Subcellular Localization of Full-Length Rev-erbB in HEK293 Cells
Cooperrider, Holly E.
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Nuclear receptors (NRs) are eukaryotic transcription factors that regulate many physiological processes including growth, inflammation, and circadian rhythms. Rev-erbβ is a NR that regulates genes involved with metabolism and circadian rhythm, effectively linking these two critical processes. The endogenous ligand of Rev-erbβ, heme, is thought to bind Rev-erbβ in the nucleus and cause the recruitment of the NR co-repressor (NCoR) to repress target gene transcription. Curiously, previous studies have disagreed on the subcellular localization of Rev-erbβ by demonstrating that NCoR can bind Rev-erbα (a Rev-erbβ isoform) in the absence of heme. This suggested a more complex role of heme in NCoR recruitment. To explain the literature discrepancy, we developed the present study hypothesis that Rev-erbβ was binding heme in the cytoplasm before translocation into the nucleus and formation of a complex with NCoR. Using cell culture techniques, Western blotting and fluorescence spectroscopy, this hypothesis was refuted by demonstrating that the subcellular localization of Rev-erbβ was independent of intracellular heme concentration. Since Rev-erbβ did not bind heme in the cytosol, heme’s interaction with Rev-erbβ and Rev-erbβ’s interaction with NCoR was determined to be limited to the nucleus. Future experiments using co-immunoprecipitation techniques, such as those conducted with Rev-erbβ and its known cofactors Tip60 and ZNHIT-1, could be used to determine if there is an unknown protein bound to Rev-erbβ in the nucleus that modulates heme reactivity. This protein could be displaced when heme binds to Rev-erbβ, allowing for the recruitment of NCoR. Present study results and future research on Rev-erbβ and other NRs, attractive pharmacological drug targets, could be used to discover connections between NRs and metabolic and circadian dysfunction.