Transcriptional regulation by the thyroid hormone receptor and its viral counterpart : the P75gag-V-erbA oncoprotein

Sammanfattning: Thyroid hormone binds to specific DNA binding receptors (TRs) in the cell nucleus and thus regulates transcription during development and in some pathological disorders. The gene of the viral TR homologue, v-erbA, is one of two oncogenes encoded in the genome of avian erythroblastosis virus (AEV). Its protein product (P75gag-V-erbA) differs from TRa by several point mutations that result in the loss of hormone binding and in dominant negative effects on gene activation. TR and p75gag-v-erbA belong to a superfamily of steroid hormone receptors which also includes the retinoic acid receptors, RAR and RXR. The regulatory functions of TR and p75gag-v-erbA are pivotal in many regulatory pathways and biological processes. I and my coworkers have studied these functions by in vitro and in vivo methods. We have studied the DNA sequences through which TR and p75gag-v-erbA regulate transcription. Different in vitro binding assays and a PCR based selection approach showed that TR monomers bind with low affinity to DNA and that homodimers maintain a low cooperativity and relatively poor affinity for DNA. In contrast, TR/RAR and in particular TR/RXR heterodimers show a strong interaction with DNA response elements containing two hexameric half-sites in a head to head orientation (direct repeats). A direct repeat element spaced by 4 nucleotides confers the strongest TR/RXR binding and the best transcriptional activation when the spacer consists of a cytidine in the first and a pyrimidine in the third position. The data suggest that TR activates transcription as a heterodimer, and that it has a limited preference for the structure of the target genes. To understand the role of P75gag-v-erbA in oncogenesis, we studied a chicken erythroblast cell line which had been transformed by AEV. We showed that in order to repress TR mediated activation, P75gag-v-erbA depends on the structure of the response elements. P75gag-v-erbA represses activation as a heterodimer with RXR or possibly as a homodimer. P75gag-v-erbA only interacts with RXR on DNA, which is in contrast to TR which also form heterodimers in solution. The strongest repression was seen with an everted repeat. An in vitro selection showed that P75gag-V-erbA/RXR heterodimers have a relaxed specificity regarding the spacer between the two half-sites in both direct and inverted orientations. The results suggest that the target genes for P75gag-v-erbA is of the everted type. We have studied the ability of TR and P75gag-v-erbA to crosstalk with an RXR specific pathway. The results obtained by transient transfections show that unliganded TR and p75gag-v-erbA repress RXR mediated transactivation in chorion carcinoma cells. The response element from the promoter of the cellular retinol binding protein (CRBPII) was likewise repressed by TR, but only to 50% by P75gag-v-erbA We propose that these homologues inhibit gene activation by different mechanisms. Possible mechanisms include the formation of inactive RXR heterodimers, the physical exclusion of co-factors and/or interference with the basal transcription machinery.