The long-term objectives of this research are to elucidate the mechanisms by which hormones regulate the expression of thyroglobulin (TG) gene and to gain further understanding of the problems of control and regulation of this gene in human thyroid tumors. The differentiated functions of the thyroid gland are regulated mainly by the pituitary gland hormone, thyrotropin (TSH). TSH enhances almost every aspect of thyroid cell metabolism including the synthesis of thyroglobulin (TG), the key precursor in the biosynthesis of thyroid hormones. In some thyroid tumors as well as in one thyroid rat cell line (FRTL-5R), the normal regulatory mechanism is altered and the synthesis of TG is independent of TSH. In this application, we propose to study the regulatory mechanisms of TG gene transcription by TSH and other hormones in normal rat thyroid FRTL-5 cells. We will construct genes by fusing the regulatory regions of TG gene with the bacterial chloramphenicol acetyltransferase (CAT) gene. Following transfer of the fused gene to FRTL-5 cells, synthesis of CAT under the control of TG gene will be investigated. The DNA regulatory elements for TSH, cAMP, insulin and any other hormone will be identified by systematic deletions of the TG regulatory sequences and investigation of the deletion mutants on hormone regulation of the CAT gene. We will also investigate the presence of nuclear factors which interact with these elements by DNA mobility shift and DNase I footprinting assay. The possible involvement of the regulatory subunits (RI and RII) of cAMP- dependent protein kinase in interaction with the TG hormonal- regulatory elements will also be investigated. The information obtained from this cell line will be applied to investigation of alterations of hormonal regulatory mechanisms of TG gene in human thyroid tumors and in the variant FRTL-5 cell line (FRTL- 5R). We will examine the possible alterations in (1) chromatin structure; (2) hormone regulatory factors and; (3) sequences of the TG regulatory elements in the variant FRTL-5R cells as well as in human thyroid tumors.
