FUNCTIONAL SYNERGISM BETWEEN MULTIPLE THYROID-HORMONE RESPONSE ELEMENTS REGULATES HEPATIC EXPRESSION OF THE RAT S-14 GENE

Hepatic expression of the rat S-14 gene is markedly and rapidly induce d in response to T-3. Previously, three contiguous restriction fragmen ts of the S-14 gene with thyroid hormone response activity were mapped to a region 2.5-3.0 kilobases upstream from the start of transcriptio n [Far Upstream Regulatory region (FUR)]. To further investigate the m olecular basis of the thyroid hormonal control of Sis gene expression, we have mapped the functional TRE sequences in the FUR region of the S-14 gene. In vitro translated thyroid hormone receptor (TR) and retin oid X receptor were used in the gel retardation assays to map receptor binding sites in the S-14 gene. Three TR-binding sequences were ident ified in the FUR region of the S-14 gene and designated: FUR10 (from - 2718 to -2694), FUR11 (from -2632 to -2595), and FUR12 (from -2582 to -2558). Each binding site contains two or more elements related to the consensus monomer binding motif 5'- Pu-GGTCA. In FUR10 and FUR12, the se motifs were arranged as direct repeats with 4 base pair spacing, wh ile in FUR11 a more complex arrangement occurred. From mutagenesis exp eriments, all three TR-binding sequences in the S-14 gene were found t o play a role and synergize with each other in the responsiveness to T -3. The importance of this functional synergy is also shown by the obs ervations that at least two TR-binding sites are required for TI induc tion in hepatocytes. In addition, synergy occurs between TR and additi onal regulatory sequences present in the FUR region and provides the m aximal T-3 response of the S-14 gene.