Antecedent enhancer activity predicts future susceptibility to seizures in mice

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, 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 novel 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. Calling Cards viral reagents were injected transcranially into neonatal mouse brains. At one month of age, acute seizures were induced with PTZ and brains were collected. Cortical tissue was split into up to six pieces, from which bulk Calling Cards libraries were prepared independently. Sample names contain animal ID as the first two integers, followed by tissue piece number. e.g. CT14-5_piece_2 indicates animal CT14-5, tissue piece 2.