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Active Repression by Thyroid Hormone Receptor Splicing Variant 2 Requires Specific Regulatory Elements in the Context of Native Triiodothyronine-Regulated Gene Promoters

National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Genetics and Biochemistry Branch (A.F., J.L., M.P., R.L., V.M.N.), Bethesda, Maryland 20892-1766; Institute of Experimental Medicine, Consiglio Nazionale Delle Richerche (A.F.) and Institute of Medical Pathology (A.P.), Catholic University, Rome 00168, Italy

Address all correspondence to: Antonella Farsetti (present address: Institute of Experimental Medicine, CNR, c/o IRE-CRS Molecular Oncogenesis Laboratory, Via delle Messi D’Oro 156, 00158 Roma, Italy). Requests for material should be sent to V.M.N. E-mail: Farsetti{at}dotto.ifo.it

Structural requirements for the inhibitory action of thyroid hormone receptor splicing variant 2 (TR2) on T₃/TRß1-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/TRß1 transactivation of MBP₂₅₆-chloramphenicol acetyl transferase (CAT) and ME₃₁₅-CAT constructs was inhibited and unaffected by TR2, respectively. In electrophoretic mobility shift assays, TR2 bound MBP-thyroid response element (TRE) as a monomer but failed to interact with ME-TRE. Mutations of ME-TRE allowed TR2 binding but not inhibition of T₃/TRß1-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 TR2 dominant negative action. Simultaneous introduction of both MBP-TRE and MBP TATA-like box in the context of ME promoter, however, triggered TR2 inhibition of T₃/TRß1 transactivation, indicating that these regulatory elements are necessary, but not individually sufficient, to mediate TR2 dominant negative activity. Functional studies at low TR2/TRß1 ratios revealed that binding to TRE facilitates TR2 dominant negative action while prevention of DNA interaction by altering TR2 P-box structure preserved TR2 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 TR2 dominant negative activity.

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