Endocrine Abstracts

Thyroid hormones (TH) are important regulators of human metabolism and development, which modulate expression of target genes via nuclear thyroid hormone receptors (TRs). Different isoforms of these classical TRs including TRβ isoforms TRβ1 and TRβ2 as well as TRα1 were shown to be functional TH-responsive transcription factors and have been extensively studied. In contrast, the cellular function of TRα2, an alternative splice variant of TRα1, is poorly understood and it is even speculated whether the TRα2 protein is of biological relevance in vivo at all or the mRNA might only act as transcriptional sink to quickly regulate TRα1 production. Characterization of specific isoform functions has in particular been hampered by the lack of reliable antibodies. We aim to address the function and physiological relevance of the poorly characterised TRα2 isoform by using a tagged TRα2 variant. ChIP-sequencing analyses will be applied to generate genome-wide DNA binding profiles specific for both TRα isoforms, and different proteomic approaches will be used to identify isoform-specific protein interactions mediating TRα1 and TRα2 functions in gene regulation. These analyses will not only unravel the role of TRα2 for TH signalling but also elucidate whether this isoform may be a possible target to modulate TH action. To gain first insights into TRα2-specific functions, co-immunoprecipitation followed by mass spectrometric analysis of peptides bound by GFP-tagged TRα isoforms transiently overexpressed in HEK293 cells has been performed. In this analysis, well-known interacting proteins such as TR-corepressor NCOR1 were found, confirming the validity of the experimental approach. Moreover, novel putative shared as well as isoform-specific binding proteins have been identified that are suggesting a potential so far unknown specific function of TRα2. Interestingly, during first cellular analyses, we also observed different subcellular localisation of the two isoforms. While TRα1 appears to be evenly distributed in the cell nucleus, TRα2 accumulated in distinct nuclear speckles, which we will further investigate. To uncover cell-type specific functions of TRα2, protein interactions and DNA binding profiles will next be analysed in other cell types. Moreover, we aim to verify the identified DNA binding profile and protein interactions found for ectopic expression of TRα2 by performing the same analyses in different induced pluripotent stem cell-derived cellular models expressing endogenously tagged TRα isoforms.