In vivo siRNA delivery to the mouse hypothalamus confirms distinct roles of TR beta isoforms in regulating TRH transcription.

RNA interference mediated by small interfering RNAs (siRNAs) is a powerful tool for evaluating gene function in vivo. In particular it should be able to provide tissue-specific and developmental stage-specific knock-down of target genes in physiological contexts. However, demonstrations of its use on neuronal specific genes in vivo are lacking. We examined whether a recently developed cationic lipid based approach was applicable to study the differential effects of the two beta thyroid hormone receptor (TR) isoforms, TRbeta1 and TRbeta2, on T3-transcriptional repression of the hypothalamic gene, TRH. The cationic lipid based technique used, JetSI/DOPE, was previously shown to efficiently knock-down reporter gene mRNA in vivo. Here we now show that its use to vectorise siRNA against TRbeta1 and TRbeta2 mRNA abrogates T3-mediated repression of hypothalamic TRH transcription. In particular, when using siRNA against either TRbeta1 or TRbeta2 differential effects are revealed. siRNA directed against TRbeta1 blocks both T3 independent activation and T3 dependent modulation of TRH transcription. In contrast, siRNA directed against TRbeta2 abrogates only T3 repression of transcription. These results corroborate our previous findings obtained in mutant TRbeta(-/-) mice, showing that the TRbeta1 and TRbeta2 isoforms have differential effects on T3-TRH transcription. The data thus show that the cationic lipid-based siRNA strategy can effectively be used to reveal fine, tissue specific and isoform specific effects on neuronal gene transcription in vivo.

The co-chaperone XAP2 is required for activation of hypothalamic thyrotropin-releasing hormone transcription in vivo.

Transcriptional control of hypothalamic thyrotropin-releasing hormone (TRH) integrates central regulation of the hypothalamo-hypophyseal-thyroid axis and hence thyroid hormone (triiodothyronine (T(3))) homeostasis. The two beta thyroid hormone receptors, TRbeta1 and TRbeta2, contribute to T(3) feedback on TRH, with TRbeta1 having a more important role in the activation of TRH transcription. How TRbeta1 fulfils its role in activating TRH gene transcription is unknown. By using a yeast two-hybrid screening of a mouse hypothalamic complementary DNA library, we identified a novel partner for TRbeta1, hepatitis virus B X-associated protein 2 (XAP2), a protein first identified as a co-chaperone protein. TR-XAP2 interactions were TR isoform specific, being observed only with TRbeta1, and were enhanced by T(3) both in yeast and mammalian cells. Furthermore, small inhibitory RNA-mediated knockdown of XAP2 in vitro affected the stability of TRbeta1. In vivo, siXAP2 abrogated specifically TRbeta1-mediated (but not TRbeta2) activation of hypothalamic TRH transcription. This study provides the first in vivo demonstration of a regulatory, physiological role for XAP2.