Abstract
In a previous study, we have shown that in vivo expression of the cerebellar Purkinje cell-specific gene Pcp-2 is regulated by thyroid hormone (T3) during neonatal development. In addition, transient cotransfection studies using thyroid hormone receptors (TRs) and a Pcp-2-lacZ construct pointed to direct regulation of Pcp-2 gene expression by T3. Therefore, we have initiated the following series of studies to define more precisely the location of the thyroid hormone regulatory elements in the Pcp-2 gene. By transfection and in vitro receptor binding analyses, 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-sites. When these elements were ligated to the modified mouse mammary tumor virus promoter (delta MMTV), both induced a 8-14-fold expression of the reporter gene, but only in the presence of T3. Gel mobility assays demonstrated that both A1 and B1 bind TRs in the presence of thyroid hormone receptor auxiliary proteins or the retinoid X beta receptor. Mutations of the G residues to T within the individual half-site sequences of A1 caused a variable decrease in the transactivation of the MMTV-CAT construct and a corresponding reduction in TR binding in vitro. Thus, mutational analysis of A1 pointed to the interaction of the flanking half-site motifs with the central AGGTCA half-site. Interestingly, lengthening of the A1 sequence at its 3'-end caused a progressive dampening of the T3 response. The results suggest that the neighboring sequence may function as a silencer of the A1 element. Since thyroid hormone regulation of Pcp-2 is manifest only during the first 2 weeks after birth, we hypothesize that A1 and B1 act as T3-dependent response elements operative only during early neonatal Purkinje cell development and that their function is suppressed by a neighboring silencer element operative when expression of Pcp-2 becomes hormone-independent.
Highlights
PCP-2is regulated by thyroid hormone (T,) during neonatal development
The fourthgene, myelinbasic protein, is expressed in oligodendrocytes and contains a previously defined thyroid hormone response element [5]. The rise of these mRNAs occurs in the neonate,immediately preceded by a 40fold surge in themRNA for the TRplisoform and the accompanying rise in the level of brain triiodothyronine [6]. These findings have prompted us to speculate that thTe,.TRpl complex plays a pivotal role in mediating the effects of thyroid hormone in brain development
An examination of the early time course of the three Purkinjecell mRNAs and themyelin basic protein mRNA in euthyroid pups and in pups rendered functionally athyroidal revealed the presence of both T,dewithin the individual half-site sequences of A1 caused a pendent andT,independent components in the increasegdene variable decrease in the transactivation of the MMW- expression observed during neonatal development
Summary
Fragmentsof the Pep-2 gene were preparedby PCR a s described under “Material and Methods.”. AMMTV-CAT, and the T, responsiveness was examined by transient transfection of CHO cells. Ideal AGGTCA half-sib 5’ to a second potential half-site with Nuclear Extract 4-nucleotide spacing and, on the same strand,a second pair of. We tested the potentialactivity of these sequences as TREs by synthesizing corresponding oligonucleotides and inserting them into themodified AMMTV promoter construct fused to a CAT reporter. The site of insertion in MMTV was the deleted glucocorticoid response elements (-88/-190) of the MMTV long terminal repeat [8, 18]
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