Helical metaphase chromatid coiling is conserved

The higher-order metaphase chromosome organization has been under controversial discussion already for 140 years. Classical light and electron microscopy proposed chromatids to be composed of helically organized chromatin fibers, so-called chromonemata. More recently also non-helical models were suggested. We studied the chromosome organization in barley by interdisciplinary cutting-edge approaches, such as chromosome sorting, chromosome conformation capture, oligonucleotide-fluorescence in situ hybridization, base analog incorporation, super-resolution microscopy, and polymer simulation to elucidate the arrangement of chromatids of large mitotic metaphase chromosomes. Our data provide cumulative evidence for the presence of a helically arranged 400 nm chromatin fiber representing the chromonema within the chromatid arms. The number of turns is positively correlated with the arm length. Turn size and chromatin density decrease towards the telomeres. Due to the specialized functions of centromeres and nucleolus-organizing regions, the helical organization is interrupted at these regions, which display several thinners and straight chromatin fibers. Based on our findings and re-analyzing previously published data from other plant and non-plant species we conclude that the helical turning of metaphase chromatid arms is a conserved feature of large eukaryotic chromosomes.

Uncoupling of Chromatin Assembly from DNA Replication in Sciara Reveals a Domain of Postreplicative Immature Chromatin

DNA replication in dividing eukaryotic cells imposes a requirement for the faithful recreation on the newly synthesized chromatids of the nucleoprotein architecture of parent chromosomes. Practically nothing is known about the structure of postreplicative immature chromatin (a very short-lived entity of <30 min.). We report here the unexpected discovery that during DNA amplification of locus II/9A in salivary gland polytene chromosomes of the fungus fly Sciara coprophila, DNA replication fork passage is uncoupled from postreplicative chromatin assembly; this enables visualization and analysis of chromatin fibers disassembled by DNA replication. We used electron microscopy to visualize a wealth of low nucleosome density immature chromatin fibers in preparations of Sciara chromatin from amplification-stage tissue. Remarkably, as gauged by high sensitivity to micrococcal nuclease and an unusually short length of DNA associated with each histone octamer, we found that locus II/9A which undergoes amplification and is replicated once every 4-6 hrs. (but not the bulk genome or a replicatively quiescent DNA stretch) was maintained in such an ummature fiber for ca. 24 hrs. Following amplification, locus II/9A assumed conventional chromatin organization, indicating that the epigenetic mark targeting nascent DNA to the chromatin assembly machinery is stable for several hours. We propose that this very unusual prolonged maintenance of a segment of the genome in immature chromatin facilitates access by the basal transcriptional machinery to the amplified DNA, and thus is an evolutionary adaptation to the demand for high transcription from genes that reside in the amplified loci.

Nucleosome induced homology recognition in chromatin

ABSTRACTOne of the least understood properties of chromatin is the ability of its similar regions to recognise each other through weak interactions. Theories based on electrostatic interactions between helical macromolecules suggest that the ability to recognize sequence homology is an innate property of the non-ideal helical structure of DNA. However, this theory does not account for nucleosomal packing of DNA. Can homologous DNA sequences recognize each other while wrapped up in the nucleosomes? Can structural homology arise at the level of nucleosome arrays? Here we present a theoretical investigation of the recognition-potential-well between chromatin fibers sliding against each other. This well is different to the one predicted and observed for bare DNA; the minima in energy do not correspond to literal juxtaposition, but are shifted by approximately half the nucleosome repeat length. The presence of this potential-well suggests that nucleosome positioning may induce mutual sequence recognition between chromatin fibers and facilitate formation of chromatin nanodomains. This has implications for nucleosome arrays enclosed between CTCF-cohesin boundaries, which may form stiffer stem-like structures instead of flexible entropically favourable loops. We also consider switches between chromatin states, e.g., through acetylation/deacetylation of histones, and discuss nucleosome-induced recognition as a precursory stage of genetic recombination.

A critical role for linker DNA in higher-order folding of chromatin fibers

Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.

