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 induced in response to T-3. Previously, three contiguous restriction fragments 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 transcription [Far Upstream Regulatory region (FUR)]. To further investigate the molecular 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 retinoid X receptor were used in the gel retardation assays to map receptor binding sites in the S-14 gene. Three TR-binding sequences were identified 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, these motifs were arranged as direct repeats with 4 base pair spacing, while in FUR11 a more complex arrangement occurred. From mutagenesis experiments, all three TR-binding sequences in the S-14 gene were found to 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 observations that at least two TR-binding sites are required for TI induction in hepatocytes. In addition, synergy occurs between TR and additional regulatory sequences present in the FUR region and provides the maximal T-3 response of the S-14 gene.