Monday, April 29, 2024
4/29/2024
PROJECT SUMMARY Type II topoisomerases (Top2) alter chromatin topology by forming transient double-strand breaks (DSBs) in the DNA, allowing a second DNA segment to pass through the DSB, and then rejoining the broken strands. Mutations in the topoisomerase Top2B have been linked to neurodevelopmental delay and autism in humans. Studies in mice have shown that Top2B regulates the transcription of developmentally regulated and neuronal activity- dependent genes. A key mechanism by which Top2B regulates gene expression in neurons is by forming long- lasting stimulus-dependent DSBs within the promoters of specific genes. However, how Top2B is regulated is unknown. The proposed research attempts to bridge these knowledge gaps. Recently we published that in response to neuronal stimulation, calcium influx through NMDA receptors activates the phosphatase calcineurin, to dephosphorylate Top2B at residues S1509 and S1511 within its C-terminal domain (CTD), which induces Top2B to form DSBs promoting the transcription of immediate early genes (IEGs). Exposing mice to a fear learning paradigm also triggers DSB formation and Top2B dephosphorylation at S1509 and S1511 in the hippocampus. Additional to these dephosphorylations, we observed other phosphorylation changes at the CTD induced by neuronal activity that we have yet to study. Together, these observations provide the first insights into the regulation of Top2B by its CTD. However, since the CTD is not part of the catalytic domains and is not necessary for Top2B relaxation activity in vitro, it is unknown how the CTD regulates the activity of the catalytic core in response to neuronal activity. It is critical to study how the CTD regulates Top2B because mutations at the CTD cause late-onset sensorineural hearing loss in humans. The proposed research will use CRISPR on the auditory neuroblast cell line US/VOT-N33 (N33) and/or the sensorineural HEI-OC1, to produce cell lines with the mutations reported in patients (D1613H and K1435del) or without the serine residues that undergo dynamic changes in phosphorylation in response to neuronal activity. The cells will be differentiated into sensory hair cells or ganglion neurons and the effects of mutations on the formation of DSBs, and the regulation of IEG transcription will be assessed. Then we will purify Top2B from the differentiated cells to study relaxation, cleavage, and decatenation activity. We will also study the effect of the mutation on protein-protein interaction by coprecipitation and mass spectrometry. The allosteric effects of the CTD on the catalytic core of Top2B will be examined using cross-linking mass spectrometry. Finally, we will assess the distribution by subcellular fractionation and immunocytochemistry, and genome-wide distribution by ChIP of active Top2B. Together, these efforts will illuminate how Top2B is regulated by the CTD in response to stimulation and address how the mutations that cause hearing loss impair this regulation, which is relevant to understand this disease.
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