Mechanisms regulating the formation and repair of neuronal activity-induced DNA breaks in vivo and their effects on chromatin architecture and neuronal physiology

Ut Southwestern Medical Center, Dallas TX

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Abstract

Project Summary Exposure to new sensory experiences activates new gene transcription programs in neurons and the products of these genes mediate the development of lasting adaptive behaviors. Defects in neuronal activity-dependent transcription programs manifest in neurodevelopmental diseases, intellectual disability, and autism spectrum disorders. Understanding how neuronal activity-dependent transcription is regulated is therefore significant. An intriguing finding in this regard is that neuronal activity induces the DNA topoisomerase, Top2B, to form DNA double strand breaks (DSBs) within the promoters of key early response genes (ERGs), such as Fos, Npas4, and Egr1, and that DSB formation in this manner facilitates the rapid transcription of these ERGs. Yet precisely how neuronal activity-induced DSBs are orchestrated to occur at specific positions, how they facilitate the transcription of associated genes, and how activity-induced DSBs are repaired are all poorly understood. To address these issues, various signaling pathways that affect synapse-to-nucleus communication in cultured mouse cortical neurons were perturbed. These efforts identified that activity-induced DSB formation is controlled by the phosphatase, calcineurin, which dephosphorylates Top2B and induces it to form DSBs upon neuronal stimulation. These signaling events are spatially compartmentalized to occur at the nuclear periphery and sites that incur activity-induced DSBs also preferentially localize to the nuclear periphery. Based on this, proposed experiments will employ high-resolution imaging and biochemical methods to determine whether similar molecular events also govern DSB formation in the hippocampus following relevant physiological stimulation and how radial gene position affects the recruitment of neurons into functional ensembles. Preliminary data suggest that while calcineurin regulates Top2B at the nuclear periphery, additional mechanisms preclude Top2B from forming DSBs at ectopic sites. Proposed experiments will decipher these mechanisms. Chromosome conformation capture experiments indicate that DSBs are necessary and sufficient to stimulate enhancer- promoter contacts at ERGs. Moreover, recurrent DSB formation progressively potentiate ERG transcription by pruning interactions between ERG promoters and heterochromatin while relatively stabilizing interactions with enhancers. Recent reports suggest that DSB repair mechanisms drive chromatin reorganization at DSB sites, yet exactly how activity-induced DSBs are repaired is unknown. Preliminary data suggest that the enzyme, TDP2, catalyzes the repair of activity-induced DSBs and the proposed experiments will investigate how TDP2 loss affects activity-dependent gene transcription and chromosome organization during recurrent stimulation of hippocampal neurons in vivo. Mutations in TDP2 cause the disease, SCAR23, which is characterized by intellectual disability and seizures, but the underlying mechanisms are unknown. Ablating Tdp2 increased the duration of UP states in acute cortical slices and the proposed experiments will use electrophysiology and assess intrinsic neuronal excitability and synaptic function to determine how TDP2 loss affects neuronal function.