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Dynamic Behavior of the RNA Polymerase II and the Ubiquitin Proteasome System During the Neuronal DNA Damage Response to Ionizing Radiation

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Abstract

Neurons are highly vulnerable to genotoxic agents. To restore genome integrity upon DNA lesions, neurons trigger a DNA damage response (DDR) that requires chromatin modifications and transcriptional silencing at DNA damage sites. To study the reorganization of the active RNA polymerase II (Pol II), which transcribes all mRNA-encoding genes, and the participation of the ubiquitin-proteasome system (UPS) in the neuronal DDR, we have used rat sensory ganglion neurons exposed to X-rays (4 Gy) ionizing radiation (IR). In control neurons, Pol II appears concentrated in numerous chromatin microfoci identified as transcription factories by the incorporation of 5′-fluorouridine into nascent RNA. Upon IR treatment, numerous IR-induced foci (IRIF), which were immunoreactive for γH2AX and 53BP1, were observed as early as 30 min post-IR; their number progressively reduced at 3 h, 1 day, and 3 days post-IR. The formation of IRIF was associated with a decrease in Pol II levels by both immunofluorescence and Western blotting. Treatment with the proteasome inhibitor bortezomib strongly increased Pol II levels in both control and irradiated neurons, suggesting that proteasome plays a proteolytic role by clearing stalled Pol II complexes at DNA damage sites, as a prelude to DNA repair. Neuronal IRIF recruited ubiquitylated proteins, including ubiquitylated histone H2A (Ub-H2A), and the catalytic proteasome 20S. Ub-H2A has been associated with transcriptional silencing at DNA damage sites. On the other hand, the participation of UPS in neuronal DDR may be essential for the ubiquitylation of Pol II and other proteasome substrates of the DNA repair machinery and their subsequent proteasome-mediated degradation.

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Acknowledgments

This work was supported by the following grants: “Dirección General de Investigación” of Spain (BFU2011-23983; BFU2014-54754-P) and “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED; CB06/05/0037)” from Spain.

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    Authors and Affiliations

    1. Department of Anatomy and Cell Biology and “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)”, University of Cantabria-IDIVAL, Cardenal Herrera Oria s/N, Santander, 39011, Spain

      Iñigo Casafont, Ana Palanca, Jorge Mata-Garrido, Maria T. Berciano & Miguel Lafarga

    2. Laboratorio de Inestabilidad Genómica, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain

      Vanesa Lafarga

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    1. Iñigo Casafont
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    2. Ana Palanca
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    5. Maria T. Berciano
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    Correspondence to Miguel Lafarga.

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    All procedures were approved by the Bioethical Committee of the University of Cantabria and were carried out according to the directives of the Council of the European Communities and current Spanish legislation.

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    Iñigo Casafont and Ana Palanca contributed equally to this work.

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    Casafont, I., Palanca, A., Lafarga, V. et al. Dynamic Behavior of the RNA Polymerase II and the Ubiquitin Proteasome System During the Neuronal DNA Damage Response to Ionizing Radiation. Mol Neurobiol 53, 6799–6808 (2016). https://doi.org/10.1007/s12035-015-9565-8

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