![]() ![]() ![]() Gene ontology analysis revealed a significant enrichment of biological processes related to synaptic transmission and synaptic function within loci that incur DSBs in the hippocampus following CFC. Interestingly however, we also observed DSB accumulation at many new loci that were not detected in cultured primary neurons. Results: Our ChIP-seq studies in vivo recapitulated our previous observations in cultured primary neurons, and indicated that physiological learning behaviors also cause DSB formation within the promoters of neuronal IEGs. Finally, molecular mechanisms that regulate the formation of Top2B-mediated DSBs were investigated through a combination of targeted mass spectrometry, mutagenesis of Top2B, and imaging-based assays to detect the formation of stimulus-induced DSBs in cultured primary neurons. To understand whether the formation of activity-induced DSBs affects learning behaviors, two month-old male Top2bCKO mice (12 animal per group) were subjected to various behavioral paradigms, including open-field and light-dark tests, object recognition and object location tasks, and contextual and cued fear conditioning tests. The resultant Top2bCKO mice were subject to CFC at 8 weeks of age, following which hippocampal lysates were prepared and γH2AX levels were assessed by western blotting. To understand whether DSBs formed in vivo are also a result of Top2B activity, conditional Top2bf/f mice were crossed with CaMKIIα-Cre mice, which causes for the deletion of Top2b from excitatory forebrain neurons in adult mice. We have obtained a mouse model (Top2bf/f mice) in which the expression of Cre recombinase allows for the conditional deletion of endogenous Top2b. Methods: To assess whether stimulus-induced DSBs are also formed at specific genomic loci in vivo, we subjected two-month old C57BL/6 mice to a contextual fear conditioning (CFC) paradigm, following which we dissected the hippocampi and performed ChIP-seq with antibodies against the DSB marker, γH2AX. ![]() Together, these results raise intriguing questions about the mechanisms that regulate the formation of stimulus-induced DSBs at specific genomic loci and whether the formation of these DSBs has a role in neuronal functions, including in the development of adaptive behaviors. These activity-induced DSBs are generated by the type II topoisomerase, topoisomerase IIβ (Top2B), and we showed surprisingly that Top2B-mediated DSBs facilitate the rapid induction of these aforementioned IEGs. ![]() Recently, we reported that activity-dependent stimulation of cultured primary neurons triggers the formation of DNA double strand breaks (DSBs) in the promoters of a subset of immediate early genes, including Fos, Npas4, and Egr1. While immediate early genes are primed for rapid induction, the specific impediments to their expression under basal conditions, and the mechanisms that relieve these constraints are still poorly understood. University of Texas Southwestern Medical Center, Dallas, Texas, United Statesīackground: Neuronal activity triggers the rapid expression of immediate early genes that play important roles in experience-driven synaptic changes, learning, and memory. ![]()
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