Abstract
Boros BD, Gachechiladze MA, Guo J, Galloway DA, Mueller SM, Shabsovich M, Yen A, Chen X, Cammack AJ, Shen T, Mitra RD, Dougherty JD, Miller TM. Nat Commun. 2026, Jan 8;17(1):300. https://doi.org/10.1038/s41467-025-65346-2 Wide variation of responses to identical stimuli presented to genetically inbred mice suggests the hypothesis that stochastic non-genetic variation, such as in chromatin state or enhancer activity during neurodevelopment, can mediate such phenotypic differences. However, this hypothesis is largely untested since capturing pre-existing molecular states requires non-destructive, longitudinal recording. Therefore, we tested the potential of Calling Cards (CC) to record transient neuronal enhancer activity during postnatal development in mice, and thereby associate such non-genetic variation with a subsequent phenotypic presentation – degree of seizure response to the pro-convulsant pentylenetetrazol. We show that recorded differences in enhancer activity at 243 loci predict a severe vs. mild response, and that these are enriched near genes associated with human epilepsy. We also validated pharmacologically a seizure-modifying role for two previously unassociated genes, Htr1f and Let7c. This proof-of-principle supports using CC broadly to discover predisposition loci for other neuropsychiatric traits and behaviors. Finally, as human disease is also influenced by non-genetic factors, similar epigenetic predispositions are possible in humans.
Commentary
Epilepsy is a heterogeneous condition with many genetic and environmental etiologies. 1 Even among monogenic forms of epilepsy, patients with the same primary mutations can experience vastly different clinical courses. 2 In addition, large-scale genetic studies have identified numerous genetic variants that individually do not result in monogenic epilepsy but appear to contribute to risk of epilepsy development and/or severity.3,4 Environmental exposures (eg, infections, head trauma, sleep deprivation, and fever, among others) can also provoke seizures in some individuals but not others, with a fraction ultimately developing epilepsy. 1 Mouse models of epilepsy also exhibit strain-dependent seizure susceptibility, differing significantly in seizure threshold and severity, mortality, and downstream neuropathological changes depending on genetic background. 5 Even effectively isogenic mice exhibit significant variability in seizure response to chemoconvulsant treatment. 6 Despite these well-documented phenomena, underlying drivers of differential susceptibility to seizure induction and epilepsy development remain poorly understood, especially in the context of isogenic mice, where genetics and environment are ostensibly identical between mice with widely varying responses to seizure induction.
In this study, Boros and colleagues 7 address the possibility of non-genetic, non-environmental factors that influence seizure susceptibility by studying the antecedent epigenetic state of isogenic mice receiving intraperitoneal pentylenetetrazol (PTZ) at a dose of 65 mg/kg. This dose was carefully selected to achieve generalized tonic-clonic activity (modified Racine score of 5) in approximately 50% of mice, allowing for adequate sample sizes of mice with mild (defined as modified Racine scale < 5) and severe (modified Racine scale > 5) seizure response. To characterize the epigenetic landscape preceding seizure induction, Boros et al utilized “Calling Cards” (CC),8,9 a transposon-transposase-based technology that creates a permanent record of transient transcription factor-DNA interactions. They used this technology to indirectly label active enhancer regions by leveraging hyperPiggyBac, a transposase with natural affinity for BRD4 binding sites. As BRD4 is a histone acetylation reader with preferential binding at active enhancers and promoters, hyperPiggyBac inserting donor self-reporting transposons at BRD4 binding sites effectively, permanently, and cumulatively tags these sites as active enhancer regions that can be identified via sequencing of CC libraries. This strategy allowed for cumulative characterization of active enhancer sites in neurons during early development, from P0-1 (when AAV viral vectors containing Cre-dependent transposase and barcoded self-reporting transposons were infused into cortex of mice expressing pan-neuronal Cre) to PTZ induction at P28. Mice were monitored for 15 min following PTZ induction—long enough to stratify seizure responses via a modified Racine scale, but not long enough for seizures to influence CC insertion and expression. This latter point was confirmed in vitro, where transposition in a cell line constitutively expressing transposase was only detectable by 2-5 h following electroporation with a transposon construct.
By stratifying mice into mild and severe seizure responders and characterizing the preceding epigenomic landscape in these mice, this study identified 110 regions (corresponding to 326 nearby genes) enriched for CC binding in mild responders and 133 regions (corresponding to 381 nearby genes) in severe responders. Importantly, these gene sets (independently and in combination) significantly overlapped with existing epilepsy-related gene lists, as well as intellectual disability and autism gene lists, but not a Parkinson's disease gene list or gene lists associated with disease states unrelated to the nervous system. These results provide external validity to the CC method used here to identify modifiers of seizure response. Notably, many CC-identified loci did not contain known epilepsy genes, offering new potential targets for disease-modifying therapeutics. To demonstrate the therapeutic promise of targeting these newly identified genes, and to improve internal validity, the authors used pharmacology or antisense oligonucleotide-mediated reduction of expression to manipulate candidate targets enriched in either mild or severe responders. Indeed, the authors were able to push and pull on seizure latency and/or severity by manipulating these newly identified gene targets, demonstrating that this approach identifies relevant epilepsy-related targets.
This study ultimately addressed a crucial but often overlooked question in mouse research: How can isogenic mice raised in the same environment respond so differently to different experimental manipulations? One answer, beautifully illustrated in this article, is stochastic epigenetic changes during development. Crucially, the authors addressed one aspect of the epigenome—sites of active enhancers, proxied by BRD4 binding (which the authors concede is imperfect, as BRD4 can also act as a repressor, depending on context). 10 Further investigations should probe other aspects of the epigenome, such as DNA methylation and other histone modifications, perhaps by fusing the transposase used in the CC method to particular DNA-methylating enzymes, histone-modifying proteins, or transcription factors to determine how stochastic differences in these aspects of the epigenome can affect seizure susceptibility. By combining CC technology with other genetic techniques, such as neuronal subtype or glia-specific Cre mouse lines, or by using inducible-Cre mouse lines, future studies can target the epigenomic landscape with more spatiotemporal precision to determine when and what cell types have the most epigenetic influence on seizure susceptibility.
However, potentially the most exciting aspect of this article is the proof-of-concept that CC technology can be used in vivo to detect meaningful differences in epigenomic landscape between otherwise isogenic individuals that ultimately result in differing phenotypic responses, demonstrating both the power of CC technology itself and of epigenetic changes during development to ultimately influence behavioral outcomes. This breakthrough comes nearly two decades after the first description of CC technology in yeast 8 and clearly shows the influence that epigenetic changes can exert on seizure susceptibility. With the sequencing revolution of the 21st century, genomic sequencing was made accessible and affordable, resulting in breakthroughs in our understanding of genetic influence on a myriad of disease states, including epilepsy. With wide application of CC technology, we can more effectively and precisely target the epigenome as an important regulator of seizure susceptibility and epilepsy development, and potentially unlock new therapeutic avenues to effectively treat epilepsy.
