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

• Fluorescence from DCFH-DA was observed in hemeprotein-expressing cells.
• We examined heme-binding of transcriptional factors, NPAS2 and REV-erbα in vivo.
• DCF fluorescence was observed in the nucleus of NPAS2 and REV-erbα expressing cells.
• DCFH reacted with heme of NLS-cytoglobin localized in nuclei.
• DCFH-DA is able to detect the heme moiety of hemeprotein in vivo.

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.

Structured summary of protein interactionsClock and Bmal1colocalize by fluorescence microscopy (View interaction)Npas2 and Bmal1colocalize by fluorescence microscopy (View interaction)