Nucleosome placement and spacing: from mechanism to function (2023)

Heterochromatin is a physical state of the chromatin fiber that maintains gene repression during cell development. Although there is evidence for the molecular mechanisms involved in heterochromatin formation, a more detailed structural mechanism of heterochromatin formation needs to be better understood. We use a simple Monte Carlo simulation model with explicit representation of key molecular events to observe molecular self-assembly leading to heterochromatin formation. Our simulations provide a structural interpretation of several important features of the heterochromatinization process. Specifically, this study represents a representation of how small amounts of HP1 can induce a highly condensed chromatin state through HP1 dimerization and sequential bridging of distant nucleosomes. It also elucidates the structural roots of a still poorly understood phenomenon of the non-deterministic nature of heterochromatin formation and subsequent gene repression. experimental chromatinI liveThe assay provides an unbiased estimate of the time scale of the repressive response to a heterochromatin triggering event.

Spt4 is a transcription elongation factor with homologues in organisms with nucleosomes. your structuralin vitroStudies implicate Spt4 in nucleosome transcription, yet theI liveThe function of Spt4 is not clear. Here we investigate the precise position of Spt4 during transcription and the consequences of loss of Spt4 on RNA polymerase II (RNAPII) dynamics and nucleosome positioning inSaccharomyces cerevisiae. In the absence of Spt4, the distance between nucleosomes in the gene body is increased and RNAPII accumulates upstream of the nucleosome dyad, most dramatically in the +2 nucleosome. Spt4 associates with RNAPII elongation at the start of transcription and its association changes dynamically depending on nucleosome positions. Together, our data indicate that Spt4 regulates early elongation dynamics, is involved in co-transcriptional nucleosome positioning, and promotes the movement of RNAPII through the gene body nucleosomes, specifically the +2 nucleosome.

Journal of Molecular Biology, Volume 433, Number 6, 2021, Article 166744

Gene regulatory programs establish cellular identity and depend on dynamic changes in the structural packaging of genomic DNA. DNA is packaged in chromatin, which is formed from nucleosome assemblies that have varying degrees of compactness and varying lengths of internucleosomal binding DNA. The nucleosome is the repeating unit of chromatin and is formed by wrapping 145-147 base pairs of DNA around an octamer of histone proteins. Each of the four histones is duplicated and has a structured core and intrinsically disordered tails. Chromatin dynamics is triggered by inter- and intranucleosomal movements, which are controlled by the DNA sequence, by the interactions between the histone core and DNA, and by the conformations, positions, and interactions of the DNA tails of the histones. histones. Understanding chromatin dynamics requires examining all of these features at the highest possible resolution. For this, molecular dynamics simulations can be used as a powerful complement or alternative to experimental approaches, where it is often very difficult to characterize the structural features and atomic interactions that control nucleosome movements. Molecular dynamics simulations can be performed at different resolutions, granulating the molecular system with different levels of detail. Here we review the achievements and remaining challenges in applying atomic resolution simulations to study the structure and dynamics of nucleosomes and their complexes with interacting partners.

Biophysical Journal, Band 114, August 10, 2018, S. 2279-2289

The compact structure of the nucleosome restricts accessibility to DNA and inhibits the binding of most sequence-specific proteins. Nucleosomes are not randomly arranged in DNA, but instead are positioned relative to the DNA sequence, suggesting models in which critical binding sites are either exposed on the ligand, leading to activation, or buried within. of a nucleosome, leading to repression. Therefore, the mechanisms that determine the positioning of nucleosomes are of paramount importance for understanding gene regulation and other events that occur in chromatin, such as transcription, replication, and repair. Here we review our current understanding of the key determinants of nucleosome positioning: DNA sequence, non-histone DNA-binding proteins, chromatin remodeling enzymes, and transcription. We describe the main challenges ahead: elucidating the precise mechanisms of chromatin opening and promoter activation, identifying the complexes that promoters occupy, and understanding the multiscale problem of chromatin fiber organization.

