Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin
Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin
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DOI：
10.1073/pnas.1504483112
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发表时间：
2015-08-18
影响因子：
11.1
通讯作者：
Muir, Tom W.
中科院分区：
文献类型：
--
作者：
Holt, Matthew T.;David, Yael;Muir, Tom W.
组蛋白H2 B在赖氨酸120处的泛素化（H2 B-Ub）在转录延伸、染色质构象以及特定组蛋白H3甲基化的调节中起关键作用。在此，我们报告了一种策略，用于泛素的位点特异性化学连接到预组装的核小体。这允许加速结构活性研究H2 B-Ub如何通过甲基转移酶人Dot 1调节H3 K79甲基化。通过对泛素表面的丙氨酸扫描，我们确定了泛素上的一个功能热点，这是体外刺激人Dot 1所必需的。重要的是，这一结果通过使用合成生物学策略在来自分离的细胞核的染色质中得到验证，该合成生物学策略允许将热点缺陷型泛素突变体选择性地掺入H2 B中。泛素热点还影响ySet 1介导的H3 K4甲基化的调节，但不是H2 B-Ub诱导的染色质纤维致密化损伤所必需的。这些数据证明了将化学连接技术应用于预组装染色质的实用性，并描绘了泛素作为组蛋白翻译后修饰的多功能性。
Ubiquitylation of histone H2B at lysine 120 (H2B-Ub) plays a critical role in transcriptional elongation, chromatin conformation, as well as the regulation of specific histone H3 methylations. Herein, we report a strategy for the site-specific chemical attachment of ubiquitin to preassembled nucleosomes. This allowed expedited structure-activity studies into how H2B-Ub regulates H3K79 methylation by the methyltransferase human Dot1. Through an alanine scan of the ubiquitin surface, we identified a functional hotspot on ubiquitin that is required for the stimulation of human Dot1 in vitro. Importantly, this result was validated in chromatin from isolated nuclei by using a synthetic biology strategy that allowed selective incorporation of the hotspot-deficient ubiquitin mutant into H2B. The ubiquitin hotspot additionally impacted the regulation of ySet1-mediated H3K4 methylation but was not required for H2B-Ub-induced impairment of chromatin fiber compaction. These data demonstrate the utility of applying chemical ligation technologies to preassembled chromatin and delineate the multifunctionality of ubiquitin as a histone post-translational modification.