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Since both lack of T3 and lack of TRb1 mechanisms failed to
explain the low level of target gene expression, we considered
the possibility that other specific developmentally regulatory
factors governed the responsivity of these genes to thyroid
hormone. Gel shift studies employing the upstream region
containing the TRE region clearly showed the presence of a
binding protein which was prominent in the fetal brain before
birth and rapidly fell in the first weeks after birth, in a
pattern inversely related to that exhibited by the response of
T3-sensitive target genes.
A number of findings led us to speculate on the possibility that the fetal gene expression inhibitor might be related to one of the COUP-TFs. These proteins are members of the nuclear receptor superfamily. They have no known binding ligands. They have been cloned from a broad spectrum of species. They bind to variations of the sequence AGGTCA either as homo- or heterodimers. They are expressed in the developing rat and mouse brain. Lastly and most importantly, they are known to act as repressors of hormone activation by competing for DNA binding with other nuclear receptors. The following studies were carried out to test the hypothesis that COUP-TF or a closely related protein acted as an inhibitor of T3 action. Three distinct bands appeared when 32P-labeled Pcp was exposed to fetal nuclear extract. Supershift studies with specific antibodies to COUP-TF supported the idea that COUP-TF could be the responsible protein. Furthermore, COUP-TF represses T3 transactivation of the Pcp2 promoter in neuro 2A cells. The results of transactivation studies employing the Pcp2 promoter and a CAT reporter show that COUP can in fact significantly reduce the fold activation by T3, from 10.2-fold to 4.0-fold. One of the assumptions made in our analysis is that the COUP protein can be found in the Purkinje cells and its fetal precursor. Evidence supporting this assumption came from immunocytochemical studies which demonstrated a high concentration of COUP in Purkinje precursor cells. On the first postnatal day the distribution of both COUP and calbindin was still clearly concentrated in Purkinje cells. After the fourth postnatal day, however, there was a marked fall in the level of COUP in Purkinje cells whereas the level of calbindin, which is also concentrated in the Purkinje cells, was maintained. Figure 1:
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