The middle region of an HP1-binding protein, HP1-BP74, associates with linker DNA at the entry/exit site of nucleosomal DNA

Kayoko Hayashihara, Susumu Uchiyama, Shigeru Shimamoto, Shouhei Kobayashi, Miroslav Tomschik, Hidekazu Wakamatsu, Daisuke No, Hiroki Sugahara, Naoto Hori, Masanori Noda, Tadayasu Ohkubo, Jordanka Zlatanova, Sachihiro Matsunaga, Kiichi Fukui. The Middle Region of an HP1-binding Protein, HP1-BP74, Associates with Linker DNA at the Entry/Exit Site of Nucleosomal DNA. Journal of Biological Chemistry, Volume 285, Issue 9, 2010, Pages 6498-6507. https://doi.org/10.1074/jbc.M109.092833

Single-epitope recognition imaging of native chromatin

© 2008 Wang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Single-Pair FRET Microscopy Reveals Mononucleosome Dynamics

We applied spFRET microscopy for direct observation of intranucleosomal DNA dynamics. Mononucleosomes, reconstituted with DNA containing a FRET pair at the dyad axis and exit of the nucleosome core particle, were immobilized through a 30 bp DNA tether on a polyethyleneglycol functionalized slide and visualized using Total Internal Reflection Fluorescence microscopy. FRET efficiency time-traces revealed two types of dynamics: acceptor blinking and intramolecular rearrangements. Both Cy5 and ATTO647N acceptor dyes showed severe blinking in a deoxygenated buffer in the presence of 2% βME. Replacing the triplet quencher βME with 1 mM Trolox eliminated most blinking effects. After suppression of blinking three subpopulations were observed: 90% appeared as dissociated complexes; the remaining 10% featured an average FRET efficiency in agreement with intact nucleosomes. In 97% of these intact nucleosomes no significant changes in FRET efficiency were observed in the experimentally accessible time window ranging from 10 ms to 10’s of seconds. However, 3% of the intact nucleosomes showed intervals with reduced FRET efficiency, clearly distinct from blinking, with a lifetime of 120 ms. These fluctuations can unambiguously be attributed to DNA breathing. Our findings illustrate not only the merits but also typical caveats encountered in single-molecule FRET studies on complex biological systems

Tetrameric Structure of Centromeric Nucleosomes in Interphase Drosophila Cells

Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3). However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical “beads-on-a-string” appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity

Nucleosome Chiral Transition under Positive Torsional Stress in Single Chromatin Fibers

Using magnetic tweezers to investigate the mechanical response of single chromatin fibers, we show that fibers submitted to large positive torsion transiently trap positive turns, at a rate of one turn per nucleosome. A comparison with the response of fibers of tetrasomes (the (H3-H4)2 tetramer bound with ~50 bp of DNA) obtained by depletion of H2A-H2B dimers, suggests that the trapping reflects a nucleosome chiral transition to a metastable form built on the previously documented righthanded tetrasome. In view of its low energy, <8 kT, we propose this transition is physiologically relevant and serves to break the docking of the dimers on the tetramer which in the absence of other factors exerts a strong block against elongation of transcription by the main RNA polymerase.Comment: 33 pages (double spacing), 7 figure

AFM Imaging of SWI/SNF action: mapping the nucleosome remodeling and sliding

We propose a combined experimental (Atomic Force Microscopy) and theoretical study of the structural and dynamical properties of nucleosomes. In contrast to biochemical approaches, this method allows to determine simultaneously the DNA complexed length distribution and nucleosome position in various contexts. First, we show that differences in the nucleo-proteic structure observed between conventional H2A and H2A.Bbd variant nucleosomes induce quantitative changes in the in the length distribution of DNA complexed with histones. Then, the sliding action of remodeling complex SWI/SNF is characterized through the evolution of the nucleosome position and wrapped DNA length mapping. Using a linear energetic model for the distribution of DNA complexed length, we extract the net wrapping energy of DNA onto the histone octamer, and compare it to previous studies.Comment: 25 pages,5 figures, to appear in Biophysical Journa

Nucleosome accessibility governed by the dimer/tetramer interface

Nucleosomes are multi-component macromolecular assemblies which present a formidable obstacle to enzymatic activities that require access to the DNA, e.g. DNA and RNA polymerases. The mechanism and pathway(s) by which nucleosomes disassemble to allow DNA access are not well understood. Here we present evidence from single molecule FRET experiments for a previously uncharacterized intermediate structural state before H2A–H2B dimer release, which is characterized by an increased distance between H2B and the nucleosomal dyad. This suggests that the first step in nucleosome disassembly is the opening of the (H3–H4)2 tetramer/(H2A–H2B) dimer interface, followed by H2A–H2B dimer release from the DNA and, lastly, (H3–H4)2 tetramer removal. We estimate that the open intermediate state is populated at 0.2–3% under physiological conditions. This finding could have significant in vivo implications for factor-mediated histone removal and exchange, as well as for regulating DNA accessibility to the transcription and replication machinery

On the spontaneous stochastic dynamics of a single gene: complexity of the molecular interplay at the promoter

International audienceBACKGROUND: Gene promoters can be in various epigenetic states and undergo interactions with many molecules in a highly transient, probabilistic and combinatorial way, resulting in a complex global dynamics as observed experimentally. However, models of stochastic gene expression commonly consider promoter activity as a two-state on/off system. We consider here a model of single-gene stochastic expression that can represent arbitrary prokaryotic or eukaryotic promoters, based on the combinatorial interplay between molecules and epigenetic factors, including energy-dependent remodeling and enzymatic activities. RESULTS: We show that, considering the mere molecular interplay at the promoter, a single-gene can demonstrate an elaborate spontaneous stochastic activity (eg. multi-periodic multi-relaxation dynamics), similar to what is known to occur at the gene-network level. Characterizing this generic model with indicators of dynamic and steady-state properties (including power spectra and distributions), we reveal the potential activity of any promoter and its influence on gene expression. In particular, we can reproduce, based on biologically relevant mechanisms, the strongly periodic patterns of promoter occupancy by transcription factors (TF) and chromatin remodeling as observed experimentally on eukaryotic promoters. Moreover, we link several of its characteristics to properties of the underlying biochemical system. The model can also be used to identify behaviors of interest (eg. stochasticity induced by high TF concentration) on minimal systems and to test their relevance in larger and more realistic systems. We finally show that TF concentrations can regulate many aspects of the stochastic activity with a considerable flexibility and complexity. CONCLUSIONS: This tight promoter-mediated control of stochasticity may constitute a powerful asset for the cell. Remarkably, a strongly periodic activity that demonstrates a complex TF concentration-dependent control is obtained when molecular interactions have typical characteristics observed on eukaryotic promoters (high mobility, functional redundancy, many alternate states/pathways). We also show that this regime results in a direct and indirect energetic cost. Finally, this model can constitute a framework for unifying various experimental approaches. Collectively, our results show that a gene - the basic building block of complex regulatory networks - can itself demonstrate a significantly complex behavior