Chu Ci, Quinn Jeffrey, Chang Howard Y
Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine.
J Vis Exp. 2012 Mar 25(61):3912. doi: 10.3791/3912.
Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression (1,2,3,4,5,6,7). The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion (8,9). While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation (10,11). However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide( 3,12,13), but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex (14); HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex (13). Prior studies mapping RNA occupancy at chromatin have revealed substantial insights (15,16), but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution (17). This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.
长链非编码RNA是重要生物学过程中染色质状态的关键调节因子，如剂量补偿、印记和发育基因表达(1,2,3,4,5,6,7)。最近发现数千种lncRNA与特定的染色质修饰复合物相关联，比如介导组蛋白H3赖氨酸27三甲基化(H3K27me3)的多梳抑制复合物2(PRC2)，这表明众多lncRNA在以基因特异性方式管理染色质状态方面具有广泛作用(8,9)。虽然一些lncRNA被认为在顺式作用下影响邻近基因，但其他lncRNA则通过反式作用来调控远距离的基因。例如，果蝇lncRNA roX1和roX2结合雄性细胞X染色体上的众多区域，对剂量补偿至关重要(10,11)。然而，其结合位点的精确位置在高分辨率下尚不清楚。同样，人类lncRNA HOTAIR可在全基因组范围内影响数百个基因上PRC2的占据情况(3,12,13)，但特异性是如何实现的尚不清楚。lncRNA还可作为模块化支架来募集多种蛋白质复合物的组装。经典的反式作用RNA支架是TERC RNA，它作为端粒酶复合物的模板和支架(14);HOTAIR也可作为PRC2和H3K4去甲基化酶复合物的支架(13)。先前绘制染色质上RNA占据情况的研究已揭示了大量见解(15,16)，但每次仅针对单个基因座。大多数lncRNA的占据位点尚不清楚，lncRNA在染色质调控中的作用大多是从lncRNA干扰的间接效应推断出来的。正如随后进行微阵列或深度测序的染色质免疫沉淀(分别为ChIP-chip或ChIP-seq)极大地增进了我们在基因组水平上对蛋白质-DNA相互作用的理解一样，在此我们阐述一种最近发表的在全基因组范围内以高分辨率绘制长链RNA占据情况的策略(17)。这种方法，即通过RNA纯化进行染色质分离(ChIRP)(图1)，基于用平铺的反义寡核苷酸亲和捕获靶标lncRNA:染色质复合物，然后以数百个碱基的分辨率生成基因组结合位点图谱，具有高灵敏度和低背景。ChIRP适用于许多lncRNA，因为鉴于RNA序列，亲和探针的设计很简单，并且不需要了解RNA的结构或功能域。
