Chemically synthesized histone H2A Lys13 di-ubiquitination promotes binding of 53BP1 to nucleosomes
Chemically synthesized histone H2A Lys13 di-ubiquitination promotes binding of 53BP1 to nucleosomes
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DOI：
10.1038/cr.2018.6
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发表时间：
2018-01
期刊：
影响因子：
44.1
通讯作者：
Jiabin Li;Yun-Kun Qi;Qiaoqiao He;H. Ai;Sanling Liu;Jiaxing Wang;Ji‐Shen Zheng;Lei Liu;C. Tian
中科院分区：
文献类型：
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作者：
Jiabin Li;Yun-Kun Qi;Qiaoqiao He;H. Ai;Sanling Liu;Jiaxing Wang;Ji‐Shen Zheng;Lei Liu;C. Tian
亲爱的编辑，p53结合蛋白1（53 BP 1）是细胞对DNA双链断裂（DSB）反应的关键调节因子[1]。为了实现其修复功能，53 BP 1必须被募集到DSB位点周围的染色质中，所述DSB位点在Lys 20处携带H4甲基化并且在Lys 15处携带H2 A泛素化[2-5]。该识别过程的结构基础最近通过与含有Lys 20-二甲基化H4（H4 K20 me 2）和Lys 15-单泛素化H2 A（H2 AK 15 monoUb）的核小体核心颗粒（NCP）结合的53 BP 1的复杂结构揭示[6]。值得注意的是，泛素连接酶RNF 168不仅在Lys 15上泛素化H2 A，而且在Lys 13上也没有选择性地泛素化H2 A，并且携带K15 Q突变的H2 A在体内仍在Lys 13处多聚泛素化[2-4，7]。这就引出了两个问题。首先，53 BP 1也是H2 A Lys 13泛素标记的阅读器吗？第二，多聚泛素化在53 BP 1募集事件中是多余的吗？在以前的研究中，53 BP 1被认为是H2 AK 15 monoUb的特异性阅读器，而不是H2 AK 13 monoUb [5，6]。在这里，使用化学定义的核小体，我们提出的第一个证据表明，53 BP 1也可以识别H2 A Lys 13二泛素标记。我们首先开发了一种可行的策略，用于全化学合成单/双泛素化组蛋白以制备核小体。令人惊讶的是，我们发现含有Lys 13-或Lys 15-二泛素化H2 A的NCP被53 BP 1有效识别。此外，在H2 AK 13 diUb中，53 BP 1优先与远端泛素而不是近端泛素相互作用。进一步的研究表明，H4 K20 me 2和核小体的酸性补丁是必不可少的相互作用。总之，我们的研究表明，H2 A Lys 13-poly-ubiquitination也可以招募53 BP 1来应对DNA损伤。为了解释H2 A泛素化在53 BP 1识别中的作用，产生位点特异性泛素化的H2 A是关键。然而，基于RNF 168的体外酶促反应不能区分相邻的Lys位点[6]，并且主要产生单泛素化的组蛋白[2，8]。因此，酶促方法可能不适合于产生具有确定连接和长度的同质聚泛素化H2 A [4]，而化学方法（如补充信息，数据S1中所述）可以制备具有分子同质性的泛素化组蛋白[9]。然而，目前还没有关于制备双泛素化组蛋白的合成方法的报道。因此，我们的研究从泛素化H2 As的全化学合成开始，首先进行了H2 AK 13 monoUb的化学合成。其序列可分为1、2、3、4、5五段。通过会聚策略组装这些片段，并应用肽酰肼的酶介导的连接以实现位点特异性泛素化（补充信息，图S1-S3）。首先，进行片段1和2之间的连接以提供泛素酰肼6。随后，将肽6与片段3连接，随后除去辅助物，得到分支肽7。平行地，区段4和5的连接产生肽8。在肽7和8的缩合以及随后的脱硫之后，获得H2 AK-13 monoUb，总分离产率为15.7%（图1B和1C）。由于H2 A的K27连接的多聚泛素化对于修复信号传导是必需的[2-4]，我们接下来制备了在Lys 13或Lys 15处携带K27连接的双泛素化的H2 A（H2 AK 13 diUb和H2 AK 15 di-Ub）。按照上述方案，我们首先合成了截短的单泛素修饰的H2 A13（图1A）。平行的，分叉的...
