Telomere-specific chromatin capture using a pyrrole\u2013imidazole polyamide probe for the identification of proteins and non-coding RNAs

Satoru Ide,Toshikazu Bando,Kazuhiro Maeshima,Asuka Sasaki,Yusuke Kawamoto,Hiroshi Sugiyama

doi:10.1186/s13072-021-00421-8

Abstract

BackgroundKnowing 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.ResultsWe 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.ConclusionPI-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.

Highlights

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
PI polyamide probe for affinity purification of telomeric repeats DNA We developed a method, PI-PRICh, which allows unbiased high-throughput identifications of telomeric chromatin-bound proteins and RNAs (Fig. 1a)
We first confirmed TH59-DB efficiently pulled down a plasmid containing a 750-bp telomeric repeats fragment, whereas the empty plasmid was not retrieved (Fig. 1c, d)

Summary

Introduction

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. All of them have some shortcomings in identifying non-coding RNA associated with DNA regulatory elements. While chromatin immunoprecipitation (ChIP) is used to map protein–DNA interactions, genome-wide analyses of ncRNA-binding sites, such as CHART, ChIRP and RAP, have been developed to map chromatin-associated RNAs [9,10,11]. These latter assays utilize antisense oligonucleotide probes to retrieve specific ncRNAs with bound DNA sequences. Methods for comprehensive and unbiased identification of ncRNAs associated with specific DNA regulatory elements are still in need

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