Genome Dynamics Laboratory, National Institute of Genetics, ROIS, Mishima, Shizuoka, 411-8540, Japan.
Department of Genetics, School of Life Science, SOKENDAI, Mishima, Shizuoka, 411-8540, Japan.
Epigenetics Chromatin. 2021 Oct 9;14(1):46. doi: 10.1186/s13072-021-00421-8.
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.
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.
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.
在细胞增殖、分化和发育过程中，了解特定 DNA 调控元件的染色质成分对于理解该元件的工作机制至关重要。区域特异性染色质纯化是剖析特定区域全面染色质组成的一种非常有价值的方法。已经开发了几种方法（例如 PICh、enChIP、CAPTURE 和 CLASP）来分离和分析染色质成分。然而，所有这些方法在识别与 DNA 调控元件相关的非编码 RNA 方面都存在一些缺点。
我们开发了一种使用 N-甲基吡咯（P）-N-甲基咪唑（I）（PI）聚酰胺探针亲和纯化特定染色质片段的新方法，该探针通过 Watson-Crick 碱基配对作为小沟结合物与双链 DNA 中的特定序列结合。这种新技术称为分离染色质片段的蛋白质组学和 RNA 组学（PI-PRICh）。使用 PI-PRICh 分离小鼠和人类端粒成分时，我们发现了庇护蛋白、众所周知的端粒酶 RNA 成分（TERC）和端粒重复 RNA（TERRA）的富集。当对具有高度重组性端粒的端粒延长替代（ALT）细胞进行 PI-PRICh 时，除了常规的端粒染色质外，我们还获得了包含端粒重复插入物散布在基因组中的染色质区域及其相关 RNA。
PI-PRICh 靶向端粒重复时，可重复性地鉴定端粒染色质的蛋白质和 RNA 成分。PI 聚酰胺是一种很有前途的替代方法，可以在天然条件下同时分离序列特异性染色质区域的相关蛋白质和 RNA，从而更好地理解细胞内染色质的组织和功能。
