IDENTIFICATION OF A THYROID-HORMONE RESPONSE ELEMENT IN THE MOUSE MYOGENIN GENE - CHARACTERIZATION OF THE THYROID-HORMONE AND RETINOID-X RECEPTOR HETERODIMERIC BINDING-SITE

Thyroid hormones are positive regulators of muscle development in vivo . Triiodo-L-thyronine (T-3) treatment of myogenic cell lines results i n the precocious expression of myogenin, a muscle specific, helix-loop -helix factor that can trans-activate muscle specific gene expression (G. Carnac et al., Mol. Endocrinol., 6: 1185-1194, 1992). We have iden tified a T-3 response element (TRE) in the mouse myogenin (MM) promote r between nucleotide positions -526 and -494 (5' GTGGTAGGTCTTTAGGGGTCT CATGGACTGACA 3'). This sequence conferred appropriate hormonal regulat ion to an enhancerless SV40 promoter. Electrophoretic mobility shift a nalysis experiments showed that thyroid hormone receptor alpha (TR alp ha) and retinoid X receptor alpha (RXR alpha) formed a heterodimeric c omplex on the MM TRE that was specifically competed by classical TREs and not by other response elements. Analyses of this heterodimer with a battery of steroid hormone response elements indicated that the comp lex was efficiently competed by a direct repeat of the AGGTCA motif se parated by 4 nucleotides, as predicted by the 3-4-5 rule. Electrophore tic mobility shift analysis experiments showed that the myogenin, grow th hormone, and myosin heavy chain TREs interacted with an identical n uclear factor(s) in muscle cells that was constitutively expressed dur ing myogenesis. Mutagenesis of the MM TRE indicated that the sequence of the direct repeats (AGGTCA) and the 4-nucleotide gap were necessary for efficient binding to the TR alpha/RXR alpha heterodimeric complex . In conclusion, our data suggest that the MM TRE is a target for dire ct cross-talk between two different hormonal signals (T-3 and 9-cis-re tinoic acid) at the receptor level. To our knowledge, this is the firs t identification and characterization of a hormonal response element i n the muscle specific helix-loop-helix family that directly interacts with TR alpha and RXR alpha. These results indicate that the thyroid h ormones, which are positive regulators of myogenic terminal differenti ation, directly target the hierarchical regulators of myogenesis.