Department of Molecular Genetics, Oncode Institute, Cancer Genomics Netherlands, Erasmus Medical Centre, Rotterdam, The Netherlands.
Department of Proteomics, Erasmus Medical Centre, Rotterdam, The Netherlands.
Sci Rep. 2017 Nov 10;7(1):15353. doi: 10.1038/s41598-017-15194-y.
The DNA damage response (DDR), comprising distinct repair and signalling pathways, safeguards genomic integrity. Protein ubiquitylation is an important regulatory mechanism of the DDR. To study its role in the UV-induced DDR, we characterized changes in protein ubiquitylation following DNA damage using quantitative di-Gly proteomics. Interestingly, we identified multiple sites of histone H1 that are ubiquitylated upon UV-damage. We show that UV-dependent histone H1 ubiquitylation at multiple lysines is mediated by the E3-ligase HUWE1. Recently, it was shown that poly-ubiquitylated histone H1 is an important signalling intermediate in the double strand break response. This poly-ubiquitylation is dependent on RNF8 and Ubc13 which extend pre-existing ubiquitin modifications to K63-linked chains. Here we demonstrate that HUWE1 depleted cells showed reduced recruitment of RNF168 and 53BP1 to sites of DNA damage, two factors downstream of RNF8 mediated histone H1 poly-ubiquitylation, while recruitment of MDC1, which act upstream of histone H1 ubiquitylation, was not affected. Our data show that histone H1 is a prominent target for ubiquitylation after UV-induced DNA damage. Our data are in line with a model in which HUWE1 primes histone H1 with ubiquitin to allow ubiquitin chain elongation by RNF8, thereby stimulating the RNF8-RNF168 mediated DDR.
DNA 损伤反应（DDR）由不同的修复和信号通路组成，可保护基因组完整性。蛋白质泛素化是 DDR 的重要调节机制。为了研究其在 UV 诱导的 DDR 中的作用，我们使用定量二甘肽蛋白质组学方法研究了 DNA 损伤后蛋白质泛素化的变化。有趣的是，我们在多个组蛋白 H1 上鉴定到了 UV 损伤后发生泛素化的位点。我们表明，UV 依赖性的多个赖氨酸上的组蛋白 H1 泛素化是由 E3 连接酶 HUWE1 介导的。最近的研究表明，多泛素化的组蛋白 H1 是双链断裂反应中的一个重要信号中间体。这种多泛素化依赖于 RNF8 和 Ubc13，它们将预先存在的泛素修饰延伸到 K63 连接的链上。在这里，我们证明 HUWE1 耗尽的细胞中，RNF8 介导的组蛋白 H1 多泛素化下游的两个因子 RNF168 和 53BP1 向 DNA 损伤位点的募集减少，而 MDC1 的募集不受影响，MDC1 是组蛋白 H1 泛素化的上游因子。我们的数据表明，组蛋白 H1 是 UV 诱导的 DNA 损伤后泛素化的一个主要靶标。我们的数据与 HUWE1 用泛素对组蛋白 H1 进行“预修饰”以允许 RNF8 进行泛素链延伸的模型一致，从而刺激了 RNF8-RNF168 介导的 DDR。