Journal of Molecular Biology, Volume 433, Number 6, 2021, Article 166720

Chromatin is the epigenomic platform for several central processes, such as DNA repair, replication, transcription, telomere and centromere function. In cancer cells, mutations in key processes lead to DNA amplification, chromosomal translocations, and chromothripsis, severely distorting the natural state of chromatin. In normal and disease states, dozens of chromatin effectors alter the physical integrity and dynamics of chromatin at the level of individual nucleosomes and assemblies of nucleosomes folded into three-dimensional shapes. Integration of these length scales, from the 10 nm nucleosome to mitotic chromosomes, while being pushed around in the crowded environment of the cell, cannot yet be achieved with a single technology. In this review, we discuss tools that have proven effective in studying the dynamics of chromatin fibers and nucleosomes. We also offer a deeper focus on atomic force microscopy (AFM) applications that can span multiple duration and time scales. Using time course AFM, we observe that chromatin condensation by H1.5 is dynamic, while using nanoimprinting force spectroscopy, we observe that both histone variants and nucleosome binding partners change the material properties of individual nucleosomes. . Finally, we show how high-speed AFM can visualize plasmid DNA dynamics, intermittent nucleosome-nucleosome contacts, and changes in nucleosome phases along a contiguous chromatin fiber. Overall, the development of innovative technologies promises to reveal the secret life of nucleosomes and potentially fill in the gaps in our understanding of how chromatin functions in living cells and tissues.

Molecular Cell, Volume 65, Number 3, 2017, pp. 565-577.e3

Micrococcal nuclease (MNase) is commonly used to map nucleosomes throughout the genome, but nucleosome maps are affected by the degree of digestion. It has been suggested that many yeast promoters are not nucleosomeless, but are instead occupied by easily digestible, "fragile" and unstable nucleosomes. We analyzed the histone content of all MNase-sensitive complexes by MNase-ChIP-seq and sonication-ChIP-seq. We found that yeast promoters are predominantly bound by non-histone protein complexes with little evidence of fragile nucleosomes. We discovered MNase-sensitive nucleosomes in other parts of the genome, including at transcription termination sites. However, they are high in A/T, suggesting that MNase sensitivity does not indicate instability, but rather that MNase prefers A/T-rich DNA, so A/T-rich nucleosomes are digested. faster than G/C-rich nucleosomes. We confirmed our observations by analyzing ChIP-exo, chemical mapping, and ATAC-seq data from other laboratories. Therefore, histone ChIP-seq experiments are essential to distinguish nucleosomes from other DNA-binding proteins that protect against MNase.

Trends in Biochemical Sciences, Band 45, August 1, 2020, S. 13-26

Gene regulation in eukaryotes requires the controlled access of sequence-specific transcription factors (TFs) to their sites on a nucleosome-dominated chromatin landscape. Nucleosomes are resistant to TF binding and often need to be removed from regulatory regions. Recent genomic studies along within vitroMeasurements suggest that the nucleosome barrier to TF binding is modulated by dynamic nucleosome unpacking driven by ATP-dependent chromatin remodelers. Genome-wide occupancy and regulation of subnucleosomal intermediates has recently attracted attention through the application of high-resolution approaches to accurately map protein-DNA interactions. We summarize here recent discoveries on nucleosome substructures and TF binding dynamics, and show how unpackaged nucleosomal intermediates produce a new signature of active chromatin.

Molecular Cell, Volume 79, Issue 3, 2020, pp. 371-375

Considering that the core of the nucleosome serves as a packaging device for coiling and contraction of the length of genomic DNA, we propose that it serves primarily to regulate transcription. A nucleosome in a promoter prevents the initiation of transcription. The association of nucleosomes with most genomic DNA prevents the initiation of cryptic promoters. Thus, the nucleosome serves not only as a general gene pressor, but also as a repressor of all transcription (genetic, intragenic, and intergenic). The core of the nucleosome plays a crucial regulatory role along with histone "tails" that modulate gene activity.