Effect of A-tract-mediated linker histone orientation on trinucleosome compaction

Linker histones (LH) have been shown to preferentially bind to AT-rich DNA, and specifically to A-tracts, contiguous stretches of adenines. Recently, using spFRET (single pair Foerster/Fluorescence Resonance Energy Transfer), we showed that the globular domain (gH) of Xenopus laevis H1.0b LH orients towards A-tracts present on the linker-DNA (L-DNA) in LH:mononucleosome complexes. We investigated the impact of this A-tract mediated orientation of the gH on the compaction of higher-order structures by studying trinucleosomes as minimal models for chromatin. Two 600 bp DNA sequences were constructed containing three Widom 601 core sequences separated by about 40 bp linkers and A-tracts inserted on either the outer or the inner L-DNAs flanking the 1st and the 3rd Widom 601 sequences. The two inner L-DNAs were fluorescently labelled at their midpoints. Trinucleosomes were reconstituted using the doubly-labelled 600 bp DNA, core histone octamers and the full-length H1.0b LH. SpFRET was performed for a range of NaCl concentrations. While the LH compacted the trinucleosomes, surprisingly, the extent of compaction was similar for trinucleosomes with A-tracts either on the two outer or on the two inner L-DNAs. Modeling constrained by the FRET efficiency suggests that the trinucleosomes adopt a zig-zagged conformation with the 1st and 3rd nucleosomes stacked on top of each other. Even though we expect that the gH of neighbouring (1st and 3rd) LHs are oriented towards the A-tracts, our models suggest that they are not sufficiently close to dimerize and affect compaction. Thus, despite differences in A-tract placements, the LH compacts trinucleosomes similarly.

Histone sequence variation in divergent eukaryotes facilitates diversity in chromatin packaging

The histone proteins defining nucleosome structure are highly conserved in common model organisms and are frequently portrayed as uniform chromatin building blocks. We surveyed over 1700 complete eukaryotic genomes and confirm that almost all encode recognisable canonical core histones. Nevertheless, divergent eukaryotes show unrecognised diversity in histone sequences and offer an opportunity to observe the potential for nucleosome variation. Recombinant histones for Plasmodium falciparum, Giardia lamblia, Encephalitozoon cuniculi and Leishmania major were prepared alongside those for human, Xenopus laevis and Saccharomyces cerevisiae. All could be assembled into nucleosomes in vitro on sequences known to direct positioning with metazoan histones. P. falciparum histones refolded into very stable nucleosomes consistent with a highly regulated transcriptional programme. In contrast, G. lamblia and E. cuniculi histones formed less stable nucleosomes and were prone to aggregation as H3-H4 tetramers. Inspection of the histone fold dimer interface residues suggested a potential to form tetrasomal arrays consistent with polymerisation. DNA binding preferences observed using systematic evolution of ligands by exponential enrichment (SELEX) for human, P. falciparum and E. cuniculi histone octamers were highly similar and reflect a shared capability to package diverse genomic sequences. This demonstrates that nucleosomal organisation is retained across eukaryotes and can accommodate genome variation, but histone protein sequences vary more than commonly recognised to provide the potential for diversity of chromatin features.

CHD4 slides nucleosomes by decoupling entry- and exit-side DNA translocation

SummaryChromatin remodellers hydrolyse ATP to move nucleosomal DNA against histone octamers. The mechanism, however, is only partially resolved, and unclear if it is conserved among the four remodeller families. Here we use single-molecule assays to examine the mechanism of action of CHD4, which is part of the least well understood family of remodellers. We demonstrate that the binding energy for CHD4-nucleosome complex formation – even in the absence of nucleotide – triggers significant conformational changes in DNA at the entry side, effectively priming the system for remodelling. During remodelling, flanking DNA enters the nucleosome in a continuous, gradual manner but exits in concerted 4–6 base-pair steps. This decoupling of entry- and exit-side translocation suggests that ATP-driven movement of entry-side DNA builds up strain inside the nucleosome that is subsequently released at the exit side by DNA expulsion. We propose a mechanism for nucleosome sliding based on these and published data.

