Activation of myoD gene transcription by 3,5,3'-triiodo-L-thyronine: a direct role for the thyroid hormone and retinoid X receptors.
Thyroid hormones are major determinants of skeletal muscle differentiation in vivo. Triiodo-L-thyronine treatment promotes terminal muscle differentiation and results in increased MyoD gene transcription in myogenic cell lines; furthermore myoD and fast myosin heavy chain gene expression are activated in rodent slow twitch muscle fibers (Molecular Endocrinology 6: 1185-1194, 1992; Development 118: 1137-1147, 1993). We have identified a T3 response element (TRE) in the mouse MyoD promoter between nucleotide positions -337 and -309 (5' CTGAGGTCAGTACAGGCTGGAGGAGTAGA 3'). This sequence conferred an appropriate T3 response to an enhancerless SV40 promoter. In vitro binding studies showed that the thyroid hormone receptor alpha (TR alpha) formed a heterodimeric complex, with either the retinoid X receptor alpha or gamma 1 isoforms (RXR alpha, RXR gamm), on the MyoD TRE that was specifically competed by other well characterised TREs and not by other response elements. Analyses of this heterodimer with a battery of steroid hormone response elements indicated that the complex was efficiently competed by a direct repeat of the AGGTCA motif separated by 4 nucleotides as predicted by the 3-4-5 rule. EMSA experiments demonstrated that the nuclear factor(s) present in muscle cells that bound to the myoD TRE were constitutively expressed during myogenesis; this complex was competed by the myosin heavy chain, DR-4 and PAL-0 TREs in a sequence specific fashion. Western blot analysis indicated that TR alpha 1 was constitutively expressed during C2C12 differentiation. Mutagenesis of the myoD 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 TRE in the helix loop helix gene, myoD, is a target for the direct heterodimeric binding of TR alpha and RXR alpha/gamma. These results provide a molecular mechanism/model for the effects of triiodo-L-thyronine on in vitro myogenesis; the activation of myoD gene expression in the slow twitch fibres and the cascade of myogenic events regulated by thyroid hormone.
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University of Queensland, Centre for Molecular Biology and Biotechnology, Ritchie Research Laboratories, St. Lucia, Australia.
Mynett-Johnson
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Dowhan
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Downes
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Griggs
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D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects*; D051379:Mice; D008969:Molecular Sequence Data; D017570:MyoD Protein / Q000235:genetics* / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism / Q000502:physiology*; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone / Q000378:metabolism / Q000502:physiology*; D012045:Regulatory Sequences, Nucleic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000187:drug effects*; D014284:Triiodothyronine / Q000494:pharmacology*
D017570:MyoD Protein; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D011988:Receptors, Thyroid Hormone; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014284:Triiodothyronine