Trimethylation of histone H3 at K9 by the lysine methyltransferase, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) plays a pivotal role in silencing tissue-specific genes and retrotransposable elements. In mammalian cells, SETDB1 undergoes monoubiquitination in the insertion region of the SET domain in an E3 ubiquitin ligase-independent manner. This ubiquitination has been shown to enhance the histone H3-K9 methyltransferase activity of SETDB1; however, the molecular mechanism underlying SETDB1 activation by ubiquitination is unknown. In this study, we developed an Escherichia coli ubiquitination plasmid for the preparation of ubiquitinated SETDB1. Western blotting and mutational analyses showed that co-expression of the SET domain of SETDB1 with the proteins encoded by the ubiquitination plasmid led to site-specific monoubiquitination of the SET domain at K867. An in vitro histone H3 methylation assay demonstrated that the ubiquitinated SET domain of SETDB1 acquired enzymatic activity. Taken together, these findings demonstrate successful preparation of the active form of SETDB1 with the E.coli ubiquitination system, which will aid biochemical and structural studies of ubiquitinated SETDB1.

中文翻译：

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在大肠杆菌中制备泛素化触发活性形式的 SETDB1 用于生化和结构分析

赖氨酸甲基转移酶在 K9 处对组蛋白 H3 进行三甲基化，SET 域分叉组蛋白赖氨酸甲基转移酶 1 (SETDB1) 在沉默组织特异性基因和反转录转座因子中起关键作用。在哺乳动物细胞中，SETDB1 在 SET 结构域的插入区域以不依赖 E3 泛素连接酶的方式进行单泛素化。这种泛素化已被证明可以增强 SETDB1 的组蛋白 H3-K9 甲基转移酶活性。然而，通过泛素化激活 SETDB1 的分子机制尚不清楚。在这项研究中，我们开发了一种用于制备泛素化 SETDB1 的大肠杆菌泛素化质粒。蛋白质印迹和突变分析表明，SETDB1 的 SET 结构域与泛素化质粒编码的蛋白质的共表达导致 K867 上 SET 结构域的位点特异性单泛素化。体外组蛋白 H3 甲基化测定表明 SETDB1 的泛素化 SET 结构域获得酶活性。综上所述，这些发现证明了利用大肠杆菌泛素化系统成功制备了活性形式的 SETDB1，这将有助于泛素化 SETDB1 的生化和结构研究。