The apparent loss of PRC2 chromatin occupancy as an artefact of RNA depletion
The apparent loss of PRC2 chromatin occupancy as an artefact of RNA depletion
复制标题
PRC2 染色质占据的明显丧失是 RNA 耗尽的结果
DOI：
10.1101/2023.08.16.553488
复制
发表时间：
2024
期刊：
影响因子：
--
通讯作者：
C. Davidovich
中科院分区：
文献类型：
--
作者：
Evan Healy;Qi Zhang;Emma H. Gail;Samuel C. Agius;Gui;Michael J. Bullen;Varun Pandey;P. P. Das;J. Polo;C. Davidovich
RNA与染色质修饰剂的募集有关，以前的研究提供了支持和反对这一观点的证据。RNA酶处理染色质是研究RNA介导的染色质修饰剂调节的常用工具，但这种方法的局限性仍不清楚。染色质免疫沉淀过程中的RNA酶A处理（RNase-ChIP或rChIP）降低了H3 K27 me 3甲基转移酶PRC 2的染色质占有率。这导致了PRC 2和染色质之间的“RNA桥”的建议。在这里，我们表明，RNA酶A处理染色质免疫沉淀过程中导致的所有兼性异染色质，包括PRC 2和H3 K27 me 3全基因组的明显损失。这种现象在小鼠胚胎干细胞、人类癌细胞和人类诱导多能干细胞中持续存在。我们跟踪这一观察结果，从非靶向染色质获得DNA，以兼性异染色质的DNA为代价进行测序，这减少了ChIP信号。我们的研究结果指出了使用RNase A处理绘制RNA依赖性染色质占有率的实质性局限性，并使先前基于该测定为PRC 2建立的结论无效。ChIP-seq过程中的RNA降解不足以从染色质中取代PRC 2。RNA降解导致多个细胞系中ChIP-seq信号的人工耗尽。ChIP-seq信号的减少可以通过非靶向DNA的增加来解释。RNA在实验过程中维持染色质的溶解度至关重要。
RNA has been implicated in the recruitment of chromatin modifiers, and previous studies have provided evidence in favour and against this idea. RNase treatment of chromatin is a prevalent tool for the study of RNA-mediated regulation of chromatin modifiers, but the limitations of this approach remain unclear. RNase A treatment during chromatin immunoprecipitation (RNase-ChIP or rChIP) reduces chromatin occupancy of the H3K27me3 methyltransferase PRC2. This led to suggestions of an “RNA bridge” between PRC2 and chromatin. Here we show that RNase A treatment during chromatin immunoprecipitation leads to the apparent loss of all facultative heterochromatin, including both PRC2 and H3K27me3 genome wide. This phenomenon persists in mouse embryonic stem cells, human cancer cells and human-induced pluripotent stem cells. We track this observation to a gain of DNA from non-targeted chromatin, sequenced at the expense of DNA from facultative heterochromatin, which reduces ChIP signals. Our results point to substantial limitations in using RNase A treatment for mapping RNA-dependent chromatin occupancy and invalidate conclusions that were previously established for PRC2 based on this assay. Highlights RNA degradation during ChIP-seq is insufficient to displace PRC2 from chromatin. RNA degradation led to the artificial depletion of ChIP-seq signals in multiple cell lines. Artificially reduced ChIP-seq signals are explained by a gain of non-targeted DNA. RNA is critical in maintaining the solubility of chromatin during experimentation.
PRC2 和染色质之间的明显 RNA 桥是 RNA 降解时非特异性染色质沉淀的产物
DOI：
10.1101/2023.08.16.553503
发表时间：
2023
期刊：
--
影响因子：
--
作者：
Hickman A
通讯作者：
Hickman A
影响因子：
16
作者：
Creamer KM;Kolpa HJ;Lawrence JB
通讯作者：
Lawrence JB