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Functional Inactivation of the Transcription Factor Pax8 through Oligomerization Chain Reaction

Istituto di Endocrinologia ed Oncologia Sperimentale-Consiglio Nazionale delie Ricerche and Dipartimento di Biologia e Patologia Cellulare e Molecolare (B.D., R.I., T.D.P., R.N., L.N., R.D.L., M.Z.), Italy; S.E.M.M. European School of Molecular Medicine (M.G.B.), and CEINGE biotecnologie avanzate (M.G.B., R.D.L.) 80131 Naples, Italy

Address all correspondence and requests for reprints to: Mariastella Zannini, Istituto di Endocrinologia ed Oncologia Sperimentale-Consiglio Nazionale delie Ricerche, c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita’ di Napoli Federico II, via Pansini, 5, 80131 Napoli, Italy. E-mail: stella{at}szn.it.

Among the approaches used to provide a functional inactivation of a target protein, we have chosen the recently described oligomerization chain reaction (OCR) strategy to functionally inactivate the transcription factor Pax8, a member of the Pax gene family expressed in thyroid cells. The OCR strategy is based on the fusion of the self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to target proteins that are able to self-associate naturally or that form heterocomplexes. In the thyroid tissue, the transcription factor Pax8 is involved in the morphogenesis of the gland and in the transcriptional regulation of thyroid-expressed genes. We have recently demonstrated that in thyroid cells Pax8 interacts biochemically and functionally with the transcription factor TTF-1 (thyroid transcription factor 1), and that such interaction leads to the synergistic activation of thyroglobulin (Tg) gene expression. Fusion of the CC domain to Pax8 leads to the formation of aberrant, nonfunctional high-molecular mass complexes to which TTF-1 is also recruited. The CC-Pax8 chimera inhibits the transcriptional activity of Pax8 and of TTF-1 on both synthetic and physiological promoters and prevents the synergistic activation of the Tg promoter mediated by these two transcription factors. Furthermore, the expression of the CC-Pax8 chimera in differentiated thyroid cells leads to the down-regulation of the endogenous expression of several differentiation markers such as Tg, sodium/iodide symporter, Foxe1, TTF-1, and thyroid oxidase 2. These results demonstrate that the OCR is a useful tool to functionally inactivate a transcription factor. Moreover, by this approach, we identified Foxe1, TTF-1, and thyroid oxidase 2 as new direct targets of Pax8 or TTF-1.

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