Abstract
Histone deacetylases (HDACs) modulate chromatin structure by removing acetyl groups from histones. Upon DNA double-strand breaks (DSBs), deacetylation of H3K56 and H4K16 by HDACs occurs immediately at break sites, and is crucial for DSB repair. Here we describe two assays that examine defective DSB repair caused by HDAC inhibition in primary cortical neurons: single-cell gel electrophoresis to assay DNA integrity (the comet assay) and western blot analysis for γH2AX, a phosphorylated histone variant associated with DSBs.
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References
Cerna D, Camphausen K, Tofilon PJ (2006) Histone deacetylation as a target for radiosensitization. Curr Top Dev Biol 73:173–204. https://doi.org/10.1016/S0070-2153(05)73006-4
Miller KM, Tjeertes JV, Coates J, Legube G, Polo SE, Britton S, Jackson SP (2010) Human HDAC1 and HDAC2 function in the DNA-damage response to promote DNA nonhomologous end-joining. Nat Struct Mol Biol 17(9):1144–1151. https://doi.org/10.1038/nsmb.1899
Dobbin MM, Madabhushi R, Pan L, Chen Y, Kim D, Gao J, Ahanonu B, Pao PC, Qiu Y, Zhao Y, Tsai LH (2013) SIRT1 collaborates with ATM and HDAC1 to maintain genomic stability in neurons. Nat Neurosci 16(8):1008–1015. https://doi.org/10.1038/nn.3460
Wang WY, Pan L, Su SC, Quinn EJ, Sasaki M, Jimenez JC, Mackenzie IR, Huang EJ, Tsai LH (2013) Interaction of FUS and HDAC1 regulates DNA damage response and repair in neurons. Nat Neurosci 16(10):1383–1391. https://doi.org/10.1038/nn.3514
Kim D, Frank CL, Dobbin MM, Tsunemoto RK, Tu W, Peng PL, Guan JS, Lee BH, Moy LY, Giusti P, Broodie N, Mazitschek R, Delalle I, Haggarty SJ, Neve RL, Lu Y, Tsai LH (2008) Deregulation of HDAC1 by p25/Cdk5 in neurotoxicity. Neuron 60(5):803–817. https://doi.org/10.1016/j.neuron.2008.10.015
Thurn KT, Thomas S, Raha P, Qureshi I, Munster PN (2013) Histone deacetylase regulation of ATM-mediated DNA damage signaling. Mol Cancer Ther 12(10):2078–2087. https://doi.org/10.1158/1535-7163.MCT-12-1242
Fairbairn DW, Olive PL, O'Neill KL (1995) The comet assay: a comprehensive review. Mutat Res 339(1):37–59
Olive PL, Banath JP (2006) The comet assay: a method to measure DNA damage in individual cells. Nat Protoc 1(1):23–29. https://doi.org/10.1038/nprot.2006.5
Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ (2001) ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem 276(45):42462–42467. https://doi.org/10.1074/jbc.C100466200
Gyori BM, Venkatachalam G, Thiagarajan PS, Hsu D, Clement MV (2014) OpenComet: an automated tool for comet assay image analysis. Redox Biol 2:457–465. https://doi.org/10.1016/j.redox.2013.12.020
Sciarretta C, Minichiello L (2010) The preparation of primary cortical neuron cultures and a practical application using immunofluorescent cytochemistry. Methods Mol Biol 633:221–231. https://doi.org/10.1007/978-1-59745-019-5_16
Acknowledgments
This work was supported by NIA grant (AG046174), NINDS grant (NS102730), and Glenn award for research in biological mechanism of aging.
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Pao, PC., Penney, J., Tsai, LH. (2019). Examining the Role of HDACs in DNA Double-Strand Break Repair in Neurons. In: Brosh, Jr., R. (eds) Protein Acetylation. Methods in Molecular Biology, vol 1983. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9434-2_13
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DOI: https://doi.org/10.1007/978-1-4939-9434-2_13
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