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“Lesser-appreciated” Histone Modifications - Monoaminylation, the CLOCK factor & Neuronal Gene Expression

By Stuart P. Atkinson, Ph.D.

July 29, 2025

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In Support of “Lesser-appreciated” Histone Modifications

Histone methylation, acetylation, and ubiquitination represent relatively well-understood post-translational modifications; a wide range of studies have explored their dynamic regulation by “writers” and “eraser” enzymes, their recognition by “reader” modules, and have begun to understand their impact on the three-dimensional architecture of chromatin and gene transcription. Monoaminylation modifications of histone H3 Gln5 generate a family of more recently appreciated epigenetic modifications that play crucial roles in gene expression in the brain (Girault 2022 and Al-Kachak & Maze); however, they remain less well-described than some of their more “illustrious” epigenetic predecessors.

Researchers from the laboratories of Haitao Li, Yael David, and Ian Maze have published a plethora of exciting studies describing how transglutaminase 2 (TG2) catalyzes the serotonylation (H3Q5ser) and dopaminylation (H3Q5dop) of Histone H3 Gln5 to alter local and global chromatin states and regulate gene expression (Lorand & Graham and Fesus & Piacentini). Their subsequent research, published recently in Nature, sought to define the enzymatic regulatory mechanisms controlling monoaminylation dynamics and exactly how they impact gene transcription in neurons in the brain (Zheng et al.), given the central role of serotonin and dopamine as monoamine neurotransmitters. In doing so, the team discovered that histamine - which also plays neuromodulatory roles (Tiligada & Ennis) - serves as a donor for histone H3Q5 monoaminylation, with the resultant modification (H3Q5his) displaying rhythmic expression patterns and contributing toward the regulation of circadian gene expression and the modulation of mouse behavior.

Of note, this exciting new study took full advantage of the wealth of epigenetic products available from Active Motif; the products involved in this study included recombinant MLL1, MLL2, MLL3, MLL4, SETD1A, and SETD1B proteins, reconstituted unmodified and K4me3-modified mononucleosomes, and an anti-H3K4me2 antibody.

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TG2: An Epigenetic Writer and Eraser that Regulates H3 Monoaminylation

Initial experiments in human cancer cells that tracked histone serotonylation identified TG2 as an H3 monoaminylation writer and eraser, while subsequent experiments employing modified peptides confirmed TG2 as an H3 monoaminylation exchanger that possessed the ability to erase serotonin/dopamine modification via nucleophilic attack (supporting the formation of another monoaminylation adduct or monoamine removal). An examination of an additional monoamine species - histamine (Tiligada & Ennis) - as a donor for histone H3Q5 monoaminylation supported the existence of TG2-mediated addition of histamine to H3 peptides at Gln5 (H3Q5his; which agrees well with previous studies (Lai & Greenberg); furthermore, TG2 converted H3Q5his to H3Q5ser or H3Q5dop on modified peptides in the presence of serotonin or dopamine. Additional analysis of H3Q5his dynamics and regulation by TG2 via in vitro competition assays with reconstituted nucleosome core particles suggested that TG2 could regulate nucleosomal H3 monoaminylation in a physiologically relevant context.

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H3Q5his Antagonizes WDR5 Binding and H3K4 Methylation

Previous studies revealed that H3Q5ser influences the deposition, maintenance, and readout of the transcriptionally permissive H3K4me3 modification (Farrelly et al. and Zhao et al.). An investigation of the functional consequences of the presence of H3Q5his compared to H3Q5ser in this sense revealed the differential binding of the K4me3 reading domain of WDR5-WD40 (a core member of the H3K4 methyltransferase complexes MLL1–4 and SETD1A/B). While H3Q5ser favored interactions, unmodified H3 and H3Q5his inhibited interactions and, as such, decreased the H3K4 methyltransferase activity of MLL1-4 and SETD1A/B. Immunoprecipitation assays employing recombinant WDR5 or a reconstituted MLL1 complex demonstrated the ability of H3Q5ser/H3K4me3/H3K4me3Q5ser to potentiate and H3Q5his to reduce interactions with H3. CUT&RUN-seq with antibodies recognizing H3Q5his in the presence or absence of H3K4me3 or the combination of H3K4me3Q5his demonstrated concordant genome-wide enrichment patterns, with more than 85% of peaks identified as lying within genic regions.

