Interaction between the thyroid hormone receptor and co-factors on the promoter of the gene encoding phospho enol pyruvate carboxykinase.
Using transient transfection studies we localized a thyroid hormone-responsive element on the promoter of the rat phospho-enol pyruvate carboxykinase gene between 355 and 174 bp upstream of the transcription start site. DNAse 1 footprinting analysis within this region showed that a 28 bp fragment at position -324 to -297 was protected by the thyroid-hormone receptor. This receptor was overexpressed in HeLa cells using a vaccinia virus expression vector. DNA-binding assays with this receptor-enriched nuclear HeLa cell extract revealed that only 20% of the thyroid hormone receptor was able to bind the target-sequence with high affinity (4 nM). Titration with nuclear extract from hepatocytes increased the percentage of c-erbA molecules able to bind to this thyroid hormone-responsive element 4-fold without a change in affinity. Our data show that the complex of the thyroid hormone responsive element of the promoter of the phosphoenol pyruvate carboxykinase gene and the thyroid hormone receptor contains only a single receptor molecule suggesting the formation of a heterodimer complex. Accordingly, this thyroid hormone receptor/DNA complex is formed only in the presence of co-factors that are present in limiting amounts in the hepatocyte nucleus.
Department of Anatomy and Embryology, AMC, Amsterdam, Netherlands.
E D
ED
Schmidt
M
M
van Beeren
C K
CK
Glass
W M
WM
Wiersinga
W H
WH
Lamers
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000502:physiology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013724:Teratoma; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D010729:Phosphoenolpyruvate Carboxykinase (GTP)