Thyroid hormone-responsive elements of the prolactin gene:evidence for both positive and negative regulation

To analyze the regulation of PRL gene expression by thyroid hormone (T₃), fusion gene constructs containing various lengths of the rat PRL gene 5′-flank-ing sequence linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were transfected into the GH3 cell line. Thyroid hormone had no effect on basal or cAMP-stimulated CAT expression in constructs containing more than 1.7 kilobasepairs of the 5′-sequence. However, deletion to 1.5 or 0.6 kilobasepairs resulted in an inhibition of both basal and cAMP-stimulated expression by T₃. A construct containing the proximal enhancer region (positions-292 to -38 basepairs) linked to the herpes simplex thymidine kinase promoter (TK) and the CAT reporter gene also responded to T₃with inhibition of basal and cAMP-induced CAT expression. The distal enhancer region (positions -1714 to -1495) linked to thymidine kinase promoter CAT responded to T₃with a stimulation of CAT expression, and the response was additive with the stimulatory response to cAMP. Deletion analysis of the distal enhancer region revealed that the sequence between positions -1530 and -1565 was required for the stimulatory response to T₃. The stimulatory response to T₃was additive with the response to estradiol, suggesting distinct elements, but deletion to position-1565 abolished the response to estradiol and permitted an inhibitory response to T₃. Mutation of the estrogen response element prevents the response to estradiol, but only blunted the response to T₃. Mutation of the sequence GGTCA at positions -1555 to -1551 resulted in an inhibitory response to T₃, implicating this sequence in the stimulatory response. The interaction of [¹²⁵l]T₃-labeled receptor with specific DNA sequences demonstrated that the distal enhancer region binds T₃receptor specifically and that sequences downstream of position -1565 are required. The proximal enhancer region did not bind T₃receptor. The results support the role of both stimulatory and inhibitory T₃-responsive elements in regulation of the expression of the PRL gene in GH₃cells.