In Vivo Mapping of FACT-Histone Interactions Identifies a Role of Pob3 C-terminus in H2A-H2B Binding
In Vivo Mapping of FACT-Histone Interactions Identifies a Role of Pob3 C-terminus in H2A-H2B Binding
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DOI：
10.1021/acschembio.5b00493
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发表时间：
2015-12-01
影响因子：
4
通讯作者：
Neumann, Heinz
中科院分区：
文献类型：
--
作者：
Hoffmann, Christian;Neumann, Heinz
组蛋白分子伴侣协助核小体重排以促进DNA和RNA聚合酶通过染色质。FACT（促进染色质转录）复合物是一种保守的组蛋白伴侣，参与转录、复制和修复。该复合物由哺乳动物和酵母中的两个主要亚基Spt 16和SSRP 1/Pob 3组成，它们通过多次接触与组蛋白和DNA结合。然而，FACT功能的确切机制在很大程度上尚不清楚。在这里，我们使用基因安装的紫外线可激活的交联剂氨基酸对苯甲酰苯丙氨酸（pBPA）的FACT在活酵母中的相互作用网络图。出乎意料的是，我们发现Pob 3的酸性C-末端最有效地与组蛋白H2 A和H2 B形成交联。该观察结果与所进行的交联化学无关，因为使用对叠氮苯丙氨酸（pAzF）获得了类似的组蛋白交联。进一步的分析鉴定了Pob 3中的C-末端核定位序列。其与Importin-alpha的相互作用干扰H2 A H2 B结合，这表明FACT募集到染色质中可能具有调节作用。从Pob 3 C-末端删除酸性残基在芽殖酵母中产生了对羟基脲敏感的表型，表明该结构域在DNA复制中的潜在作用。
Histone chaperones assist nucleosomal rearrangements to facilitate the passage of DNA and RNA polymerases through chromatin. The FACT (facilitates chromatin transcription) complex is a conserved histone chaperone involved in transcription, replication, and repair. The complex consists of two major subunits, Spt16 and SSRP1/Pob3 in mammals and yeast, which engage histones and DNA by multiple contacts. However, the precise mechanism of FACT function is largely unclear. Here, we used the genetically installed UV-activatable cross-linker amino acid p-benzoylphenylalanine (pBPA) to map the interaction network of FACT in living yeast. Unexpectedly, we found the acidic C-terminus of Pob3 forming cross-links to histone H2A and H2B most efficiently. This observation was independent of the performed cross-linking chemistry since similar histone cross-links were obtained using p-azidophenylalanine (pAzF). Further analyses identified a C-terminal nuclear localization sequence in Pob3. Its interaction with Importin-alpha interfered with H2A H2B binding, which suggests a possible regulatory role in FACT recruitment to chromatin. Deletion of acidic residues from the Pob3 C-terminus creates a hydroxyurea-sensitive phenotype in budding yeast, suggesting a potential role for this domain in DNA replication.