超声处理对PRC2相关蛋白染色质免疫共沉淀测序结果的影响

Effect of sonication on results of ChIP-seq experiments involving PRC2 related proteins

摘要:

超声处理是染色质免疫共沉淀 (Chromatin immunoprecipitation，ChIP) 实验过程中染色质片段化的重要手段之一。选用多梳抑制复合物2 (Polycomb repressive complex 2，PRC2) 相关蛋白组蛋白甲基转移酶EZH2及其催化产物H3K27me3为代表，研究不同分子量大小的蛋白在不同超声处理时间下对染色质免疫共沉淀测序(Chromatin immunoprecipitation sequencing，ChIP-seq)实验的影响，结果表明在启动子区或非启动子区，不同超声时间下小分子量组蛋白H3K27me3结合位点的注释基因均无明显差异，说明超声时间对组蛋白ChIP-seq数据影响不大。与组蛋白不同，超声时间从10 min延长至20 min后启动子区EZH2新增结合位点的注释基因能够显著聚类在与肌动蛋白丝组装等相关通路上。超声20 min相比10 min，非启动子区EZH2新增基因的GO (Gene ontology) 聚类通路要远多于丢失基因，且超声30 min相比20 min，非启动子区丢失基因的GO聚类通路要远多于新增基因，这些通路大多与RNA聚合酶Ⅱ (RNA polymerase Ⅱ，RNAPII)、器官发育、细胞形态发生相关。这表明超声时间不足或过长均会导致EZH2的基因组定位信息的不全。另外，超声主要影响启动子区中的PRC2非结合区域及二价启动子区域的EZH2结合位点，还影响非启动子区中PRC2结合区域、PRC2不结合区域以及活化态增强子区域的EZH2结合位点。综上，建议对大分子量的染色质修饰相关蛋白优化超声处理时间，使形成的染色质片段聚集在100–500 bp可获得比较全面的基因组信息。对小分子量的组蛋白来说，超声时间对ChIP-seq结果影响不大。

Abstract:

Sonication is one of the essential strategies of chromatin fragmentation in Chromatin immunoprecipitation (ChIP) assay. The impact of proteins with different molecular weights generated under different duration of sonication on the results of Chromatin immunoprecipitation sequencing (ChIP-seq) experiments involving the Polycomb Repressive Complex 2 (PRC2) related protein EZH2, which is a histone methyltransferase, and its product H3K27me3 was investigated. The results indicate that in the promoter region or nonpromoter region, there were hardly any differences among the H3K27me3 peaks annotated genes from different duration of sonication, which suggesting that the duration of sonication had little effect on histone ChIP-seq results. In contrast, in the promoter region newly gained EZH2 peaks annonated genes at 20 min sonication time were significantly clustered in the gene ontology (GO) pathways related to actin filament bundle compared with 10 min. In the nonpromoter region, compared with 10 min, the GO pathways of newly gained EZH2 peaks annonated genes at 20 min sonication time is much more than that of lost EZH2 peaks annonated genes. And in the nonpromoter region, compared with 20 min, the GO pathways of lost EZH2 peaks annonated genes at 30 min sonication time is much more than that of newly gained EZH2 peaks annonated genes. Most of these pathways are associated with RNA polymerase II (RNAPII), organ development and cell morphogenesis. These suggest that the genomic information of EZH2 will be lost if the duration of sonication is not enough or too long. Different duration of sonication mainly affect the EZH2 peaks in PRC2 unoccupied region and the bivalent promoter in the promoter region, as well as the PRC2 occupied region, PRC2 unoccupied region and the activated enhancer in the nonpromoter region. Therefore, the sonication for the chromatin related proteins with high molecular weights need to be optimized to make chromatin fragments size vary from 100 bp to 500 bp, which will yield relatively comprehensive genomic information of the target protein. For histones, which are of small molecular weights, duration of sonication has little effect on them.