Entropic Competition between Supercoiled and Torsionally Relaxed Chromatin Fibers Drives Loop Extrusion through Pseudo-Topologically Bound Cohesin

We propose a model for cohesin-mediated loop extrusion, where the loop extrusion is driven entropically by the energy difference between supercoiled and torsionally relaxed chromatin fibers. Different levels of negative supercoiling are controlled by varying imposed friction between the cohesin ring and the chromatin fiber. The speed of generation of negative supercoiling by RNA polymerase associated with TOP1 is kept constant and corresponds to 10 rotations per second. The model was tested by coarse-grained molecular simulations for a wide range of frictions between 2 to 200 folds of that of generic fiber and the surrounding medium. The higher friction allowed for the accumulation of higher levels of supercoiling, while the resulting extrusion rate also increased. The obtained extrusion rates for the given range of investigated frictions were between 1 and 10 kbps, but also a saturation of the rate at high frictions was observed. The calculated contact maps indicate a qualitative improvement obtained at lower levels of supercoiling. The fits of mathematical equations qualitatively reproduce the loop sizes and levels of supercoiling obtained from simulations and support the proposed mechanism of entropically driven extrusion. The cohesin ring is bound on the fibers pseudo-topologically, and the model suggests that the topological binding is not necessary.

Chromatin Fiber Folding Represses Transcription and Loop Extrusion in Quiescent Cells

Determining the conformation of chromatin in cells at the nucleosome level and its relationship to cellular processes has been a central challenge in biology. We show that in quiescent yeast, widespread transcriptional repression coincides with the local compaction of chromatin fibers into structures that are less condensed and more heteromorphic than canonical 30-nanometer forms. Acetylation or substitution of H4 tail residues decompacts fibers and leads to global transcriptional de-repression. Fiber decompaction also increases the rate of loop extrusion by condensin. These findings establish a role for H4 tail-dependent local chromatin fiber folding in regulating transcription and loop extrusion in cells. They also demonstrate the physiological relevance of canonical chromatin fiber folding mechanisms even in the absence of regular 30-nanometer structures.

The Dynamic Influence of Linker Histone Saturation within the Poly-Nucleosome Array

Linker histones bind to nucleosomes and modify chromatin structure and dynamics as a means of epigenetic regulation. Biophysical studies have shown that chromatin fibers can adopt a plethora of conformations with varying levels of compaction. Linker histone condensation, and its specific binding disposition, has been associated with directly tuning this ensemble of states. However, the atomistic dynamics and quantification of this mechanism remains poorly understood. Here, we present molecular dynamics simulations of octa-nucleosome arrays, based on a cryo-EM structure of the 30-nm chromatin fiber, with and without the globular domains of the H1 linker histone to determine how they influence fiber structures and dynamics. Results show that when bound, linker histones inhibit DNA flexibility and stabilize repeating tetra-nucleosomal units, giving rise to increased chromatin compaction. Furthermore, upon the removal of H1, there is a significant destabilization of this compact structure as the fiber adopts less strained and untwisted states. Interestingly, linker DNA sampling in the octa-nucleosome is exaggerated compared to its mono-nucleosome counterparts, suggesting that chromatin architecture plays a significant role in DNA strain even in the absence of linker histones. Moreover, H1-bound states are shown to have increased stiffness within tetra-nucleosomes, but not between them. This increased stiffness leads to stronger long-range correlations within the fiber, which may result in the propagation of epigenetic signals over longer spatial ranges. These simulations highlight the effects of linker histone binding on the internal dynamics and global structure of poly-nucleosome arrays, while providing physical insight into a mechanism of chromatin compaction.SignificanceLinker histones dynamically bind to DNA in chromatin fibers and serve as epigentic regulators. However, the extent to which they influence the gamut of chromatin architecture is still not well understood. Using molecular dynamics simulations, we studied compact octa-nucleosome arrays with and without the H1 linker histone to better understand the mechanisms dictating the structure of the chromatin fiber. Inclusion of H1 results in stabilization of the compact chromatin structure, while its removal results in a major conformational change towards an untwisted ladder-like state. The increased rigidity and correlations within the H1-bound array suggests that H1-saturated chromatin fibers are better suited to transferring long-range epigentic information.