Genome organization across scales: mechanistic insights from in vitro reconstitution studies

Abstract

Eukaryotic genomes are compacted and organized into distinct three-dimensional (3D) structures, which range from small-scale nucleosome arrays to large-scale chromatin domains. These chromatin structures play an important role in the regulation of transcription and other nuclear processes. The molecular mechanisms that drive the formation of chromatin structures across scales and the relationship between chromatin structure and function remain incompletely understood. Because the processes involved are complex and interconnected, it is often challenging to dissect the underlying principles in the nuclear environment. Therefore, in vitro reconstitution systems provide a valuable approach to gain insight into the molecular mechanisms by which chromatin structures are formed and to determine the cause-consequence relationships between the processes involved. In this review, we give an overview of in vitro approaches that have been used to study chromatin structures across scales and how they have increased our understanding of the formation and function of these structures. We start by discussing in vitro studies that have given insight into the mechanisms of nucleosome positioning. Next, we discuss recent efforts to reconstitute larger-scale chromatin domains and loops and the resulting insights into the principles of genome organization. We conclude with an outlook on potential future applications of chromatin reconstitution systems and how they may contribute to answering open questions concerning chromatin architecture.

Keywords: in vitro reconstitution; 3D genome organization; chromatin; loop extrusion; nucleosome.

Figure 1.. In vitro reconstitution of nucleosome positioning in S. cerevisiae.

(A) Stereotypical nucleosome-free region (NFR)-array pattern at transcription start sites (TSSs) in wild-type S. cerevisiae chromatin. Gray circles indicate nucleosomes. Nucleosome density is derived from micrococcal nuclease-sequencing (MNase-seq) data [23] and averaged over all TSSs. For MNase-seq, chromatin is digested with the endo- and exonuclease MNase, which predominantly cleaves nucleosome-free DNA. The protected, nucleosomal DNA is then purified and sequenced to infer the nucleosome positions. (B) Schematic overview of genome-wide in vitro reconstitution experiments to study nucleosome positioning in S. cerevisiae based on salt gradient dialysis (SGD). Nucleosomes are assembled by incubating purified histone octamers with a DNA template in a high-salt buffer, allowing for spontaneous assembly of nucleosomes as the salt is slowly dialyzed away. The positioning of nucleosomes in the SGD chromatin is DNA-intrinsic and irregular. By incubating SGD chromatin with a transcription factor (TF), ATP-dependent chromatin remodeler and ATP, regular nucleosome positioning patterns can be reconstituted and analyzed by MNase-seq. (C) Example of MNase-seq data derived from the reconstitution approach described in panel B (red line) [24]. SGD chromatin was prepared with recombinant yeast histone octamers at a high nucleosome density (histone-to-DNA ratio = 0.8) and incubated with the TF Reb1 and/or the indicated remodelers, leading to the formation of distinct nucleosome density profiles. The MNase-seq data are averaged over Reb1-bound TSSs. Comparison with the in vivo MNase-seq data (gray background) highlights differences in NFR width and nucleosome spacing between the different in vitro conditions.

Figure 2.. In vitro reconstitution of chromatin domains in S. cerevisiae.

(A) Schematic overview of an in vitro Chromosome Conformation Capture (3C) procedure to map the 3D structure of reconstituted chromatin at sub-nucleosome resolution. Reconstituted chromatin (prepared as described in Figure 1B) is cross-linked with disuccinimidyl glutarate (DSG) and formaldehyde (FA) and digested with MNase, which is followed by proximity ligation, sonication and sequencing. (B) Comparison of reconstituted chromatin that has been incubated with different remodelers shows that the positioning of nucleosomes has an important role in determining higher-order chromatin structures. In vitro 3C data [24] for two example regions are shown, with the corresponding in vivo [55] data at the top for comparison.

Figure 3.. In vitro reconstitution of loop extrusion by SMC complexes.

The top row shows a schematic overview of the experimental set-up to study loop extrusion on single DNA molecules. Biotin-labeled DNA is tethered to an avidin-coated glass slide. By applying continuous buffer flow and adding ATP and fluorescently-labeled SMC complexes, loop extrusion can be imaged in real-time. The bottom row shows a figure legend, a schematic overview of symmetric and asymmetric loop extrusion, and a brief summary of the insights that have been provided by in vitro single-molecule experiments with SMC complexes.