Reciprocal interactions of human C10orf12 and C17orf96 with PRC2 revealed by BioTAP-XL cross-linking and affinity purification
Reciprocal interactions of human C10orf12 and C17orf96 with PRC2 revealed by BioTAP-XL cross-linking and affinity purification
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DOI：
10.1073/pnas.1400648111
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发表时间：
2014-02-18
影响因子：
11.1
通讯作者：
Kuroda, Mitzi I.
中科院分区：
文献类型：
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作者：
Alekseyenko, Artyom A.;Gorchakov, Andrey A.;Kuroda, Mitzi I.
了解表观遗传调节因子的组成仍然是染色质生物学中的一个重要挑战。染色质相关复合物的传统生化分析需要在也可以破坏关键相互作用的条件下从DNA中释放它们。在这里，我们开发了一种互补的方法（BioTAP-XL），其中交联（XL）增强了蛋白质相互作用的保存，并允许在相同的串联亲和纯化（BioTAP）方案下分析DNA靶点。我们通过分析人EZH 2（polycomb抑制复合物2（PRC 2）的核心亚基）来证明BioTAP-XL的能力。我们鉴定并验证了两种强相互作用物C10 orf 12和C17 orf 96，它们显示与典型PRC 2组分（SUZ 12，EED，MTF 2，JARID 2，PHF 1和AEBP 2）相似水平的EZH 2-BioTAP富集。BioTAP标记的C10 orf 12或C17 orf 96的ChIP-seq分析揭示了每个结合模式与EZH 2和H3 K27 me 3沉默标记的位置的相似性，验证了它们与PRC 2组分的物理相互作用。有趣的是，C10 orf 12和C17 orf 96相互作用的质谱分析显示，这些蛋白质可能是互斥的PRC 2亚基，它们不能相互作用，也不能与JARID 2和AEBP 2相互作用。此外，C10 orf 1/2显示出与EHMT 1/2复合物的组分的强烈和意想不到的关联，因此可能将PRC 2连接到另一种组蛋白甲基转移酶。类似地，CBX 4-BioTAP蛋白下拉的结果与PRC 1复合物多样性的报道一致。我们的研究结果强调了多个亚基的相互分析的重要性，并表明反复使用BioTAP-XL具有很强的潜力，以揭示网络的染色质为基础的相互作用在高等生物。
Understanding the composition of epigenetic regulators remains an important challenge in chromatin biology. Traditional biochemical analysis of chromatin-associated complexes requires their release from DNA under conditions that can also disrupt key interactions. Here we develop a complementary approach (BioTAP-XL), in which cross-linking (XL) enhances the preservation of protein interactions and also allows the analysis of DNA targets under the same tandem affinity purification (BioTAP) regimen. We demonstrate the power of BioTAP-XL through analysis of human EZH2, a core subunit of polycomb repressive complex 2 (PRC2). We identify and validate two strong interactors, C10orf12 and C17orf96, which display enrichment with EZH2-BioTAP at levels similar to canonical PRC2 components (SUZ12, EED, MTF2, JARID2, PHF1, and AEBP2). ChIP-seq analysis of BioTAP-tagged C10orf12 or C17orf96 revealed the similarity of each binding pattern with the location of EZH2 and the H3K27me3-silencing mark, validating their physical interaction with PRC2 components. Interestingly, analysis by mass spectrometry of C10orf12 and C17orf96 interactions revealed that these proteins may be mutually exclusive PRC2 subunits that fail to interact with each other or with JARID2 and AEBP2. C10orf12, in addition, shows a strong and unexpected association with components of the EHMT1/2 complex, thus potentially connecting PRC2 to another histone methyltransferase. Similarly, results from CBX4-BioTAP protein pulldowns are consistent with reports of a diversity of PRC1 complexes. Our results highlight the importance of reciprocal analyses of multiple subunits and suggest that iterative use of BioTAP-XL has strong potential to reveal networks of chromatin-based interactions in higher organisms.