Introduction

The transcriptional activation of thyroid hormone receptors (TRs) involves the recruitment of nuclear coregulatory proteins. There is evidence for tissue-specific recruitment of some coregulators, leading to diverging tissue-dependent TR functions. Considering the role of TH in brain development, we aimed to identify the coregulators that are involved in neuronal TR activity by studying interactomes for TRs in a neuronal cell line.

Methods

Flag/HA-tagged TRα1 and TRβ1 were stably expressed in SHSY5Y cells. TR-interacting proteins were purified from nuclear extracts after 24 hours 0/100 nM T3 stimulation using sequential purification on anti-FLAG and anti-HA resins. Isolated proteins were identified by LC/MS-MS.

Results

Over a hundred nuclear proteins were co-purified with FH-TRs. Known TR-interacting proteins, such as RXRs, regardless of T3, the NCoR1/HDAC3 repressors in the absence of T3, and SRCs and the Mediator complex in the presence of T3, were identified, confirming their roles in neuronal TR activity. Strikingly, many other identified proteins are members of complexes that are involved in chromatin remodeling but have not been described as coregulators of TRs, for instance, the nucleosome remodeling deacetylase (NuRD) and the Spt-Ada-Gcn5-Acetyltransferase (SAGA) complexes, both of which were recruited in a T3-dependent manner. Most of the identified proteins were found to be associated with both TR isoforms, suggesting that the action of TRs in SHSY5Y cells is mainly regulated by common coregulators. However, eight proteins were found to be only associated with TRα1 (such as MLL5, UTX, and MBD3) and one only with TRβ1 (PDE4D).

Conclusion

We demonstrated that TRs interact with several potential novel binding partners in SHSY5Y cells. In addition, a subset of distinct nuclear proteins seems to interact with TRs in an isoform-specific manner. Further studies are needed to elucidate the relevance of these novel interactions.