Long Yicheng, Hwang Taeyoung, Gooding Anne R, Goodrich Karen J, Hanson Skylar D, Vallery Tenaya K, Rinn John L, Cech Thomas R
Department of Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, CO, USA.
Howard Hughes Medical Institute, University of Colorado, Boulder, CO, USA.
bioRxiv. 2024 May 2:2023.08.17.553776. doi: 10.1101/2023.08.17.553776.
Polycomb Repressive Complex 2 (PRC2), an important histone modifier and epigenetic repressor, has been known to interact with RNA for almost two decades. In our previous publication (Long, Hwang et al. 2020), we presented data supporting the functional importance of RNA interaction in maintaining PRC2 occupancy on chromatin, using comprehensive approaches including an RNA-binding mutant of PRC2 and an rChIP-seq assay. Recently, concerns have been expressed regarding whether the RNA-binding mutant has impaired histone methyltransferase activity and whether the rChIP-seq assay can potentially generate artifacts. Here we provide new data that support a number of our original findings. First, we found the RNA-binding mutant to be fully capable of maintaining H3K27me3 levels in human induced pluripotent stem cells. The mutant had reduced methyltransferase activity in vitro, but only on some substrates at early time points. Second, we found that our rChIP-seq method gave consistent data across antibodies and cell lines. Third, we further optimized rChIP-seq by using lower concentrations of RNase A and incorporating a catalytically inactive mutant RNase A as a control, as well as using an alternative RNase (RNase T1). The EZH2 rChIP-seq results using the optimized protocols supported our original finding that RNA interaction contributes to the chromatin occupancy of PRC2.
多梳抑制复合体2（PRC2）是一种重要的组蛋白修饰因子和表观遗传抑制因子，近二十年来一直被认为可与RNA相互作用。在我们之前的出版物（Long、Hwang等人，2020年）中，我们通过包括PRC2的RNA结合突变体和rChIP-seq分析在内的综合方法，提供了支持RNA相互作用在维持PRC2在染色质上占据的功能重要性的数据。最近，有人对RNA结合突变体是否损害组蛋白甲基转移酶活性以及rChIP-seq分析是否可能产生假象表示担忧。在这里，我们提供了新的数据来支持我们最初的一些发现。首先，我们发现RNA结合突变体完全能够在人诱导多能干细胞中维持H3K27me3水平。该突变体在体外甲基转移酶活性降低，但仅在早期时间点对某些底物有影响。其次，我们发现我们的rChIP-seq方法在不同抗体和细胞系中产生了一致的数据。第三，我们通过使用较低浓度的核糖核酸酶A并加入催化失活的突变体核糖核酸酶A作为对照，以及使用另一种核糖核酸酶（核糖核酸酶T1）进一步优化了rChIP-seq。使用优化方案的EZH2 rChIP-seq结果支持了我们最初的发现，即RNA相互作用有助于PRC2在染色质上的占据。
