Kim S W, Ahn I M, Larsen P R
Thyroid Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Cell Biol. 1996 Aug;16(8):4465-77. doi: 10.1128/MCB.16.8.4465.
We studied the effects of thyroid hormone (T3) on nuclear protein-DNA interactions by using dimethyl sulfate (DMS) and DNase I ligation-mediated PCR footprinting. We examined an endogenous gene the growth hormone (GH) gene, and a stably transfected plasmid containing the chicken lysozyme silencer (F2) T3 response element (TRE) gene, F2-TRE-TK-CAT, both in pituitary tumor (GC) cells. The 235-1 cell line, which expresses prolactin (PRL) and Pit-1, but not the T3 receptor (TR) or GH, was used as a control. DMS and DNase I footprinting identified protected 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 the tripartite GH TRE at -180 bp against either DMS or DNase I in the absence or presence of T3 in either cell line. However, T3 increased protection of the Pit-1 and Sp1 binding sites against DMS in GC cells. In GC cells stably transfected with a plasmid containing F2-TRE-TK-CAT or TRalpha, chloramphenicol acetyltransferase expression was T3 inducible and DMS footprinting revealed both F2 TRE TR-binding half sites in a pattern suggesting the binding of TR homodimers before and during T3 exposure. We conclude that the GH gene is accessible to specific nuclear proteins in GC, but not in 235-1, cells and that T3 enhances 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-affinity F2 TRE before and during T3 exposure suggests that reversible interaction of T3 with DNA-bound TRs, rather than transient T3-TR contact with TREs, determines the level of T3-stimulated transcriptional activation.
我们通过使用硫酸二甲酯（DMS）和DNase I连接介导的PCR足迹法研究了甲状腺激素（T3）对核蛋白-DNA相互作用的影响。我们在垂体肿瘤（GC）细胞中检测了一个内源性基因——生长激素（GH）基因，以及一个稳定转染的含有鸡溶菌酶沉默子（F2）T3反应元件（TRE）基因的质粒，即F2-TRE-TK-CAT。表达催乳素（PRL）和Pit-1但不表达T3受体（TR）或GH的235-1细胞系用作对照。DMS和DNase I足迹法在GC细胞中鉴定出GH基因的Pit-1、Sp1和Zn-15结合位点中有受保护的G残基，但在235-1细胞中未鉴定出。在任一细胞系中，无论有无T3，-180 bp处的三联体GH TRE对DMS或DNase I均无特异性保护作用。然而，T3增加了GC细胞中Pit-1和Sp1结合位点对DMS的保护作用。在稳定转染了含有F2-TRE-TK-CAT或TRα质粒的GC细胞中，氯霉素乙酰转移酶表达是T3诱导型的，DMS足迹法显示两个F2 TRE TR结合半位点的模式表明在T3暴露之前和期间TR同二聚体的结合。我们得出结论，GH基因在GC细胞中可被特定核蛋白识别，但在235-1细胞中不能，并且T3增强了这种相互作用，尽管没有证据表明TR与低亲和力的大鼠GH TRE结合。在T3暴露之前和期间TR与高亲和力F2 TRE的结合表明，T3与DNA结合的TRs的可逆相互作用，而不是T3-TR与TREs的瞬时接触，决定了T3刺激的转录激活水平。
