Regulation of the rat thyrotropin receptor gene by the methylation-sensitive transcription factor GA-binding protein.
The GA-binding protein (GABP), a transcription factor with a widespread tissue distribution, consists of two subunits, a and beta1, and acts as a potent positive regulator of various genes. The effect of GABP on transcription of the TSH receptor (TSHR) gene in rat FRTL-5 thyroid cells has now been investigated. Both deoxyribonuclease I footprint analysis and gel mobility-shift assays indicated that bacterially expressed glutathione S-transferase fusion proteins of GABP subunits bind to a region spanning nucleotides (nt) -116 to -80 of the TSHR gene. In gel mobility-shift assays, nuclear extracts of FRTL-5 cells and FRT cells yielded several specific bands with a probe comprising nt -116 to -80. Supershift assays with antibodies to GABPalpha and to GABPbeta1 showed that GABP was a component of the probe complexes formed by the nuclear extracts. Immunoblot analysis confirmed the presence of both GABP subunits in the nuclear extracts. A reporter gene construct containing the TSHR gene promoter was activated, in a dose-dependent manner, in FRTL-5 cells by cotransfection with constructs encoding both GABPalpha and GABPbeta1. Both GABP binding to and activation of the TSHR gene promoter were prevented by methylation of CpG sites at nt -93 and -85. These CpG sites were highly methylated (>82%) in FRT cells and completely demethylated in FRTL-5 cells, consistent with expression of the TSHR gene in the latter, but not the former. These results suggest that GABP regulates transcription of the TSHR gene in a methylation-dependent manner and that methylation of specific CpG sites and the methylation sensitivity of GABP contribute to the failure of FRT cells to express the endogenous TSHR gene.
Third Department of Internal Medicine, Yamanashi Medical University, Tamaho, Japan.
N
N
Yokomori
M
M
Tawata
T
T
Saito
H
H
Shimura
T
T
Onaya
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002457:Cell Extracts; D018899:CpG Islands; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051268:GA-Binding Protein Transcription Factor; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D051381:Rats; D011989:Receptors, Thyrotropin / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013961:Thyroid Gland / Q000166:cytology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D051268:GA-Binding Protein Transcription Factor; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D014157:Transcription Factors