Opposing roles of H3- and H4-acetylation in the regulation of nucleosome structure­­a FRET study.

Using FRET in bulk and on single molecules, we assessed the structural role of histone acetylation in nucleosomes reconstituted on the 170 bp long Widom 601 sequence. We followed salt-induced nucleosome disassembly, using donor­acceptor pairs on the ends or in the internal part of the nucleosomal DNA, and on H2B histone for measuring H2A/H2B dimer exchange. This allowed us to distinguish the influence of acetylation on salt-induced DNA unwrapping at the entry­exit site from its effect on nucleosome core dissociation. The effect of lysine acetylation is not simply cumulative, but showed distinct histone-specificity. Both H3- and H4-acetylation enhance DNA unwrapping above physiological ionic strength; however, while H3-acetylation renders the nucleosome core more sensitive to salt-induced dissociation and to dimer exchange, H4-acetylation counteracts these effects. Thus, our data suggest, that H3- and H4-acetylation have partially opposing roles in regulating nucleosome architecture and that distinct aspects of nucleosome dynamics might be independently controlled by individual histones.

From Sampling to Sequencing: A Liquid Biopsy Pre-Analytic Workflow to Maximize Multi-Layer Genomic Information from a Single Tube.

Liquid biopsies hold great promise for the management of cancer. Reliable liquid biopsy data depend on stable and reproducible pre-analytical protocols that comply with quality measures, irrespective of the sampling and processing site. We established a workflow for plasma preservation, followed by processing, cell-free nucleic acid isolation, quantification, and enrichment of potentially tumor-derived cell-free DNA and RNA. Employing the same input material for a direct comparison of different kits and protocols allowed us to formulate unbiased recommendations for sample collection, storage, and processing. The presented workflow integrates the stabilization in Norgen, PAX, or Streck tubes and subsequent parallel isolation of cell-free DNA and RNA with NucleoSnap and NucleoSpin. Qubit, Bioanalyzer, and TapeStation quantification and quality control steps were optimized for minimal sample use and high sensitivity and reproducibility. We show the efficiency of the proposed workflow by successful droplet digital PCR amplification of both cell-free DNA and RNA and by detection of tumor-specific alterations in low-coverage whole-genome sequencing and DNA methylation profiling of plasma-derived cell-free DNA. For the first time, we demonstrated successful parallel extraction of cell-free DNA and RNA from plasma samples. This workflow paves the road towards multi-layer genomic analysis from one single liquid biopsy sample.

Structural variability of nucleosomes detected by single-pair Förster resonance energy transfer: histone acetylation, sequence variation, and salt effects.

Nucleosomes were reconstituted from 170 bp long fragments of 5S rDNA and an optimal positioning sequence, the Selex 601, with recombinant histones. In free-solution single pair Förster resonance energy transfer (spFRET) measurements of the distance between fluorescently labeled bases in the nucleosomal DNA, the samples exhibited structural diversity. The structural heterogeneity correlated with the stability of the complexes and depended on the DNA sequence and histone acetylation. The stability of the nucleosomes was assessed via dilution-driven disruption: histone acetylation decreased nucleosome stability. The spFRET experiments used a new approach for data acquisition and analysis that we term "deliberately detuned detection" (D3). This permits the separation of subpopulations in the samples even for the low-FRET regime characteristic for the linker-DNA labeled nucleosomes. Thus, it became possible to study in more detail histone acetylation- and salt-dependent structural variations using either end- or internally labeled DNAs on the nucleosome. We found that the distance distribution of the fluorophore pairs on the linker DNA ends was much more sensitive to histone acetylation or sequence variation than that of labels on the internal part of the DNA, which was more tightly associated with the histone core. spFRET on freely diffusing nucleosomes allows us therefore to localize the influence of histone modifications and DNA sequence variations on the nucleosome structure and dynamics.

Effects of charge-modifying mutations in histone H2A α3-domain on nucleosome stability assessed by single-pair FRET and MD simulations.

Nucleosomes are important for chromatin compaction and gene regulation; their integrity depends crucially on the structural properties of the histone tails. Recent all-atom molecular dynamics simulations revealed that removal of the N-terminal tails of histone H3, known to destabilize nucleosomes, causes a rearrangement of two arginines of histone H2A, namely R81 and R88 by altering the electrostatic environment of the H2A α3 domain. Whether this rearrangement is the cause or the effect of decreased stability, is unclear. Here, we emulate the altered electrostatic environment that was found after H3 tail clipping through charge-modifying mutations to decouple its impact on intranucleosomal interactions from that of the histone tails. Förster resonance energy transfer experiments on recombinant nucleosomes and all-atom molecular dynamics simulations reveal a compensatory role of those amino acids in nucleosome stability. The simulations indicate a weakened interface between H2A-H2B dimers and the (H3-H4)2 tetramer, as well as between dimers and DNA. These findings agree with the experimental observations of position and charge dependent decreased nucleosome stability induced by the introduced mutations. This work highlights the importance of the H2A α3 domain and suggests allosteric effects between this domain and the outer DNA gyre as well as the H3 N-terminal tail.

Trinucleosome compaction studied by fluorescence energy transfer and scanning force microscopy.

The effect of the salt concentration, linker histone H1, and histone acetylation on the structure of trinucleosomes reconstituted on a 608 bp DNA containing one centered nucleosome positioning signal was studied. Fluorescence resonance energy transfer (FRET) in solution and scanning force microscopy (SFM) measurements in liquid were done on the same samples. The distance between the DNA ends decreases under the effect of an increasing salt concentration and also by the incorporation of the H1 linker histone. A decrease of internucleosomal center-to-center (cc) distances by H1 was observed that was limited to a minimal value of about 20 nm. The distribution of the angle formed between consecutive nucleosomes was narrowed by H1. The effect of acetylation of all histones leads to decompaction, measured as an increased distance between the DNA ends, and also increased the internucleosomal distances. Selective acetylation of histone H4, however, compacts the structure as measured by FRET.