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

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.