IDENTIFICATION OF THYROID-HORMONE RESPONSE ELEMENTS IN RODENT PCP-2, A DEVELOPMENTALLY-REGULATED GENE OF CEREBELLAR PURKINJE-CELLS

In a previous study, we have shown that in vivo expression of the cere bellar Purkinje cell-specific gene Pcp-2 is regulated by thyroid hormo ne (T-3) during neonatal development. In addition, transient cotransfe ction studies using thyroid hormone receptors (TRs) and a Pcp-2-lacZ c onstruct pointed to direct regulation of Pcp-2 gene expression by T-3. Therefore, we have initiated the following series of studies to defin e more precisely the location of the thyroid hormone regulatory elemen ts in the Pcp-2 gene. By transfection and in vitro receptor binding an alyses, we have identified two thyroid hormone response elements, A1 ( -295/-268) and B1 (+207/+227). A1 contains a central half site flanked by two similar half-sites. B1 contains two pairs of alternate half-si tes. When these elements were ligated to the modified mouse mammary tu mor virus promoter (Delta MMTV), both induced a 8-14-fold expression o f the reporter gene, but only in the presence of T-3. Gel mobility ass ays demonstrated that both A1 and B1 bind TRs in the presence of thyro id hormone receptor auxiliary proteins or the retinoid X beta receptor . Mutations of the G residues to T within the individual half-site seq uences of A1 caused a variable decrease in the transactivation of the MMTV-CAT construct and a corresponding reduction in TR binding in vitr o. Thus, mutational analysis of A1 pointed to the interaction of the f lanking half-site motifs with the central AGGTCA half-site. Interestin gly, lengthening of the A1 sequence at its 3'-end caused a progressive dampening of the T-3 response. The results suggest that the neighbori ng sequence may function as a silencer of the A1 element. Since thyroi d hormone regulation of Pcp-2 is manifest only during the first 2 week s after birth, we hypothesize that A1 and B1 act as T-3-dependent resp onse elements operative only during early neonatal Purkinje cell devel opment and that their function is suppressed by a neighboring silencer element operative when expression of Pcp-2 becomes hormone-independen t.