Intercalation of small molecules into DNA in chromatin is primarily controlled by superhelical constraint.

Rosevalentine Bosire,Árpád Szöőr,Beatrix Dienes,Attila Kovács,Gábor Szabó,Ralf Seidel,György Vámosi,Péter Nánási,László Imre,Anett Mázló,Ronald Hancock

doi:10.1371/journal.pone.0224936

Abstract

The restricted access of regulatory factors to their binding sites on DNA wrapped around the nucleosomes is generally interpreted in terms of molecular shielding exerted by nucleosomal structure and internucleosomal interactions. Binding of proteins to DNA often includes intercalation of hydrophobic amino acids into the DNA. To assess the role of constrained superhelicity in limiting these interactions, we studied the binding of small molecule intercalators to chromatin in close to native conditions by laser scanning cytometry. We demonstrate that the nucleosome-constrained superhelical configuration of DNA is the main barrier to intercalation. As a result, intercalating compounds are virtually excluded from the nucleosome-occupied regions of the chromatin. Binding of intercalators to extranucleosomal regions is limited to a smaller degree, in line with the existence of net supercoiling in the regions comprising linker and nucleosome free DNA. Its relaxation by inducing as few as a single nick per ~50 kb increases intercalation in the entire chromatin loop, demonstrating the possibility for long-distance effects of regulatory potential.

Highlights

The nucleosomes interspersed with linker DNA and the linker histones form the basic repeating unit of chromatin serving, from a structural point-of-view, to compact and package eukaryotic DNA [1]
By incubating live HeLa cells with the intercalating dye ethidium bromide (EBr), we observed that the dye is taken up and its fluorescence is observed in the cytoplasm and nucleoli but not in the chromatin. (Fig 1A–1C)
In addition to the barrier presented by the plasma membrane of viable cells, the chromatin itself resists staining with the intercalating dye EBr (Fig 1)

Summary

Introduction

The nucleosomes interspersed with linker DNA and the linker histones form the basic repeating unit of chromatin serving, from a structural point-of-view, to compact and package eukaryotic DNA [1]. Accessibility of DNA within the chromatin to regulatory factors controls transcription, replication, recombination and repair [2]. A combination of nuclease hypersensitivity assays with generation sequencing (NGS) techniques have been used to characterize chromatin accessibility genome-wide. The most widely used techniques include MNase-seq [3], DNase-seq [4], ATAC-seq [5] and NOMe-seq [6].

Methods

Results

Conclusion