Plasmodium falciparum bromo-domain containing epigenetic protein complexes recruited to modified histone tails as identified by histone peptide pulldown (HPP)-MS/MS and further characterized by GFP-IP-MS/MS and HA-IP-MS/MS

In order to identify epigenetic protein complexes recruited to histone PTMs in the deadly human malaria parasite Plasmodium falciparum, we set out to perform histone peptide pulldowns with various histone peptides. Biotinylated peptides corresponding to P. falciparum histone variant H2A.Z, H2B.Z and H3.3, canonical P. falciparum histone H4 or human histone H4 tail sequences (differing on position 21 from the P. falciparum peptide) bearing multiple histone post-translational modifications (acetylations and/or methylations) as well as the unmodified control peptides were coupled to beads and incubated with native nuclear extract obtained from mixed-stage asexual cultures. Proteins preferentially binding to the modified histone peptide were identified by quantitative proteomics using di-methyl labelling. This yielded 6 out of 7 P. falciparum bromo-domain proteins preferentially binding to the acetylated histone peptides, as well as many putative bromo-domain protein interactors. In addition, a PHD-domain containing protein was found to be recruiting a PfSAGA-like complex to H3K4 di- and tri-methylated peptides. 7 reader-domain containing proteins (BDP1, BDP2, BDP4, TAF1, GCN5, PHD1, PHD2) and 5 putative interactors (PF3D7_0306100, PF3D7_1124300, PF3D7_1128000, PF3D7_1225200, PF3D7_1451200) were endogenously tagged by GFP or HA and used in quantitative GFP-/HA-IP-MS/MS experiments to further characterize epigenetic reader-complex composition. As negative control, GFP- and HA-IPs were performed on native nuclear extract not encoding tagged protein. Most HPP experiment employed a 3-label setup, while few only relied on “light” and “heavy”-label sample pools (labels and conditions listed below). GFP- and HA-IP experiments were using a 2-label “light” and “heavy” di-methyl setup only. For all experiments technical replicates were performed using a label-swap approach (called F (forward) and R (reverse)) and independent biological replicates were performed for each. For GFP- and HA-IP forward reactions are set-up as: GFP-/HA-beads = “heavy” di-methyl label, control-beads = “light” di-methyl label. Reverse reactions are set-up as: GFP-/HA-beads = “light” di-methyl label, control-beads = “heavy” di-methyl label. For GFP-/HA-IP raw files the bait and tag used for fishing are included in the file name.

为了鉴定致死性人类疟原虫——恶性疟原虫（Plasmodium falciparum）中被招募至组蛋白翻译后修饰（histone post-translational modification, PTM）的表观遗传蛋白质复合物，我们拟通过多种组蛋白肽段开展组蛋白肽段下拉实验。将对应于恶性疟原虫组蛋白变体H2A.Z、H2B.Z、H3.3，经典型恶性疟原虫组蛋白H4，或人组蛋白H4尾巴序列（其第21位氨基酸与恶性疟原虫对应肽段存在差异）的生物素标记肽段，连同携带多种组蛋白翻译后修饰（乙酰化和/或甲基化）的肽段以及未修饰对照肽段，偶联至磁珠后，与从混合期无性繁殖培养物中提取的天然核提取物共同孵育。采用二甲基标记法开展定量蛋白质组学分析，以此鉴定优先结合修饰组蛋白肽段的蛋白质。该实验共筛选出7种恶性疟原虫溴结构域（bromo-domain）蛋白中的6种，它们可优先结合乙酰化组蛋白肽段，同时还鉴定出大量推定的溴结构域蛋白互作因子。此外，我们发现1种含PHD结构域（PHD-domain）的蛋白质可将PfSAGA样复合物招募至H3K4二甲基化和三甲基化肽段。我们通过绿色荧光蛋白（green fluorescent protein, GFP）或血凝素（hemagglutinin, HA）对内源的7种含读码结构域的蛋白质（BDP1、BDP2、BDP4、TAF1、GCN5、PHD1、PHD2）及5种推定互作因子（PF3D7_0306100、PF3D7_1124300、PF3D7_1128000、PF3D7_1225200、PF3D7_1451200）进行标记，并利用定量免疫沉淀-串联质谱（immunoprecipitation-tandem mass spectrometry, IP-MS/MS）实验进一步表征表观遗传阅读器复合物的组成。以未携带标记蛋白的天然核提取物作为阴性对照，开展GFP和HA免疫沉淀实验。大多数组蛋白肽段下拉实验采用3标记体系，少数仅依赖“轻”、“重”标记样本池（标记物及实验条件列于下文）。而GFP和HA免疫沉淀实验仅采用2标记的“轻”、“重”二甲基标记体系。所有实验均通过标记互换法（分为正向F与反向R实验）设置技术重复，并独立开展生物学重复。对于GFP和HA免疫沉淀实验，正向实验设置为：GFP磁珠/HA磁珠采用“重”二甲基标记，对照磁珠采用“轻”二甲基标记；反向实验设置为：GFP磁珠/HA磁珠采用“轻”二甲基标记，对照磁珠采用“重”二甲基标记。对于GFP/HA免疫沉淀的原始数据文件，用于捕获靶标的诱饵蛋白及标记标签已包含在文件名中。