A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB [ChIP-seq]

The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA. Overall design: CS5246 EZH2 ChIP in WT mESCs

多梳抑制复合体2（Polycomb Repressive Complex 2, PRC2）是一类保守的酶，可对组蛋白H3的赖氨酸27位点进行三甲基化修饰（H3K27me3），进而介导基因沉默。PRC2对特定长链非编码RNA（long noncoding RNAs, lncRNAs）的表达具有显著响应性。其中最典型的案例为：在X染色体失活过程中，长链非编码RNA Xist开始表达后不久，PRC2即被招募至X染色体上。然而，lncRNAs将PRC2招募至染色质的具体分子机制尚未阐明。本研究发现，一种广泛使用的、靶向PRC2催化亚基人类EZH2的兔单克隆抗体，在染色质免疫沉淀（chromatin immunoprecipitation, ChIP）常用的缓冲液条件下，可在小鼠胚胎干细胞（mouse embryonic stem cells, ESCs）中与RNA结合蛋白支架附着因子B（Scaffold Attachment Factor B, SAFB）发生交叉反应。研究人员通过在ESCs中敲除EZH2，经蛋白质印迹法验证该抗体仅特异性识别EZH2（无交叉反应）。同样，与已发表数据集的比对结果证实，该抗体可通过ChIP-Seq富集PRC2结合位点。但采用ChIP洗脱条件对甲醛交联的ESCs进行RNA免疫沉淀（RNA-IP）时，可检测到一类独特的RNA结合峰，该峰与SAFB结合峰共定位，且其富集信号会在SAFB敲除后消失，但在EZH2敲除后无此变化。对野生型及EZH2敲除ESCs进行免疫沉淀（immunoprecipitation, IP）及基于质谱的蛋白质组学分析，进一步证实该EZH2抗体可通过不依赖EZH2的方式富集SAFB。本研究结果凸显了在研究染色质修饰酶与RNA的相互作用时，采用正交实验方法的重要性。实验整体设计：野生型小鼠胚胎干细胞中针对CS5246 EZH2的ChIP实验。