Comparative interactome analysis of the PRE DNA-binding factors: purification of the Combgap, Zeste, Psq, and Adf1 associated proteins. Comparative interactome analysis of the PRE DNA-binding factors: purification of the Combgap, Zeste, Psq, and Adf1 associated proteins

To gain an insight into the PRE DNA-binding protein regulatory network, here, using immunoaffinity purification coupled to the high throughput mass spectrometry, we isolated factors associated with the Combgap, Psq, Zeste and Adf1 PRE DNA-binding proteins. We show that Combgap and Zeste are more tightly associated with the Polycomb repressive complex 1 (PRC1), while Psq interacts strongly with the TrxG proteins, including the BAP SWI/SNF complex. The Adf1 interactome contained Mediator subunits as the top interactors. In addition, Combgap efficiently interacted with AGO2, NELF, and TFIID. Combgap, Psq, and Adf1 have architectural proteins in their networks. We further investigated the existence of direct interactions between different PRE DNA-binding proteins and demonstrated that Combgap-Adf1, Psq-Dsp1, and Pho-Spps can interact in the yeast two-hybrid assay. Overall, our data suggest that Combgap, Psq, Zeste and Adf1 are associated with the protein complexes implicated in different regulatory activities and indicate their potential multifunctional role in the regulation of transcription. Overall design: ChIP-seq of Combgap, Zeste, Psq, and Adf1 in S2 cells

为深入解析Polycomb反应元件（Polycomb Response Element, PRE）DNA结合蛋白的调控网络，本研究采用免疫亲和纯化耦联高通量质谱的实验方法，分离得到与Combgap、Psq、Zeste及Adf1这四种PRE DNA结合蛋白相关的互作因子。
实验结果显示，Combgap与Zeste和多梳抑制复合体1（Polycomb repressive complex 1, PRC1）结合更为紧密；而Psq则可与Trithorax家族（Trithorax Group, TrxG）蛋白发生强相互作用，其中包括BAP SWI/SNF复合体。Adf1的互作组中，中介体亚基为其首要互作蛋白。
此外，Combgap可与AGO2、负延伸因子（Negative Elongation Factor, NELF）及转录因子IID（Transcription Factor IID, TFIID）高效结合。Combgap、Psq及Adf1的调控网络中均包含架构蛋白。
本研究进一步探究了不同PRE DNA结合蛋白间的直接互作关系，并通过酵母双杂交实验证实Combgap与Adf1、Psq与Dsp1以及Pho与Spps可发生相互作用。
综合以上实验数据，本研究表明Combgap、Psq、Zeste及Adf1分别与参与不同调控活动的蛋白质复合体相结合，提示它们在转录调控过程中可能发挥多方面的功能。
实验整体设计：在果蝇S2细胞中对Combgap、Zeste、Psq及Adf1进行染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）。