Histone H4 H75E mutation attenuates global genomic and Rad26-independent transcription-coupled nucleotide excision repair

Nucleotide excision repair (NER) consists of global genomic NER (GG-NER) and transcription coupled NER (TC-NER) subpathways. In eukaryotic cells, genomic DNA is wrapped around histone octamers (an H3–H4 tetramer and two H2A–H2B dimers) to form nucleosomes, which are well known to profoundly inhibit the access of NER proteins. Through unbiased screening of histone H4 residues in the nucleosomal LRS (loss of ribosomal DNA-silencing) domain, we identified 24 mutations that enhance or decrease UV sensitivity of Saccharomyces cerevisiae cells. The histone H4 H75E mutation, which is largely embedded in the nucleosome and interacts with histone H2B, significantly attenuates GG-NER and Rad26-independent TC-NER but does not affect TC-NER in the presence of Rad26. All the other histone H4 mutations, except for T73F and T73Y that mildly attenuate GG-NER, do not substantially affect GG-NER or TC-NER. The attenuation of GG-NER and Rad26-independent TC-NER by the H4H75E mutation is not due to decreased chromatin accessibility, impaired methylation of histone H3 K79 that is at the center of the LRS domain, or lowered expression of NER proteins. Instead, the attenuation is at least in part due to impaired recruitment of Rad4, the key lesion recognition and verification protein, to chromatin following induction of DNA lesions.

Mechanistic Analyses of Supercoiling Behaviors of DNA in Nucleosomes and Chromatins

Besides those in 146-base pair nucleosome core particle DNA, supercoils have been known to be present in 10-base pair arm DNA segments and naked linker DNA segments. The interacting patterns among histone octamers, histone H1, 10-base pair arm DNA segments and linker DNA have, however, not yet been elucidated. In the current report, we examine correlations among constituents of nucleosomes from the mechanistic perspectives and present molecular pathways for elucidating supercoiling behaviors of their component DNA sequences. It is our hope that our new analyses could serve as incentives to further clarify correlations between histones and DNA in the dynamic structures of chromatins in the future.

Epigenetic chromatin changes and the transcription cycle

In order to allow transcription to occur, the association of DNA with histone octamers and the compacted physical state of the chromatin fiber must be modified by the opportunistic binding of pioneer transcription factors to their cognate DNA binding sites. Once bound, pioneer factors recruit chromatin remodelers and histone-modifying enzymes for the purpose of repositioning nucleosomes and exposing regulatory regions (enhancers and gene promoters) to the components necessary for the initiation of transcription. Histone modifications, such as acetylation, methylation and ubiquitination, and the dynamic phosphorylation of specific amino acids on the major RNA polymerase II subunit activate transcription and attract the factors necessary to eliminate the pausing that normally occurs soon after initiation. Further histone modifications and the replacement of certain core histones by histone variants facilitate transcript elongation and termination. Two additional major epigenetic modifications that impact the process of transcription consist of the action of non-coding RNAs and DNA methylation.

Enhancing the Anticancer Efficacy of Immunotherapy through Combination with Histone Modification Inhibitors

In the nucleus of each cell, the DNA is wrapped around histone octamers, forming the so-called “nucleosomal core particles”. The histones undergo various modifications that influence chromatin structure and function, including methylation, acetylation, ubiquitination, phosphorylation, and SUMOylation. These modifications, known as epigenetic modifications (defined as heritable molecular determinants of phenotype that are independent of the DNA sequence), result in alterations of gene expression and changes in cell behavior. Recent work has shown that epigenetic drugs targeting histone deacetylation or methylation modulate the immune response and overcome acquired resistance to immunotherapy. A number of combination therapies involving immunotherapy and epigenetic drugs, which target histone deacetylation or methylation, are currently under various clinical/pre-clinical investigations and have shown promising anticancer efficacy. These combination therapies may provide a new strategy for achieving sustained anticancer efficacy and overcoming resistance.