It is widely accepted that cAMP regulates gene transcription principally by activating the protein kinase A (PKA)-targeted transcription factors. Here, we show that cAMP enhances the generation of 5-hydroxymethylcytosine (5hmC) in multiple cell types. 5hmC is converted from 5-methylcytosine (5mC) by Tet methylcytosine dioxygenases, for which Fe(II) is an essential cofactor. The promotion of 5hmC was mediated by a prompt increase of the intracellular labile Fe(II) pool (LIP). cAMP enhanced the acidification of endosomes for Fe(II) release to the LIP likely through RapGEF2. The effect of cAMP on Fe(II) and 5hmC was confirmed by adenylate cyclase activators, phosphodiesterase inhibitors, and most notably by stimulation of G protein-coupled receptors (GPCR). The transcriptomic changes caused by cAMP occurred in concert with 5hmC elevation in differentially transcribed genes. Collectively, these data show a previously unrecognized regulation of gene transcription by GPCR-cAMP signaling through augmentation of the intracellular labile Fe(II) pool and DNA hydroxymethylation.

Epigenetic variation reflects the impact of a dynamic environment on chromatin. However, it remains elusive how environmental factors influence epigenetic events. Here, we show that G protein-coupled receptors (GPCRs) alter H3K4 methylation via oscillatory intracellular cAMP. Activation of Gs-coupled receptors caused a rapid decrease of H3K4me3 by elevating cAMP, whereas stimulation of Gi-coupled receptors increased H3K4me3 by diminishing cAMP. H3K4me3 gradually recovered towards baseline levels after the removal of GPCR ligands, indicating that H3K4me3 oscillates in tandem with GPCR activation. cAMP increased intracellular labile Fe(II), the cofactor for histone demethylases, through a non-canonical cAMP target-Rap guanine nucleotide exchange factor-2 (RapGEF2), which subsequently enhanced endosome acidification and Fe(II) release from the endosome via vacuolar H+-ATPase assembly. Removing Fe(III) from the media blocked intracellular Fe(II) elevation after stimulation of Gs-coupled receptors. Iron chelators and inhibition of KDM5 demethylases abolished cAMP-mediated H3K4me3 demethylation. Taken together, these results suggest a novel function of cAMP signaling in modulating histone demethylation through labile Fe(II).

Epigenetic variation reflects the impact of a dynamic environment on chromatin. However, it remains elusive how environmental factors influence epigenetic events. Here, we show that G protein-coupled receptors (GPCRs) alter H3K4 methylation via oscillatory intracellular cAMP. Activation of Gs-coupled receptors caused a rapid decrease of H3K4me3 by elevating cAMP, whereas stimulation of Gi-coupled receptors increased H3K4me3 by diminishing cAMP. H3K4me3 gradually recovered towards baseline levels after the removal of GPCR ligands, indicating that H3K4me3 oscillates in tandem with GPCR activation. cAMP increased intracellular labile Fe(II), the cofactor for histone demethylases, through a non-canonical cAMP target-Rap guanine nucleotide exchange factor-2 (RapGEF2), which subsequently enhanced endosome acidification and Fe(II) release from the endosome via vacuolar H⁺-ATPase assembly. Removing Fe(III) from the media blocked intracellular Fe(II) elevation after stimulation of Gs-coupled receptors. Iron chelators and inhibition of KDM5 demethylases abolished cAMP-mediated H3K4me3 demethylation. Taken together, these results suggest a novel function of cAMP signaling in modulating histone demethylation through labile Fe(II).