Active repression by thyroid hormone receptor splicing variant alpha2 requires specific regulatory elements in the context of native triiodothyronine-regulated gene promoters

Alfredo Pontecorvi, Antonella Farsetti, J. Lazar, M. Phyillaier, R. Lippoldt, V. M. Nikodem

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)4705-4712
Number of pages8
JournalEndocrinology
Volume138
Publication statusPublished - 1997

Keywords

  • Animals
  • DNA
  • DNA, Recombinant
  • Genes, Dominant
  • Genes, Regulator
  • Genetic Variation
  • Malate Dehydrogenase
  • Myelin Basic Proteins
  • Promoter Regions, Genetic
  • Rats
  • Receptors, Thyroid Hormone
  • Transcription, Genetic
  • Triiodothyronine

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