Endocrine Abstracts

Objective: Thyroid hormone receptors (TRs) are important ligand-dependent regulators of thyroid hormone target gene expression. In contrast, the role of TRα2, a thyroid hormone receptor α splice variant that is unable to bind thyroid hormones, remains enigmatic for more than 30 years. However, its evolutionary conservation in eutherian mammals and high expression levels in some cell types as well as under ‘pathological conditions’ such as heart failure strongly indicate its physiological relevance. While in vitro data from ectopically expressed TRα2 suggested a weak antagonistic activity to other TRs, no functional data on the endogenous TRα2 isoform was available due to the lack of reliable isoform-specific antibodies. Therefore, we aimed to gain insights into isoform-specific actions in different cell types by using induced pluripotent stem cell (iPSC) lines expressing endogenously tagged TRα isoforms.

Methods and Results: To enable specific detection of TRα isoforms, we introduced two different tags into the THRA locus of human iPSC lines by a CRISPR/Cas9-based approach. As a result, the derivative cell lines are either expressing TRα1 C-terminally tagged with a 2×HA tag or TRα2 C-terminally tagged with a 3×FLAG tag. We then differentiated the iPSCs to cardiomyocytes and confirmed the presence of cardiomyocyte differentiation markers MLC2a, MLC2v, cardiac Troponin-T and α-Actinin by immunofluorescence staining as well as flow cytometry analysis. Applying a CUT&Tag (Cleavage Under Targets and Tagmentation) approach, we obtained isoform-specific DNA binding profiles in both iPSCs and cardiomyocytes. While the majority of binding sites were shared between both isoforms in hiPSCs, we surprisingly found in addition a specific set of genes to be exclusively bound by TRα2 but not TRα1 in cardiomyocytes.

Conclusions and Outlook: Our recent data reveal the first isoform-specific genome-wide DNA binding profiles of TRα at endogenous receptor levels. While the binding profiles for both isoforms were highly similar in hiPSCs, TRα2-specific binding to promoters of a specific family of genes was exclusively seen in cardiomyocytes, indicating a TRα2-specific function in the heart. How isoform-specificity of DNA binding is regulated and whether TRα2 forms distinct protein complexes regulating gene expression is currently under investigation. Based on our findings, which reveal TRα2-specific activities in cardiomyocytes, it is tempting to speculate whether TRα2 also exerts distinct physiological activities in other cell types.