IN-VIVO GENOMIC FOOTPRINTING OF THYROID HORMONE-RESPONSIVE GENES IN PITUITARY-TUMOR CELL-LINES

We studied the effects of thyroid hormone (T-3) on nuclear protein-DNA interactions by using dimethyl sulfate (DMS) and DNase I ligation-med iated PCR footprinting. We examined an endogenous gene the growth horm one (GH) gene, and a stably transfected plasmid containing the chicken lysozyme silencer (F2) T-3 response element (TRE) gene, FZ-TRE-TK-CAT , both in pituitary tumor (GC) cells. The 235-1 cell line, which expre sses prolactin (PRL) and Pit-1, but not the T-3 receptor (TR) or GH, w as used as a control. DMS and DNase I footprinting identified protecte d G residues in the Pit-1, Sp1, and Zn-15 binding sites of the GH gene in GC, but not in 235-1, cells. There was no specific protection of t he tripartite GH TRE at -180 bp against either DMS or DNase I in the a bsence or presence of T-3 in either cell line. However, T-3 increased protection of the Pit-1 and Spl binding sites against DMS in GC cells. In GC cells stably transfected with a plasmid containing F2-TRE-TK-CA T or TR alpha, chloramphenicol acetyltransferase expression was T-3 in ducible and DMS footprinting revealed both F2 TRE TR-binding half site s in a pattern suggesting the binding of TR homodimers before and duri ng T-3 exposure. We conclude that the GH gene is accessible to specifi c nuclear proteins in GC, but not in 235-1, cells and that T-3 enhance s this interaction, although there is no evidence of TR binding to the low-affinity rat GH TRE. The presence of TR binding to the high-affin ity F2 TRE before and during T-3 exposure suggests that reversible int eraction of T-3 with DNA-bound TRs, rather than transient T-3-TR conta ct with TREs, determines the level of T-3-stimulated transcriptional a ctivation.