Ministry of Education Key Laboratory of Resource Biology and Biotechnology in Western China; Shaanxi Provincial Key Laboratory of Biotechnology; School of Medicine, Northwest University, Xi'an, China.
Department of Biomedical Sciences, Jockey Club College of Veterinary Medicine and Life Sciences; The Tung Biomedical Sciences Centre, City University of Hong Kong, Hong Kong SAR, China.
Sci Adv. 2024 Aug 2;10(31):eadn1397. doi: 10.1126/sciadv.adn1397. Epub 2024 Jul 31.
Chromatin marks are associated with transcriptional regulatory activities. However, very few lncRNAs have been characterized with the role in regulating epigenetic marks, largely due to the technical difficulty in identifying chromatin-associating RNA. Current methods are largely limited by the availability of ChIP-grade antibody and the crosslinking, which generates high noise. Here, we developed a method termed Chrom-seq to efficiently capture RNAs associated with various chromatin marks in living cells. Chrom-seq jointly applies highly specific chromatin mark reader with APEX2, which catalyzes the oxidation of biotin-aniline to label the adjacent RNAs for isolation by streptavidin-coated beads. Using the readers of mCBX7/dPC, mCBX1, and mTAF3, we detected RNA species significantly associated with H3K27me3, H3K9me3, and H3K4me3, respectively. We demonstrated that Chrom-seq outperformed other equivalent methods in terms of sensitivity, efficiency, and cost of practice. It provides an antibody-free approach to systematically map RNAs at chromatin marks with potential regulatory roles in epigenetic events.
染色质标记与转录调控活性有关。然而，很少有长链非编码 RNA 被证实具有调节表观遗传标记的作用，这主要是由于鉴定与染色质相关的 RNA 的技术难度较大。目前的方法在很大程度上受到 ChIP 级抗体和交联的可用性的限制，这会产生很高的噪声。在这里，我们开发了一种称为 Chrom-seq 的方法，可有效地捕获活细胞中与各种染色质标记相关的 RNA。Chrom-seq 联合使用高度特异性的染色质标记读取器和 APEX2，APEX2 可催化生物素苯胺的氧化，以标记相邻的 RNA，然后通过链霉亲和素包被的珠子进行分离。使用 mCBX7/dPC、mCBX1 和 mTAF3 的读取器，我们分别检测到与 H3K27me3、H3K9me3 和 H3K4me3 显著相关的 RNA 种类。我们证明，Chrom-seq 在灵敏度、效率和实践成本方面均优于其他等效方法。它提供了一种无抗体的方法，可以系统地绘制在表观遗传事件中具有潜在调节作用的染色质标记处的 RNA。
