Active promoters give rise to false positive 'Phantom Peaks' in ChIP-seq experiments.
Active promoters give rise to false positive 'Phantom Peaks' in ChIP-seq experiments.
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DOI：
10.1093/nar/gkv637
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发表时间：
2015-08-18
影响因子：
14.9
通讯作者：
Becker PB
中科院分区：
文献类型：
--
作者：
Jain D;Baldi S;Zabel A;Straub T;Becker PB
染色质免疫沉淀(ChIP)技术被广泛应用于染色体结合位点的鉴定。在活细胞中，染色质蛋白与其目标序列发生交叉连接。将纯化的染色质剪切，并用特异性抗体免疫沉淀富集相关蛋白。然后通过大规模并行测序(CHIP-SEQ)确定共纯化的基因组DNA。我们应用CHIP-SEQ技术在果蝇胚胎中定位了两个含有ISWI的核小体重塑因子ACF和RSF的染色体结合位点。利用针对其标志性亚基ACF1和RSF-1的几种多克隆和单克隆抗体，获得了稳健的图谱，表明这两个重构体共同占据了大量活性启动子。进一步的验证包括使用染色质对不表达ACF1或RSF-1的突变胚胎进行对照。令人惊讶的是，CHIP-SEQ图谱没有变化，这表明它们不是由于特定的免疫沉淀。保守分析列出了大约3000个染色体座位，其中大部分是活跃的启动子，容易在芯片中非特异地浓缩，表现为“幻影峰”。这些峰不是免疫前血清获得的，在输入染色质中也不明显。挖掘modENCODE芯片序列图谱在许多表观遗传调控因子图谱中识别出潜在的幻影峰。这些图谱和其他具有显著幻影峰的芯片序列数据必须与目标蛋白质已经耗尽的细胞的染色质进行验证。
Chromatin immunoprecipitation (ChIP) is widely used to identify chromosomal binding sites. Chromatin proteins are cross-linked to their target sequences in living cells. The purified chromatin is sheared and the relevant protein is enriched by immunoprecipitation with specific antibodies. The co-purifying genomic DNA is then determined by massive parallel sequencing (ChIP-seq). We applied ChIP-seq to map the chromosomal binding sites for two ISWI-containing nucleosome remodeling factors, ACF and RSF, in Drosophila embryos. Employing several polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF-1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters. Further validation included controls using chromatin of mutant embryos that do not express ACF1 or RSF-1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and appear as ‘Phantom Peaks’. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin. Mining the modENCODE ChIP-seq profiles identifies potential Phantom Peaks in many profiles of epigenetic regulators. These profiles and other ChIP-seq data featuring prominent Phantom Peaks must be validated with chromatin from cells in which the protein of interest has been depleted.