The long-term objective of our research is the elucidation of the molecular pathways which function in thyroid hormone (T3) regulation of Na, K-ATPase subunit genes. In our research to-date, chimeric genes consisting of a portion of the 5'-flanking region of alpha2 and alpha3 subunit genes ligated to the coding region of the firefly luciferase gene have been utilized to transiently transfect neonatal cardiac myocytes. Surprisingly, we find that the expression of both alpha2 and alpha3 chimeric genes is significantly repressed by T3 in the presence of co-transfected T3 receptor. These observations are consistent with a preliminary finding of T3-mediated repression of endogenous alpha2 and alpha3 subunit gene transcription. In the proposed studies we intend to examine the underlying molecular mechanism of T3 repression of Na, K-ATPase alpha2 and alpha3 chimeric genes in rat neonatal cardiac myocytes to test the hypothesis that subunit gene transcription rates are inhibited following an interaction of the T3-receptor complex with subunit DNA sequence motifs. Accordingly,the specific aims of the proposed research are: (1) To determine the effect of T3 on the rate of alpha2 and alpha 3 subunit gene transcription by the run-on transcription assay. (2) To ascertain the location and sequence of the element responsible for T3-mediated repression of chimeric gene expression (nTRE) through an analysis of T3- responsiveness of various truncated subunit/luciferase chimeric genes. (3) To characterize the transacting factor which mediates T3 inhibition of alpha2 and alpha3 subunit chimeric gene activity by an assessment of binding of both unfractionated nuclear extracts and purified T3-receptor to subunit gene fragments containing negative TREs. We expect that the accomplishment of these research objectives will provide new and significant information concerning fundamental features of T3 action at a molecular level.
