Telomere-specific chromatin capture using a pyrrole–imidazole polyamide probe for the identification of proteins and non-coding [PI-PRICh, DNA_pulldown1]

Background: Knowing chromatin components at a DNA regulatory element at any given time is essential for understanding how the element works during cellular proliferation, differentiation and development. A region-specific chromatin purification is an invaluable approach to dissecting the comprehensive chromatin composition at a particular region. Several methods (e.g., PICh, enChIP, CAPTURE and CLASP) have been developed for isolating and analyzing chromatin components. However, all of them have some shortcomings in identifying non-coding RNA associated with DNA regulatory elements. Results: We have developed a new approach for affinity purification of specific chromatin segments employing an N-methyl pyrrole (P)-N-methylimidazole (I) (PI) polyamide probe, which binds to a specific sequence in double-stranded DNA via Watson–Crick base pairing as a minor groove binder. This new technique is called proteomics and RNA-omics of isolated chromatin segments (PI-PRICh). Using PI-PRICh to isolate mouse and human telomeric components, we found enrichments of shelterin proteins, the well-known telomerase RNA component (TERC) and telomeric repeat-containing RNA (TERRA). When PI-PRICh was performed for alternative lengthening of telomere (ALT) cells with highly recombinogenic telomeres, in addition to the conventional telomeric chromatin, we obtained chromatin regions containing telomeric repeat insertions scattered in the genome and their associated RNAs. Conclusion: PI-PRICh reproducibly identified both the protein and RNA components of telomeric chromatin when targeting telomere repeats. PI polyamide is a promising alternative to simultaneously isolate associated proteins and RNAs of sequence-specific chromatin regions under native conditions, allowing better understanding of chromatin organization and functions within the cell. Overall design: DNA regions pulled down by TH59-DB pull-down in PI-PRICh for HeLa1.3 and U2-OS cells

背景：在任意特定时刻，解析DNA调控元件的染色质组成成分，对于理解该元件在细胞增殖、分化与发育过程中的功能机制至关重要。区域特异性染色质纯化技术，是解析特定区域完整染色质组成的极具价值的研究手段。目前已开发出PICh、enChIP、CAPTURE及CLASP等多种方法，用于染色质成分的分离与分析，但上述方法在鉴定与DNA调控元件结合的非编码RNA时均存在一定局限。结果：我们开发了一种全新的特异性染色质片段亲和纯化方法，该方法采用N-甲基吡咯（P）-N-甲基咪唑（I）（PI）聚酰胺探针作为小沟结合剂，通过沃森-克里克碱基配对结合双链DNA中的特定序列。这项新技术被命名为分离染色质片段蛋白质组与RNA组学分析技术（PI-PRICh）。利用PI-PRICh分离小鼠和人类端粒组分时，我们发现shelterin蛋白、经典端粒酶RNA组分（TERC）以及端粒重复序列相关RNA（TERRA）均出现显著富集。针对端粒具有高度重组活性的端粒替代延长（ALT）细胞开展PI-PRICh实验时，除获取常规端粒染色质外，我们还获得了基因组中分散存在的端粒重复序列插入片段及其结合RNA所在的染色质区域。结论：当靶向端粒重复序列时，PI-PRICh可重复性地鉴定出端粒染色质的蛋白质与RNA两类组分。PI聚酰胺作为一种极具潜力的替代方法，可在天然条件下同时分离序列特异性染色质区域的结合蛋白与RNA，有助于更深入地解析细胞内染色质的组织形式与生物学功能。整体实验设计：针对HeLa1.3与U2-OS细胞，采用TH59-DB探针在PI-PRICh实验中富集得到的DNA区域