The human skeletal alpha-actin promoter is regulated by thyroid hormone: identification of a thyroid hormone response element.
Skeletal alpha-actin mRNA increases in the adult heart during cardiac hypertrophy after the imposition of hemodynamic overload/aortic restriction. 3,3',5-Triiodo-L-thyronine (T3) elicits a cardiac response similar to the effect of prolonged exercise and was recently shown to cause a rapid increase in the amount of skeletal alpha-actin mRNA in hearts from normal and hypophysectomized animals. We used transient transfection analysis to show that T3 induces the expression of the native skeletal alpha-actin promoter between nucleotide positions -2000 and +239 linked to the chloramphenicol acetyltransferase reporter gene in COS-1 fibroblasts and myogenic C2C12 cells. This T3 (10-100 nM)-induced transcriptional activation is dependent on the expression of the thyroid hormone receptors from transfected alpha 1 and beta 1 c-erbA complementary DNA expression vectors. Electrophoretic mobility shift assays were used to identify a thyroid hormone response element (TRE) in the human skeletal alpha-actin gene. This TRE is located between nucleotide positions -173 and -149 with respect to the start of transcription at +1 (5' TGGTCAACGCAGGGGACCCGGGCGG 3'). Electrophoretic mobility shift assay experiments showed that the putative skeletal alpha-actin TRE and defined rodent growth hormone TREs (that bind thyroid hormone receptors in vitro and in vivo) interacted with an identical nuclear factor in vitro in muscle cells that was developmentally regulated during myogenesis. Transient transfection analysis utilizing 5' unidirectional deletions of the skeletal alpha-actin promoter indicated that cis-acting sequences between nucleotide positions -432 and -153, which encompassed the TRE, were required for T3/thyroid hormone receptor-dependent trans-activation in vivo. Furthermore, we demonstrated that the skeletal alpha-actin TRE is juxtaposed next to SRF and SpI binding sites, at its 5' and 3' flanks, respectively. It is also surrounded by sequences densely populated by other SpI, SRF, and CTF binding sites. In conclusion, these results indicate that T3-induced increases in alpha-actin mRNA in animals are mediated by a direct transcriptional mechanism that may involve interactions with ubiquitous proteins.
Collie
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University of Queensland, Ritchie Research Laboratories, St Lucia, Queensland, Australia.
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D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001842:Bone and Bones / Q000737:chemistry*; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009132:Muscles / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000502:physiology; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection / Q000235:genetics; D014284:Triiodothyronine / Q000502:physiology*
D000199:Actins; D011518:Proto-Oncogene Proteins; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D066246:ErbB Receptors