Saavedra Francisco, Rivera Carlos, Rivas Elizabeth, Merino Paola, Garrido Daniel, Hernández Sergio, Forné Ignasi, Vassias Isabelle, Gurard-Levin Zachary A, Alfaro Iván E, Imhof Axel, Almouzni Geneviève, Loyola Alejandra
Fundación Ciencia & Vida, Santiago 7780272, Chile.
Munich Center of Integrated Protein Science and Biomedical Center, Ludwig-Maximilians University of Munich, Planegg-Martinsried 80336, Germany.
Nucleic Acids Res. 2017 Nov 16;45(20):11700-11710. doi: 10.1093/nar/gkx775.
Newly synthesized histones H3 and H4 undergo a cascade of maturation steps to achieve proper folding and to establish post-translational modifications prior to chromatin deposition. Acetylation of H4 on lysines 5 and 12 by the HAT1 acetyltransferase is observed late in the histone maturation cascade. A key question is to understand how to establish and regulate the distinct timing of sequential modifications and their biological significance. Here, we perform proteomic analysis of the newly synthesized histone H4 complex at the earliest time point in the cascade. In addition to known binding partners Hsp90 and Hsp70, we also identify for the first time two subunits of the histone acetyltransferase inhibitor complex (INHAT): PP32 and SET/TAF-Iβ. We show that both proteins function to prevent HAT1-mediated H4 acetylation in vitro. When PP32 and SET/TAF-Iβ protein levels are down-regulated in vivo, we detect hyperacetylation on lysines 5 and 12 and other H4 lysine residues. Notably, aberrantly acetylated H4 is less stable and this reduces the interaction with Hsp90. As a consequence, PP32 and SET/TAF-Iβ depleted cells show an S-phase arrest. Our data demonstrate a novel function of PP32 and SET/TAF-Iβ and provide new insight into the mechanisms regulating acetylation of newly synthesized histone H4.
新合成的组蛋白H3和H4会经历一系列成熟步骤，以实现正确折叠，并在染色质沉积之前建立翻译后修饰。在组蛋白成熟级联反应后期，可观察到HAT1乙酰转移酶对H4赖氨酸5和12位点的乙酰化。一个关键问题是要了解如何建立和调节连续修饰的不同时间及其生物学意义。在这里，我们在级联反应的最早时间点对新合成的组蛋白H4复合物进行了蛋白质组学分析。除了已知的结合伴侣Hsp90和Hsp70外，我们还首次鉴定出组蛋白乙酰转移酶抑制剂复合物（INHAT）的两个亚基：PP32和SET/TAF-Iβ。我们表明，这两种蛋白质在体外均起到阻止HAT1介导的H4乙酰化的作用。当PP32和SET/TAF-Iβ蛋白水平在体内下调时，我们检测到赖氨酸5和12以及其他H4赖氨酸残基上的超乙酰化。值得注意的是，异常乙酰化的H4稳定性较差，这会减少与Hsp90的相互作用。因此，PP32和SET/TAF-Iβ缺失的细胞会出现S期停滞。我们的数据证明了PP32和SET/TAF-Iβ的新功能，并为调节新合成组蛋白H4乙酰化的机制提供了新的见解。