Rhythmic Patterns of Neuronal H3Q5 Monoaminylation

In vivo analysis of the hypothalamic tuberomammillary nucleus (TMN) in mice revealed enrichment for H3Q5his compared to other non-histaminergic brain nuclei. Only the TMN contains neurons expressing the enzyme that catalyzes the formation of histamine (Lin et al.), which is known for its ability to control arousal and maintain sleep-wake cycles and energy balance (Fujita et al.). RNA-seq suggested that the TMN displays rhythmic patterns of CLOCK-associated gene expression (including known circadian genes) that require chromatin-based control (the CLOCK transcription factor represents a master mediator of circadian gene expression (King et al.)); in agreement, H3Q5his and H3K4me3Q5his displayed concordant rhythmic fluctuations in levels - high during active phases and low during inactive phases.

Further dissection of how these modifications regulate circadian gene expression employed CUT&RUN-seq for H3K4me3Q5his, additional related modifications, and WDR5, which confirmed H3K4me3Q5his rhythmicity in the TMN (high during active and low during inactive phases) and revealed overlap between circadian genes in the TMN and those genes displaying rhythmic fluctuations in H3K4me3Q5his. Comparing rhythmic patterns of H3K4me3Q5his to H3K4me3Q5ser (also circadian in the TMN) revealed that both patterns displayed enrichment during the peak of the active phase, but only H3K4me3Q5ser displayed a marked reduction when nearing the end of the active phase. H3K4me3Q5ser loss corresponded to WDR5 loss, with WDR5 enrichment during the active phase observed at permissive CLOCK–BMAL1-target genes induced during the mouse active phase (Takahashi, 2017).

Finally, CUT&RUN–seq assessments in the mouse TMN after the chemical perturbation of diurnal rhythms revealed reduced levels of H3K4me3Q5his, H3K4me3Q5ser, and WDR5 genome-wide (including at CLOCK–BMAL1-target genes); this result phenocopies the molecular regulation of these marks/proteins during inactive phases.

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Does H3Q5 monoaminylation contribute to rhythmicity?

Exploring the causal roles of H3Q5 monoaminylation in regulating molecular and/or behavioral rhythmicity employed viral transduction of the TMN to introduce a mutant histone that does not support H3 monoaminylation. RNA-seq analysis after 3 weeks revealed the disruption of circadian transcriptional regulation with disrupted genes displaying an enrichment for ontologies related to CLOCK-mediated transcription. Further analyses indicate prominent roles in circadian entrainment, neurotrophin signaling, and synaptic regulation. Finally, and perhaps most interestingly, perturbing H3Q5 monoaminylation in the TMN shifted diurnal locomotor activity in mice, particularly during transitions from inactive to active states, and vice versa.

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TG2, H3Q5his, and Neuronal Plasticity and Rhythmicity

Overall, these data provide evidence for the multifunctional chromatin regulatory nature of TG2 – catalyzing the addition and removal of histone monoaminylation modifications according to the presence of relevant factors in the microenvironment – and the importance of the H3Q5his modification in regulating the gene expression profiles that underly neuronal plasticity and rhythmicity.

What´s next in the quest to fully appreciate this epigenetic regulatory mechanism? The authors suggest the exploration of events related to rhythmicity that remain independent of WDR5 binding and the development of novel tools to study the detailed role of TG2’s genomic interactions in the brain and to accurately and simultaneously quantify H3 monoaminylation modifications within a given cell population/tissue.

For more on how histone H3 monoaminylation (and H3Q5his in particular) dynamics influences gene expression in the brain, see Nature, January 2025.

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About the author

Stuart P. Atkinson, Ph.D.

Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!

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