Binding of NF-κB to Nucleosomes: Effect of Translational Positioning, Nucleosome Remodeling and Linker Histone H1

Imtiaz Nisar Lone,Zahary Yordanov Peshev,Dimitar Angelov,John Lalith Charles Richard,Manu Shubhdarshan Shukla,Stefan Dimitrov

doi:10.1371/journal.pgen.1003830

Abstract

NF-κB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. Here we used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates. We show that NF-κB p50 homodimer is able to bind to its recognition sequence, when it is localized at the edge of the core particle, but not when the recognition sequence is at the interior of the nucleosome. Remodeling of the nucleosome by the chromatin remodeling machine RSC was not sufficient to allow binding of NF-κB to its recognition sequence located in the vicinity of the nucleosome dyad, but RSC-induced histone octamer sliding allowed clearly detectable binding of NF-κB with the slid particle. Importantly, nucleosome dilution-driven removal of H2A–H2B dimer led to complete accessibility of the site located close to the dyad to NF-κB. Finally, we found that NF-κB was able to displace histone H1 and prevent its binding to nucleosome. These data provide important insight on the role of chromatin structure in the regulation of transcription of NF-κB dependent genes.

Highlights

In eukaryotes, all DNA-templated reactions occur in the context of chromatin
Most studies of gene induction by inflammatory stimuli have focused on transcription factors that recognize specific DNA sequences and the cytoplasmic events that regulate the activation of these transcription factors
Transcriptional activation of eukaryotic genes is influenced by chromatin structure

Summary

Introduction

All DNA-templated reactions occur in the context of chromatin. The repeating structure of chromatin, the nucleosome, consists of a nucleosome core (made up of two copies of each core histone H2A, H2B, H3 and H4) around which 147 bp of DNA is wrapped [1], a linker histone and a linker DNA [2]. Sequence-specific binding of transcription factors is the key event for gene activation. Transcription factors must get access to their regulatory sites. In general the nucleosomes represent a barrier for the access of transcription factors to their cognate sequence [5]. Some transcription factors such as human glucocorticoid receptor [6,7,8], yeast PHO2/ PHO4 proteins [9], and GAL4 [10,11] have been shown to bind to their recognition sequences embedded in the nucleosomes. Other distinct transcription factors, such as Sp1, Lef-1, ETS-1 and USF have been shown to be able to invade the nucleosome and to interact with their cognate sequences [13,14]

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Conclusion