BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors
BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors
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DOI：
10.1016/j.jprot.2014.09.029
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发表时间：
2015-04-06
影响因子：
3.3
通讯作者：
Raught, Brian
中科院分区：
文献类型：
--
作者：
Dingar, Dharmendra;Kalkat, Manpreet;Raught, Brian
最近开发了基于BioID邻近性的生物素标记技术，用于表征蛋白质-蛋白质相互作用网络[1]。迄今为止，该方法已应用于培养细胞中表达的许多不同多肽。在这里，我们报告的适应BioID识别周围的c-MYC癌蛋白在标准培养条件下生长的人类细胞和小鼠肿瘤异种移植物中的蛋白质-蛋白质相互作用。值得注意的是，体内BioID产生了>100个高置信度的MYC相互作用蛋白，包括>30个已知的结合伴侣。推定的新型MYC相互作用物包括STAGA/KAT 5和SWI/SNF染色质重塑复合物的组分、DNA修复和复制因子、一般转录和延伸因子以及转录共调节因子如DNA解旋酶蛋白质染色体结构域8（CHD 8）。ENCODE ChIP-seq数据集为这些发现提供了额外的信心，突出了在此鉴定的MYC相互作用物在整个基因组中的显著一致结合，并且我们使用酵母双杂交分析和基于邻近的连接测定验证了先前未报告的MYC-CHD 8相互作用。总之，我们证明，BioID可以用来确定真正的相互作用的合作伙伴在体内的染色质相关蛋白。c-MYC（MYC）癌基因是一种转录因子，在肿瘤的发生和发展过程中起着重要的作用。MYC表达在超过50%的人类癌症中失调，但这种蛋白质在正常细胞生物学和肿瘤进展中的作用仍然没有得到很好的理解，部分原因是鉴定MYC相互作用蛋白质在技术上具有挑战性。开发一种新的策略来深入了解含MYC的蛋白质复合物将标志着癌症研究的一个关键进展。最近描述的BioID邻近标记技术代表了一种有前途的新的补充方法，用于表征培养细胞中的蛋白质-蛋白质相互作用（PPI）。在这里，我们报告BioID还可用于表征肿瘤异种移植物中染色质相关蛋白的蛋白质-蛋白质相互作用，并提供全面、高置信度的体内MYC相互作用组。本文是特刊的一部分，题为：蛋白质动态健康和疾病。客座编辑：Pierre Thibault和Anne-Claude Gingras。(C)2014爱思唯尔有限公司版权所有。
The BioID proximity-based biotin labeling technique was recently developed for the characterization of protein-protein interaction networks [1]. To date, this method has been applied to a number of different polypeptides expressed in cultured cells. Here we report the adaptation of BioID to the identification of protein-protein interactions surrounding the c-MYC oncoprotein in human cells grown both under standard culture conditions and in mice as tumor xenografts. Notably, in vivo BioID yielded >100 high confidence MYC interacting proteins, including >30 known binding partners. Putative novel MYC interactors include components of the STAGA/KAT5 and SWI/SNF chromatin remodeling complexes, DNA repair and replication factors, general transcription and elongation factors, and transcriptional co-regulators such as the DNA helicase protein chromodomain 8 (CHD8). Providing additional confidence in these findings, ENCODE ChIP-seq datasets highlight significant coincident binding throughout the genome for the MYC interactors identified here, and we validate the previously unreported MYC-CHD8 interaction using both a yeast two hybrid analysis and the proximity-based ligation assay. In sum, we demonstrate that BioID can be utilized to identify bona fide interacting partners for a chromatin-associated protein in vivo. This technique will allow for a much improved understanding of protein-protein interactions in a previously inaccessible biological setting.Biological significanceThe c-MYC (MYC) oncogene is a transcription factor that plays important roles in cancer initiation and progression. MYC expression is deregulated in more than 50% of human cancers, but the role of this protein in normal cell biology and tumor progression is still not well understood, in part because identifying MYC-interacting proteins has been technically challenging MYC-containing chromatin-associated complexes are difficult to isolate using traditional affinity purification methods, and the MYC protein is exceptionally labile, with a half-life of only similar to 30 min. Developing a new strategy to gain insight into MYC-containing protein complexes would thus mark a key advance in cancer research.The recently described BioID proximity-based labeling technique represents a promising new complementary approach for the characterization of protein-protein interactions (PPIs) in cultured cells. Here we report that BioID can also be used to characterize protein-protein interactions for a chromatin-associated protein in tumor xenografts, and present a comprehensive, high confidence in vivo MYC interactome.This article is part of a Special Issue entitled: Protein dynamics in health and disease.Guest Editors: Pierre Thibault and Anne-Claude Gingras. (C) 2014 Elsevier B.V. All rights reserved.