Thyroid hormone receptors are ligand-activated transcription factors that modulate the expression of some target genes in a tissue- and development-specific manner. In higher organisms, thyroid hormone is essential during active neurogenesis accompanied by active myelination. We have identified a novel nuclear protein from 19 day embryonic rat brain, which displays a distinct interaction pattern with thyroid hormone receptor isoforms at the level of the thyroid hormone response elements. Gel mobility shift assays showed that this interaction could be detected only with nuclear extract prepared from developing brain and it is the receptor b and response element specific. To investigate whether this brain nuclear protein is one of known retinoid like receptors (alpha, beta, gamma), we used antibody raised against common DNA-binding domain and antibodies isoform- specific in antibody supershift assays. Our results indicate that this putative thyroid hormone receptor beta heterodimerization partner is not one of the retinoid like receptor isoforms but it is most likely a new member of the retinoid like receptor subfamily, since it has been only recognized by common antibody. To clone this protein we set up the yeast-based genetic assay which is used to detect protein- protein interactions. When using a GAL4-DNA binding domain-rat thyroid hormone receptor beta1 fusion in a yeast two hybrid screening an oligo(dt) primed 19 day-old rat embryonic brain DNA fusion library in a GAL4-activating domain vector, we have identified two cDNA sequences. One of them was an already known nuclear protein' SP120 (scaffold protein). The SP120, a nuclear matrix protein, has been shown to bind to matrix-attached regions of the nuclear DNA. The other cDNA encodes a novel protein. Preliminary data from Northern analyses showed that this cDNA detected a message of about 2kb only in embryonic brain and testis. The size of this message also indicated that our clone is not a full length. Six clones ranging from 0.800- 2kb were obtained by screening a rat testis cDNA library. They have been presently subjected to DNA sequencing. Both clones, SP120 and a novel clone, will be tested in vitro assays for interactions with thyroid hormone receptors and other members of the superfamily. Similarly, they will be used in transient transfection assays, either alone or together with the other receptors to assess their function in transcriptional regulation. Clearly, many different experiments are necessary to elucidate molecular basis and functional significance of the interaction of SP120 and a novel protein with thyroid hormone receptors in the eukaryotic system.
