Klingberg Rebecca, Jost Jan Oliver, Schümann Michael, Gelato Kathy Ann, Fischle Wolfgang, Krause Eberhard, Schwarzer Dirk
Laboratory of Protein Chemistry and §Mass Spectrometry, Leibniz-Institut für Molekulare Pharmakologie , Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
ACS Chem Biol. 2015 Jan 16;10(1):138-45. doi: 10.1021/cb500563n. Epub 2014 Nov 5.
Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails.
组蛋白的多种翻译后修饰（PTM），包括丝氨酸和苏氨酸残基的位点特异性磷酸化，决定了染色质的可及性。根据组蛋白编码理论，PTM可招募调控蛋白或阻止它们与染色质结合。在此，我们报告了一种基于稳定同位素标记氨基酸定量质谱（SILAC MS）方案同时分析这两种效应的通用策略。我们应用该方法研究组蛋白H3的S10磷酸化的生化作用。用由L-和D-氨基酸合成的H3尾进行差异下拉实验发现，组蛋白乙酰转移酶1（HAT1）和成视网膜细胞瘤结合蛋白7（RBBP7）是与未修饰的H3尾相互作用的蛋白质网络的一部分。在S10处含有不可水解的磷酸丝氨酸模拟物膦酰亚甲基丙氨酸（Pma）的另一种H3衍生诱饵招募了14-3-3家族的几种异构体，并阻止了HAT1和RBBP7与未修饰的H3尾结合。我们的观察结果为H3S10磷酸化的多种功能提供了新的见解。此外，概述的方法通常适用于研究未修饰组蛋白尾的特定结合伴侣。
