Oct-1, silencer sequence, and GC box regulate thyroid hormone receptor β1 promoter

Abstract

Thyroid hormone, acting through thyroid hormone receptors (TRs), plays a crucial role in brain development and its insufficiency results in irreversible brain damage. TR α mRNA is expressed continuously from early embryonic stages, but the level of TR β1 mRNA in brain is more abundant in adult than in fetus. To identify important factors which regulate TR β1 expression, we compared mouse fetal and adult brain nuclear extracts by DNase I footprinting and electrophoretic gel mobility shift assays (EMSA) of the TR β1 promoter. We carried out transient transfection studies in COS 1 cells using the TR β1 promoter fused to Luciferase gene, and used mutated promoter vectors and various expression vectors. In DNase I footprinting using the fragment -950 to -717, fetal brain nuclear extracts protected the areas -910 to -884 and -815 to -800 more than did adult extracts. In EMSA, proteins in fetal nuclear extracts bound to a silencer sequence (−924 to -916), GC box (−901 to -887), and E box (−810 to -805), more strongly than did proteins in adult brain extracts. The bands formed on GC box were not supershifted by Sp-1, Sp-2, Sp-3, Sp-4, EGR-1, or EGR-2 antibodies. Three bands were detected on the octamer binding site probe (−913 to -906) and one protein was supershifted by Oct-1 antibody. Adult brain extracts appear to contain more Oct-1 protein than do fetal extracts. The other two bands were more intense in fetal extracts than in adult extracts, but were not supershifted by either Oct-1 or Oct-2 antibodies. Mutation of the silencer response element, mutation of the GC box, and Oct-1 over expression in COS 1 cells increased TR β1 promoter function as assayed by Luciferase reporter. Mutation of the octamer binding site, to which only Oct-1 bound in COS 1 cells, decreased Luciferase reporter activity. Thus the TR β1 promoter was regulated negatively by the proteins bound to the silencer sequence and the GC box, and positively by Oct-1. Silencer and GC box binding proteins are more abundant in fetal brain, and Oct-1 is more abundant in adult brain. The results may be responsible for increased amounts of TR β1 present in late fetal and adult brain.