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Linker histones (LHs) bind to nucleosomes with their globular domain (gH) positioned in either an on- or an off-dyad binding mode. Here, we study the effect of the linker DNA (L-DNA) sequence on the binding of a full-length LH, Xenopus laevis H1.0b, to a Widom 601 nucleosome core particle (NCP) flanked by two 40 bp long L-DNA arms, by single-pair FRET spectroscopy. We varied the sequence of the 11 bp of L-DNA adjoining the NCP on either side, making the sequence either A-tract, purely GC, or mixed with 64% AT. The labeled gH consistently exhibited higher FRET efficiency with the labeled L-DNA containing the A-tract than that with the pure-GC stretch, even when the stretches were swapped. However, it did not exhibit higher FRET efficiency with the L-DNA containing 64% AT-rich mixed DNA when compared to the pure-GC stretch. We explain our observations with a model that shows that the gH binds on dyad and that two arginines mediate recognition of the A-tract via its characteristically narrow minor groove. To investigate whether this on-dyad minor groove-based recognition was distinct from previously identified off-dyad major groove-based recognition, a nucleosome was designed with A-tracts on both the L-DNA arms. One A-tract was complementary to thymine and the other to deoxyuridine. The major groove of the thymine-tract was lined with methyl groups that were absent from the major groove of the deoxyuridine tract. The gH exhibited similar FRET for both these A-tracts, suggesting that it does not interact with the thymine methyl groups exposed on the major groove. Our observations thus complement previous studies that suggest that different LH isoforms may employ different ways of recognizing AT-rich DNA and A-tracts. This adaptability may enable the LH to universally compact scaffold-associated regions and constitutive heterochromatin, which are rich in such sequences., (Copyright © 2021. Published by Elsevier Inc.)

Background Fluorescence recovery after photobleaching (FRAP) studies can provide kinetic information about proteins in cells. Single point mutations can significantly affect the binding kinetics of proteins and result in variations in the recovery half time (t 50 ) measured in FRAP experiments. FRAP measurements of linker histone (LH) proteins in the cell nucleus have previously been reported by Brown et al. (2006) and Lele et al. (2006). Methods We performed Brownian dynamics (BD) simulations of the diffusional association of the wild-type and 38 single or double point mutants of the globular domain of mouse linker histone H1.0 (gH1.0) to a nucleosome. From these simulations, we calculated the bimolecular association rate constant (k on ), the Gibbs binding free energy (ΔG) and the dissociation rate constant (k off ) related to formation of a diffusional encounter complex between the nucleosome and the gH1.0. Results We used these parameters, after application of a correction factor to account for the effects of the crowded environment of the nucleus, to compute FRAP recovery times and curves that are in good agreement with previously published, experimentally measured FRAP recovery time courses. Conclusions Our computational analysis suggests that BD simulations can be used to predict the relative effects of single point mutations on FRAP recovery times related to protein binding. General Significance BD simulations assist in providing a detailed molecular level interpretation of FRAP data., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chromatosomes are fundamental units of chromatin structure that are formed when a linker histone protein binds to a nucleosome. The positioning of the linker histone on the nucleosome influences the packing of chromatin. Recent simulations and experiments have shown that chromatosomes adopt an ensemble of structures that differ in the geometry of the linker histone-nucleosome interaction. In this article we review the application of Brownian, Monte Carlo, and molecular dynamics simulations to predict the structure of linker histone-nucleosome complexes, to study the binding mechanisms involved, and to predict how this binding affects chromatin fiber structure. These simulations have revealed the sensitivityof the chromatosome structure to variations in DNA and linker histone sequence, as well as to posttranslational modifications, thereby explaining the structural variability observed in experiments. We propose that a concerted application of experimental and computational approaches will reveal the determinants of chromatosome structural variability and how it impacts chromatin packing.

There is renewed interest in linker histone (LH)-nucleosome binding and how LHs influence eukaryotic DNA compaction. For a long time, the goal was to uncover "the structure of the chromatosome," but recent studies of LH-nucleosome complexes have revealed an ensemble of structures. Notably, the reconstituted LH-nucleosome complexes used in experiments rarely correspond to the sequence combinations present in organisms. For a full understanding of the determinants of the distribution of the chromatosome structural ensemble, studies must include a complete description of the sequences and experimental conditions used, and be designed to enable systematic evaluation of sequence and environmental effects., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Linker histone (LH) proteins play a key role in higher-order structuring of chromatin for the packing of DNA in eukaryotic cells and in the regulation of genomic function. The common fruit fly (Drosophila melanogaster) has a single somatic isoform of the LH (H1). It is thus a useful model organism for investigating the effects of the LH on nucleosome compaction and the structure of the chromatosome, the complex formed by binding of an LH to a nucleosome. The structural and mechanistic details of how LH proteins bind to nucleosomes are debated. Here, we apply Brownian dynamics simulations to compare the nucleosome binding of the globular domain of D. melanogaster H1 (gH1) and the corresponding chicken (Gallus gallus) LH isoform, gH5, to identify residues in the LH that critically affect the structure of the chromatosome. Moreover, we investigate the effects of posttranslational modifications on the gH1 binding mode. We find that certain single-point mutations and posttranslational modifications of the LH proteins can significantly affect chromatosome structure. These findings indicate that even subtle differences in LH sequence can significantly shift the chromatosome structural ensemble and thus have implications for chromatin structure and transcriptional regulation., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Linker histones are essential for DNA compaction in chromatin. They bind to nucleosomes in a 1:1 ratio forming chromatosomes. Alternative configurations have been proposed in which the globular domain of the linker histone H5 (gH5) is positioned either on- or off-dyad between the nucleosomal and linker DNAs. However, the dynamic pathways of chromatosome assembly remain elusive. Here, we studied the conformational plasticity of gH5 in unbound and off-dyad nucleosome-bound forms with classical and accelerated molecular dynamics simulations. We find that the unbound gH5 converts between open and closed conformations, preferring the closed form. However, the open gH5 contributes to a more rigid chromatosome and restricts the motion of the nearby linker DNA through hydrophobic interactions with thymidines. Moreover, the closed gH5 opens and reorients in accelerated simulations of the chromatosome. Brownian dynamics simulations of chromatosome assembly, accounting for a range of amplitudes of nucleosome opening and different nucleosome DNA sequences, support the existence of both on- and off-dyad binding modes of gH5 and reveal alternative, sequence and conformation-dependent chromatosome configurations. Taken together, these findings suggest that the conformational dynamics of linker histones and nucleosomes facilitate alternative chromatosome configurations through an interplay between induced fit and conformational selection., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)