In vivo genomic footprinting of thyroid hormone-responsive genes in pituitary tumor cell lines.
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.
Kim
S W
SW
Thyroid Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Ahn
I M
IM
Larsen
P R
PR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D005786:Gene Expression Regulation; D013006:Growth Hormone / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013463:Sulfuric Acid Esters / Q000737:chemistry; D015534:Trans-Activators / Q000378:metabolism; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014284:Triiodothyronine / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; C494186:Pou1f1 protein, rat; D012333:RNA, Messenger; D011988:Receptors, Thyroid Hormone; D016329:Sp1 Transcription Factor; D013463:Sulfuric Acid Esters; D015534:Trans-Activators; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C489979:Zfp292 protein, rat; D014284:Triiodothyronine; D013006:Growth Hormone; D003850:Deoxyribonuclease I; C007482:dimethyl sulfate