The Quinone reductase 2 (QR2) enzyme functions as a dedicated reader of histone H3 glutamine 5 serotonylation (H3Q5ser) through FAD-dependent molecular recognition involving π-π stacking and hydrophobic interactions, and this chromatin binding inhibits QR2's enzymatic activity while facilitating allosteric recruitment of the transcription factor POU2F1 to regulate chromatin accessibility and gene expression programs essential for human neuronal maturation, including morphological development, synaptic connectivity, and electrophysiological function.

Histone H3 Gln5 serotonylation (H3Q5ser) is a recently described posttranslational modification that plays important roles in guiding transcriptional permissiveness in brain and peripheral systems. H3Q5ser has been implicated in diverse physiological and pathological processes ranging from neural differentiation to sensory processing, circadian rhythmicity, stress responsivity, placental gene regulation, and tumorigenesis. Since H3Q5ser can occur in combination with H3 Lys4 trimethylation (H3K4me3), most mechanistic studies to date have focused on H3Q5ser’s roles in modulating H3K4me3 reader interactions, where it has been shown to potentiate TAF3/TFIID binding to H3K4me3 and inhibit the recruitment of K4me3 demethylases; however, whether H3 serotonylation functions as an autonomous chromatin signaling mark through dedicated reader proteins has remained unknown. Here, using a combination of proteomic-, structural-, molecular-, epigenomic-, and cellular-based approaches, we demonstrate that the Quinone reductase 2 (QR2) enzyme reads H3Q5ser independently of H3K4me3. CRISPR-Cas9-mediated disruption of H3 serotonylation or QR2’s binding to the mark in human induced pluripotent stem cell-derived neurons impairs the establishment of neuronal transcriptional programs, alters synaptic connectivity, and disrupts electrophysiological maturation. These findings thus uncover an H3 serotonylation-dependent chromatin signaling axis that is essential for human neurodevelopment.