Imaging of heme/hemeproteins in nucleus of the living cells expressing heme-binding nuclear receptors

Several factors involved in the core circadian rhythm are PAS domain proteins, one of which, neuronal PAS2 (NPAS2), contains a heme-binding motif. It is thought that heme controls the transcriptional activity of core circadian factors BMAL1-NPAS2, and that the heme-binding nuclear receptor REV-erbα negatively regulates the expression of BMAL1. To examine the role of heme in the nucleus, we expressed nuclear hemeproteins including the nuclear localization signal-added cytoglobin, NPAS2 and REV-erbα. Then, the living cells expressing these proteins were treated with 2′,7′- dichlorodihydrofluorescin diacetate (DCFH-DA). The fluorescent signal derived from DCFH-DA was observed in the nucleus. When the cells were cultured with hemin, the signal of heme in the nucleus increased. Considering that DCFH-DA reacted with heme, we propose that the use of DCFH-DA could be useful in detection of the heme moiety of hemeprotein in vivo.