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 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.
Zou
L
L
Department of Medicine, University of Minnesota, Minneapolis 55455.
Hagen
S G
SG
Strait
K A
KA
Oppenheimer
J H
JH
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000166:cytology / Q000196:embryology; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics*; D011689:Purkinje Cells / Q000378:metabolism*; D051381:Rats; D011988:Receptors, Thyroid Hormone / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D014284:Triiodothyronine / Q000378:metabolism
D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine