Genome-in-a-Box: Building a Chromosome from the Bottom Up

Abstract

Chromosome structure and dynamics are essential for life, as the way that our genomes are spatially organized within cells is crucial for gene expression, differentiation, and genome transfer to daughter cells. There is a wide variety of methods available to study chromosomes, ranging from live-cell studies to single-molecule biophysics, which we briefly review. While these technologies have yielded a wealth of data, such studies still leave a significant gap between top-down experiments on live cells and bottom-up in vitro single-molecule studies of DNA–protein interactions. Here, we introduce “genome-in-a-box” (GenBox) as an alternative in vitro approach to build and study chromosomes, which bridges this gap. The concept is to assemble a chromosome from the bottom up by taking deproteinated genome-sized DNA isolated from live cells and subsequently add purified DNA-organizing elements, followed by encapsulation in cell-sized containers using microfluidics. Grounded in the rationale of synthetic cell research, the approach would enable to experimentally study emergent effects at the global genome level that arise from the collective action of local DNA-structuring elements. We review the various DNA-structuring elements present in nature, from nucleoid-associated proteins and SMC complexes to phase separation and macromolecular crowders. Finally, we discuss how GenBox can contribute to several open questions on chromosome structure and dynamics.

Reconstitution of Ultrawide DNA Origami Pores in Liposomes for Transmembrane Transport of Macromolecules

“…While such biomimetic pores constitute an exciting platform for investigating nucleocytoplasmic transport in vitro , they may also be employed as a building block for synthetic cells, thus contributing to the in vitro reconstitution of an artificial model of a nucleus. This will, for example, aid the study of emergent properties of liposome-confined genomes 29 by allowing the controlled injection of genome-processing proteins. Indeed, our DNA origami pores may be a great asset for building a synthetic cell as they provide a stable portal that is large enough for biological macromolecules like proteins and RNAs to enter a liposome.…”

Generating Chromosome Geometries in a Minimal Cell From Cryo-Electron Tomograms and Chromosome Conformation Capture Maps

“…Factors affecting local structure include supercoiling, plectonemic loops resulting from supercoiling, small loops formed by SMC bridging distant chromosome segments, and bending and stiffening by proteins such as histone-like protein (HU), heat-stable nucleoid structuring protein (H-NS), and integration host factor (IHF) (Dame, 2005;Ohniwa et al, 2011;Dame and Tark-Dame, 2016;Dame et al, 2019;Verma et al, 2019;Birnie and Dekker, 2021). These micro level effects can strongly affect gene expression as localized crowding affects the access of the RNA polymerase (RNAP) to genes and supercoiling and plectonemes affect the RNAP's ability to transcribe a gene (Kim et al, 2019).…”

Mechanisms for Chromosome Segregation in Bacteria

“…Thus far, it has remained difficult to disentangle the relative importance of various individual mechanisms in segregating genome-sized substrates in cells. To investigate this in an alternative way, a recently proposed bottom-up approach named “genome-in-a-box” may provide an interesting starting-point ( Birnie and Dekker, 2020 ). Here, a Mbp-long genome, stripped of all proteins is confined within a microfluidic device, and single components such as SMCs, parABS , or other genome-structuring proteins can be added to monitor their individual effects, irrespective of the additional mechanisms that are normally simultaneously at play in cells.…”