School of Life and Environmental Sciences, University of Sydney, Australia.
FEBS J. 2019 Jun;286(11):2043-2061. doi: 10.1111/febs.14800. Epub 2019 Mar 19.
The nucleosome remodelling and deacetylase complex (NuRD) is a widely conserved regulator of gene expression. The determination of the subunit composition of the complex and identification of its binding partners are important steps towards understanding its architecture and function. The question of how these properties of the complex vary across different cell types has not been addressed in detail to date. Here, we set up a two-step purification protocol coupled to liquid chromatography-tandem mass spectrometry to assess NuRD composition and interaction partners in three different cancer cell lines, using label-free intensity-based absolute quantification (iBAQ). Our data indicate that the stoichiometry of the NuRD complex is preserved across our three different cancer cell lines. In addition, our interactome data suggest ZNF219 and SLC25A5 as possible interaction partners of the complex. To corroborate this latter finding, in vitro and cell-based pull-down experiments were carried out. These experiments indicated that ZNF219 can interact with RBBP4, GATAD2A/B and chromodomain helicase DNA binding 4, whereas SLC25A5 might interact with MTA2 and GATAD2A.
核小体重塑和去乙酰化酶复合物（NuRD）是一种广泛保守的基因表达调控因子。确定复合物的亚基组成并鉴定其结合伙伴是理解其结构和功能的重要步骤。迄今为止，复合物的这些特性如何在不同细胞类型中变化的问题尚未得到详细解决。在这里，我们建立了一个两步纯化方案，结合液相色谱-串联质谱法，使用无标记强度的绝对定量（iBAQ）来评估三种不同癌细胞系中的 NuRD 组成和相互作用伙伴。我们的数据表明，NuRD 复合物的化学计量在我们的三种不同癌细胞系中是保持不变的。此外，我们的相互作用组数据表明 ZNF219 和 SLC25A5 可能是该复合物的相互作用伙伴。为了证实这一发现，进行了体外和基于细胞的下拉实验。这些实验表明，ZNF219 可以与 RBBP4、GATAD2A/B 和染色质解旋酶 DNA 结合蛋白 4 相互作用，而 SLC25A5 可能与 MTA2 和 GATAD2A 相互作用。
