Nucleolin Participates in DNA Double-Strand Break-Induced Damage Response through MDC1-Dependent Pathway

Junya Kobayashi,Hiroko Fujimoto,Jun Sato,Sandeep Burma,Shinya Matsuura,David J Chen,Ikue Hayashi,Kenshi Komatsu,Thomas G Hofmann

doi:10.1371/journal.pone.0049245

Abstract

H2AX is an important factor for chromatin remodeling to facilitate accumulation of DNA damage-related proteins at DNA double-strand break (DSB) sites. In order to further understand the role of H2AX in the DNA damage response (DDR), we attempted to identify H2AX-interacting proteins by proteomics analysis. As a result, we identified nucleolin as one of candidates. Here, we show a novel role of a major nucleolar protein, nucleolin, in DDR. Nucleolin interacted with γ-H2AX and accumulated to laser micro-irradiated DSB damage sites. Chromatin Immunoprecipitation assay also displayed the accumulation of nucleolin around DSB sites. Nucleolin-depleted cells exhibited repression of both ATM-dependent phosphorylation following exposure to γ-ray and subsequent cell cycle checkpoint activation. Furthermore, nucleolin-knockdown reduced HR and NHEJ activity and showed decrease in IR-induced chromatin accumulation of HR/NHEJ factors, agreeing with the delayed kinetics of γ-H2AX focus. Moreover, nucleolin-knockdown decreased MDC1-related events such as focus formation of 53 BP1, RNF168, phosphorylated ATM, and H2A ubiquitination. Nucleolin also showed FACT-like activity for DSB damage-induced histone eviction from chromatin. Taken together, nucleolin could promote both ATM-dependent cell cycle checkpoint and DSB repair by functioning in an MDC1-related pathway through its FACT-like function.

Highlights

DNA double-strand breaks (DSBs) are often generated in genomic DNA upon exposure to ionizing radiation, DNA damaging agents such as bleomycin and neocarzinostatin, or due to the stalling or collapse of DNA replication forks
We report here that nucleolin participates in the DNA doublestrand break-induced DNA damage response, via MDC1-dependent pathway
We indentify nucleolin as an H2AXinteracting protein and show that nucleolin can accumulate at DSB damage sites by laser micro-irradiation and Chromatin Immunoprecipitation (ChIP) analysis (Fig. 2 and S2)

Summary

Introduction

DNA double-strand breaks (DSBs) are often generated in genomic DNA upon exposure to ionizing radiation, DNA damaging agents such as bleomycin and neocarzinostatin, or due to the stalling or collapse of DNA replication forks. The yeast Ino and Swr chromatin-remodeling complexes interact with the phosphorylated form of histone H2AX (cH2AX) and facilitate DSB repair [8,9,10] Wu and his colleagues showed that knockdowns of human INO80 or a binding partner, YY1, increased cellular sensitivity toward DNA-damaging agents, and that both INO80 and YY1 are essential for HR repair [11]. Downregulation of Brg, a component of human SWI/SNF1 complex, results in inefficient DSB repair and increased DNA damage sensitivity [12] These and additional reports suggest that both histone modification and chromatin remodeling are important for the maintenance of genomic stability in the face of both endogenous and exogenous DNA damage

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Conclusion