Phase separation directs ubiquitination of gene body nucleosomes

The conserved yeast E3 ligase Bre1 and its partner E2 Rad6 monoubiquitinate histone H2B across gene bodies during the transcription cycle. While processive ubiquitination might in principle arise from Bre1/Rad6 traveling with RNA polymerase II, we provide a different explanation. Here we implicate liquid-liquid phase separation as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, whose intrinsically disordered region phase separates via dynamic, multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, accelerating H2B ubiquitination. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone modifying enzymes generate chromatin-associated reaction chambers with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, causing neurological disease when impaired.保守型酵母来源的E3泛素连接酶Bre1及其搭档E2泛素结合酶Rad6，可在转录周期中于基因体范围内对组蛋白H2B进行单泛素化修饰。尽管从原理上讲，持续性泛素化可能源于Bre1/Rad6与RNA聚合酶II共转运，但我们提出了另一种解释机制。本研究证实液-液相分离为其核心调控机制。生化重组实验表明，Bre1可与支架蛋白Lge1结合；Lge1的内在无序区域可通过动态多价相互作用发生相分离。所形成的凝聚体呈现分层结构：内层为Lge1核心，外层为Bre1催化壳。该分层液态凝聚体可招募Rad6与核小体底物，从而加速H2B的泛素化修饰。体内实验证实，若要在+1核小体下游的基因体区域完成H2B泛素化，Lge1的凝聚体形成结构域是必需的。我们的研究数据表明，组蛋白修饰酶的分层凝聚体可形成与染色质结合的反应腔室，沿基因体分布并增强催化活性。类似的过程可能也存在于人类细胞中，当其功能受损时可引发神经系统疾病。