Chrom-seq Identifies RNAs at Chromatin Marks [Chrom-seq]

Chromatin marks are associated with transcriptional regulatory activities. 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. Chrom-seq provides an antibody-free approach to systematically map RNAs at chromatin marks with potential regulatory roles in different epigenetic events. Chrom-seq leverages highly specific reader protein to bring the engineered ascorbate peroxidase APEX2 close to the modified chromatin. Upon exposure to H2O2, APEX2 catalyzes oxidation of surrounding biotin-aniline (Btn-An) which then reacts with guanosines in adjacent RNA molecules to form a covalent adduct, facilitating subsequent purification of tagged RNAs with streptavidin-conjugated beads for identification by sequencing.

染色质标记（chromatin marks）与转录调控活性紧密相关。本研究开发了一种名为Chrom-seq的方法，可在活细胞中高效捕获与各类染色质标记相结合的RNA。Chrom-seq将高特异性染色质标记识别蛋白与APEX2联合使用，APEX2可催化生物素苯胺（biotin-aniline）的氧化反应，以此标记邻近RNA，进而通过链霉亲和素包被磁珠实现分离纯化。本研究使用mCBX7/dPC、mCBX1及mTAF3三种识别蛋白，分别检测到了与H3K27me3、H3K9me3及H3K4me3显著相关的RNA物种。Chrom-seq提供了一种无需抗体的研究策略，可系统性绘制染色质标记处的RNA图谱，这类RNA在不同表观遗传事件中具备潜在调控功能。Chrom-seq借助高特异性识别蛋白，将工程化抗坏血酸过氧化物酶APEX2靶向结合至修饰后的染色质附近。当暴露于过氧化氢（H₂O₂）时，APEX2可催化周围的生物素苯胺（Btn-An）发生氧化，随后氧化产物可与邻近RNA分子中的鸟苷发生反应，形成共价加合物，进而可通过链霉亲和素偶联磁珠纯化带有标记的RNA，最终通过测序完成鉴定。