Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, Stuttgart University, Allmandring 31, 70569, Stuttgart, Germany.
Department of Biochemistry and Molecular Biology, Poznań University of Medical Sciences, Święcickiego 6 St., 60-781, Poznan, Poland.
Epigenetics Chromatin. 2018 Jan 25;11(1):4. doi: 10.1186/s13072-018-0175-3.
Protein posttranslational modifications (PTMs) occur broadly in the human proteome, and their biological outcome is often mediated indirectly by reader proteins that specifically bind to modified proteins and trigger downstream effects. Particularly, many lysine methylation sites among histone and nonhistone proteins have been characterized; however, the list of readers associated with them is incomplete.
This study introduces a modified yeast three-hybrid system (Y3H) to screen for methyllysine readers. A lysine methyltransferase is expressed together with its target protein or protein domain functioning as bait, and a human cDNA library serves as prey. Proof of principle was established using H3K9me3 as a bait and known H3K9me3 readers like the chromodomains of CBX1 or MPP8 as prey. We next conducted an unbiased screen using a library composed of human-specific open reading frames. It led to the identification of already known lysine methylation-dependent readers and of novel methyllysine reader candidates, which were further confirmed by co-localization with H3K9me3 in human cell nuclei.
Our approach introduces a cost-effective method for screening reading domains binding to histone and nonhistone proteins which is not limited by expression levels of the candidate reading proteins. Identification of already known and novel H3K9me3 readers proofs the power of the Y3H assay which will allow for proteome-wide screens of PTM readers.
蛋白质翻译后修饰（PTMs）广泛存在于人类蛋白质组中，其生物学结果通常通过专门结合修饰蛋白并触发下游效应的阅读器蛋白间接介导。特别是，组蛋白和非组蛋白中的许多赖氨酸甲基化位点已被描述；然而，与之相关的阅读器列表并不完整。
本研究引入了改良的酵母三杂交系统（Y3H）来筛选甲基赖氨酸阅读器。赖氨酸甲基转移酶与作为诱饵的靶蛋白或蛋白结构域一起表达，而人类 cDNA 文库则作为猎物。使用 H3K9me3 作为诱饵和已知的 H3K9me3 阅读器（如 CBX1 或 MPP8 的 chromodomains）作为猎物证明了原理。接下来，我们使用由人类特异性开放阅读框组成的文库进行了无偏见的筛选。它导致了已鉴定的赖氨酸甲基化依赖性阅读器和新的甲基赖氨酸阅读器候选物的鉴定，这些候选物通过与人类细胞核中 H3K9me3 的共定位进一步得到证实。
我们的方法引入了一种经济有效的筛选方法，用于筛选与组蛋白和非组蛋白结合的阅读结构域，不受候选阅读蛋白表达水平的限制。已经鉴定的和新的 H3K9me3 阅读器的鉴定证明了 Y3H 测定的有效性，它将允许对 PTM 阅读器进行全蛋白质组筛选。
