Characterization of the 5'-flanking region of the rat thyrotropin receptor gene.

Abstract

Genomic clones containing 1.7 kilobases of the 5'-flanking region of the rat TSH receptor (TSHR) plus coding sequence from the ATG initiation codon [1 basepair (bp)] to the start of the first intron (170 bp) have been isolated and characterized. RNAase protection, primer extension, and cDNA sequences cloned by the anchored polymerase chain reaction identified multiple transcriptional start sites, the major ones clustered between -89 to -68 bp. This portion of the 5'-flanking region has neither a TATA nor a CCAAT box, is GC rich but has no GC box motif, and has features of promoters seen in "housekeeping" genes. Chimeras containing 1.7 kilobases (-1707 to -2 bp) of the 5'-flanking region, or deletions thereof, and the bacterial chloramphenicol acetyltransferase (CAT) gene expressed significant CAT activity when transfected into rat thyroid cell lines, FRTL-5 and FRT, but not BRL rat liver or HeLa cells. TSH decreased CAT activity in the FRTL-5 thyroid cells that had been stably transfected with the TSHR-CAT chimeric constructs. Negative regulation of promoter activity by TSH was duplicated by 10 microM forskolin in FRT thyroid cells, which express no TSHR mRNA. Deletion analyses indicated that a "minimal" region, exhibiting promoter activity, tissue specificity, and negative regulation by TSH, is located between -195 and -39 bp; this region is highly conserved in rat and human TSHR genes. Differential digestion of genomic DNA by MspI and HpaII revealed that the TSHR promoter is methylated in FRT, but not FRTL-5, cells; methylation of the promoter may be associated with loss of endogenous TSHR gene expression in FRT cells.