Nucleosome spacing controls chromatin spatial structure and accessibility.

Recent research highlights the significance of the three-dimensional structure of chromatin in regulating various cellular processes, particularly transcription. This is achieved through dynamic chromatin structures that facilitate long-range contacts and control spatial accessibility. Chromatin consists of DNA and a variety of proteins, of which histones play an essential structural role by forming nucleosomes. Extensive experimental and theoretical research in recent decades has yielded conflicting results about key factors that regulate the spatial structure of chromatin, which remains enigmatic. By using a computer model that allows us to simulate chromatin volumes containing physiological nucleosome concentrations, we investigated whether nucleosome spacing or nucleosome density is fundamental for three-dimensional chromatin accessibility. Unexpectedly, the regularity of the nucleosome spacing is crucial for determining the accessibility of the chromatin network to diffusive processes, whereas variation in nucleosome concentrations has only minor effects. Using only the basic physical properties of DNA and nucleosomes was sufficient to generate chromatin structures consistent with published electron microscopy data. Contrary to other work, we found that nucleosome density did not substantially alter the properties of chromatin fibers or contact probabilities of genomic loci. No breakup of fiber-like structures was observed at high molar density. These findings challenge previous assumptions and highlight the importance of nucleosome spacing as a key driver of chromatin organization. These results identified changes in nucleosome spacing as a tentative mechanism for altering the spatial chromatin structure and thus genomic functions.

Cohesin and CTCF complexes mediate contacts in chromatin loops depending on nucleosome positions.

The spatial organization of the eukaryotic genome plays an important role in regulating transcriptional activity. In the nucleus, chromatin forms loops that assemble into fundamental units called topologically associating domains that facilitate or inhibit long-range contacts. These loops are formed and held together by the ring-shaped cohesin protein complex, and this can involve binding of CCCTC-binding factor (CTCF). High-resolution conformation capture experiments provide the frequency at which two DNA fragments physically associate in three-dimensional space. However, technical limitations of this approach, such as low throughput, low resolution, or noise in contact maps, make data interpretation and identification of chromatin intraloop contacts, e.g., between distal regulatory elements and their target genes, challenging. Herein, an existing coarse-grained model of chromatin at single-nucleosome resolution was extended by integrating potentials describing CTCF and cohesin. We performed replica-exchange Monte Carlo simulations with regularly spaced nucleosomes and experimentally determined nucleosome positions in the presence of cohesin-CTCF, as well as depleted systems as controls. In fully extruded loops caused by the presence of cohesin and CTCF, the number of contacts within the formed loops was increased. The number and types of these contacts were impacted by the nucleosome distribution and loop size. Microloops were observed within cohesin-mediated loops due to thermal fluctuations without additional influence of other factors, and the number, size, and shape of microloops were determined by nucleosome distribution and loop size. Nucleosome positions directly affect the spatial structure and contact probability within a loop, with presumed consequences for transcriptional activity.

Differences in nanoscale organization of regulatory active and inactive human chromatin.

Methodological advances in conformation capture techniques have fundamentally changed our understanding of chromatin architecture. However, the nanoscale organization of chromatin and its cell-to-cell variance are less studied. Analyzing genome-wide data from 733 human cell and tissue samples, we identified 2 prototypical regions that exhibit high or absent hypersensitivity to deoxyribonuclease I, respectively. These regulatory active or inactive regions were examined in the lymphoblast cell line K562 by using high-throughput super-resolution microscopy. In both regions, we systematically measured the physical distance of 2 fluorescence in situ hybridization spots spaced by only 5 kb of DNA. Unexpectedly, the resulting distance distributions range from very compact to almost elongated configurations of more than 200-nm length for both the active and inactive regions. Monte Carlo simulations of a coarse-grained model of these chromatin regions based on published data of nucleosome occupancy in K562 cells were performed to understand the underlying mechanisms. There was no parameter set for the simulation model that can explain the microscopically measured distance distributions. Obviously, the chromatin state given by the strength of internucleosomal interaction, nucleosome occupancy, or amount of histone H1 differs from cell to cell, which results in the observed broad distance distributions. This large variability was not expected, especially in inactive regions. The results for the mechanisms for different distance distributions on this scale are important for understanding the contacts that mediate gene regulation. Microscopic measurements show that the inactive region investigated here is expected to be embedded in a more compact chromatin environment. The simulation results of this region require an increase in the strength of internucleosomal interactions. It may be speculated that the higher density of chromatin is caused by the increased internucleosomal interaction strength.