeta1 transactivation, indicating that these regulatory elements are necessary, but not individually sufficient, to mediate TR alpha2 dominant negative activity. Functional studies at low TR alpha2/TRbeta1 ratios revealed that binding to TRE facilitates TR alpha2 dominant negative action while prevention of DNA interaction by altering TR alpha2 P-box structure preserved TR alpha2 inhibitory effect, although with lower potency. In conclusion, the results suggest that, in native promoters of T3-regulated genes, a dual molecular mechanism, with DNA-binding dependent and DNA-binding independent components, underlies TR alpha2 dominant negative activity.
Farsetti
A
A
National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Genetics and Biochemistry Branch, Bethesda, Maryland 20892-1766, USA. Farsetti@dotto.ifo.it
Lazar
J
J
Phyillaier
M
M
Lippoldt
R
R
Pontecorvi
A
A
Nikodem
V M
VM
D000818:Animals; D004247:DNA / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics*; D005799:Genes, Dominant / Q000235:genetics; D005809:Genes, Regulator / Q000502:physiology*; D014644:Genetic Variation / Q000502:physiology*; D008291:Malate Dehydrogenase / Q000235:genetics; D004676:Myelin Basic Protein / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology*; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology; D014284:Triiodothyronine / Q000502:physiology*
D004274:DNA, Recombinant; D004676:Myelin Basic Protein; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D004247:DNA; D008291:Malate Dehydrogenase