Rev-erbα (NR1D1) and Rev-erbβ (NR1D2) are heme sensors and atypical members of the nuclear receptor superfamily. Many of the Rev-erbα target genes play important roles in regulation of metabolic processes. In addition, Rev-erbα regulates the expression of proteins that comprise the core circadian clock in mammals. GSK4112 is a functional Rev-erbα agonist in liver cells to inhibit expression of the circadian target gene bmal1 with EC50 value of 0.4 μM. In the presence of 0.5 μM hemin, recruitment of the NCoR1 ID1 peptide was reduced. In contrast, 10 μM GSK4112 increased the interaction of the receptor with the peptide above the basal level, consistent with the data from the chemical array screening. Following saturation of the receptor with hemin, the tertiary amine GSK4112 was able to reverse the suppression of the FRET (fluorescence resonance energy transfer) signal. The competitive actions of hemin and GSK4112 in the FRET assay strongly suggested that their binding as ligands to Rev-erbα was mutually exclusive. In the presence of 10 μM GSK4112, a statistically significant repression of bmal1 expression was observed. The mRNA for g6pc, pepck, and pgc1a were all repressed by 10 μM GSK4112 in HepG2 cells. A ChIP experiment in the same cells showed that a doubling of HDAC3 recruitment was observed in the presence of GSK4112 demonstrating that GSK4112 performed as a Rev-erbα agonist in cells to promote the formation of a transcriptional repressor complex with HDAC3 and repress target genes associated with the pathway of gluconeogenesis[3].
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