ACTIVE REPRESSION BY THYROID-HORMONE RECEPTOR SPLICING VARIANT ALPHA-2 REQUIRES SPECIFIC REGULATORY ELEMENTS IN THE CONTEXT OF NATIVE TRIIODOTHYRONINE-REGULATED GENE PROMOTERS

Structural requirements for the inhibitory action of thyroid hormone r eceptor splicing variant alpha 2 (TR alpha 2) on T-3/TR beta 1-mediate d transactivation were investigated in native promoters of two T-3-reg ulated genes: the brain-specific myelin basic protein (MBP) and the ho usekeeping malic enzyme (ME). T-3/TR beta 1 transactivation of MBP256- chloramphenicol acetyl transferase (CAT) and ME315-CAT constructs was inhibited and unaffected by TR alpha 2, respectively. In electrophoret ic mobility shift assays, TR alpha 2 bound MBP-thyroid response elemen t (TRE) as a monomer but failed to interact with ME-TRE. Mutations of ME-TRE allowed TR alpha 2 binding but not inhibition of T-3/TR beta 1- mediated transactivation. In the context of the MBP promoter, replacem ent of MBP-TRE with ME-TRE or exchange of MBP TATA-like box with the M E CC-rich region spanning the transcription start site abolished TR al pha 2 dominant negative action. Simultaneous introduction of both MBP- TRE and MBP TATA-like box in the context of ME promoter, however, trig gered TR alpha 2 inhibition of T-3/TR beta 1 transactivation, indicati ng that these regulatory elements are necessary, but not individually sufficient, to mediate TR alpha 2 dominant negative activity. Function al studies at low TR alpha 2/TR beta 1 ratios revealed that binding to TRE facilitates TR alpha 2 dominant negative action while prevention of DNA interaction by altering TR alpha 2 P-box structure preserved. T R alpha 2 inhibitory effect, although with lower potency. In conclusio n, the results suggest that, in native promoters of T-3-regulated gene s, a dual molecular mechanism, with DNA-binding dependent and DNA-bind ing independent components, underlies TR alpha 2 dominant negative act ivity.