Dear Editor, p53-binding protein 1 (53BP1) is a critical regulator of cellular response to DNA double-strand breaks (DSBs)[1]. To accomplish its repair function, 53BP1 must be recruited to the chromatin surrounding DSB sites that carry H4 methylation at Lys20 and H2A ubiquitination at Lys15 [2-5]. The structural basis of this recognition process was recently revealed by the complex structure of 53BP1 bound to a nucleosome core particle (NCP) containing Lys20-dimethylated H4 (H4K20me2) and Lys15-mono-ubiquitinated H2A (H2AK15monoUb)[6]. It is fascinating to note that ubiquitin ligase RNF168 ubiquitinated H2A not only on Lys15, but also on Lys13 without selectivity, and H2A bearing the K15Q mutation was still poly-ubiquitinated at Lys13 in vivo [2-4, 7]. This leads to two questions. First, is 53BP1 also a reader of H2A Lys13 ubiquitin mark? Second, is poly-ubiquitination redundant at the 53BP1 recruitment event? In previous studies, 53BP1 was considered as a specific reader of H2AK15monoUb, but not H2AK13monoUb [5, 6]. Here, using chemically defined nucleosomes, we present the first evidence that 53BP1 can also recognize the H2A Lys13 di-ubiquitin mark. We first developed a practicable strategy for the total chemical synthesis of mono-/di-ubiquitinated histones to prepare nucleosomes. Surprisingly, we found that a NCP containing either Lys13-or Lys15-di-ubiquitinated H2A was effectively recognized by 53BP1. Moreover, 53BP1 preferentially interacted with the distal ubiquitin rather than the proximal ubiquitin in the H2AK13diUb. Further studies revealed that both H4K20me2 and the nucleosomal acidic patch are essential for the interaction. Together, our study suggested that H2A Lys13-poly-ubiquitination could also recruit 53BP1 in response to DNA damage. To decipher the role of H2A ubiquitination in 53BP1 recognition, it is critical to generate site-specifically ubiquitinated H2As. However, the in vitro RNF168-based enzymatic reaction was unable to discriminate between the adjacent Lys sites [6], and generated mainly mono-ubiquitinated histones [2, 8]. Therefore, enzymatic approaches might not be suited for producing homogenous poly-ubiquitinated H2A of definite linkage and length [4], whereas chemical methods (as described in Supplementary information, Data S1) could prepare ubiquitinated histones with molecular homogeneity [9]. However, there are no reports yet about synthetic methods for preparing di-ubiquitinated histones. Accordingly, our studies commenced with the total chemical synthesis of the ubiquitinated H2As.Initially, we implemented chemical synthesis of H2AK13monoUb. Its sequence was divided into five segments, namely 1, 2, 3, 4 and 5. These segments were assembled through a convergent strategy, and auxiliary-mediated ligation of peptide hydrazide was applied to achieve site-specific ubiquitination (Supplementary information, Figures S1-S3). First, the ligation between segments 1 and 2 was conducted to furnish ubiquitin hydrazide 6. Subsequently, peptide 6 was ligated with segment 3, followed by auxiliary removal, to give the branched peptide 7. In parallel, the ligation of segments 4 and 5 produced peptide 8. After the condensation of peptides 7 and 8, and subsequent desulfurization, H2AK-13monoUb was obtained with a total isolated yield of 15.7%(Figure 1B and 1C). Since K27-linked poly-ubiquitination of H2A is essential for repair signaling [2-4], we next prepared H2A bearing K27-linked di-ubiquitination at Lys13 or Lys15 (H2AK13diUb and H2AK15di-Ub). Following the above protocols, we first synthesized truncated mono-ubiquitin-modified H2A 13 (Figure 1A). In parallel, branched …