Gallego Laura D, Ghodgaonkar Steger Medini, Polyansky Anton A, Schubert Tobias, Zagrovic Bojan, Zheng Ning, Clausen Tim, Herzog Franz, Köhler Alwin
Max F. Perutz Laboratories, Medical University of Vienna, Vienna Biocenter Campus, 1030 Vienna, Austria;
Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich 81377, Germany;
Proc Natl Acad Sci U S A. 2016 Sep 20;113(38):10553-8. doi: 10.1073/pnas.1606863113. Epub 2016 Sep 6.
Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6-Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt-Ada-Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities.
组蛋白H2B的共转录泛素化是基因调控的关键。酵母E3泛素连接酶Bre1（人类RNF20/40）与E2泛素结合酶Rad6配对，在赖氨酸123处对H2B进行单泛素化。核小体核心颗粒（NCP）上的这个单一赖氨酸残基是如何被Rad6-Bre1机制靶向的尚不清楚。利用化学交联和质谱技术，我们在一个明确的体外系统中确定了Rad6、Bre1和NCP的功能界面。Bre1的RING结构域仅与组蛋白H2B和H2A的不同区域发生交联，表明Bre1与NCP酸性补丁在空间上对齐。通过以这种独特的方向对接在NCP表面，Bre1将Rad6活性位点直接定位在H2B赖氨酸123上方。Spt-Ada-Gcn5乙酰转移酶（SAGA）的H2B去泛素化酶模块与Bre1竞争结合NCP酸性补丁，表明存在调控控制。我们的研究揭示了一种确保位点特异性NCP泛素化和对相反酶活性进行微调的机制。
