3D genome organization: a role for phase separation and loop extrusion? Maike Stam, Mariliis Tark-Dame and Paul Fransz In eukaryotes, genomic information is encoded in chromosomes, which occupy distinct territories within the nucleus. Inside these territories, chromosomes are folded in a hierarchical set of topological structures, called compartments, topologically associated domains and loops. Phase separation and loop extrusion are the mechanisms indicated to mediate the 3D organization of the genome, and gene activity and epigenetic marks determine the activity level of the formed chromatin domains. The main difference between plants and animals may be the absence of canonical insulator elements in plants. Comparison across plant species indicates that the identification of chromatin domains is affected by genome size, gene density, and the linear distribution of genes and transposable elements. Address Swammerdam Institute for Life Sciences, Universiteit van Amsterdam, 1098 XH Amsterdam, The Netherlands Corresponding author: Stam, Maike (m.e.stam@uva.nl) Current Opinion in Plant Biology 2019, 48:36–46 This review comes from a themed issue on Genome studies and molecular genetics Edited by Steven Kelly For a complete overview see the Issue and the Editorial Available online 28th April 2019 https://doi.org/10.1016/j.pbi.2019.03.008 1369-5266/ã 2019 Elsevier Ltd. All rights reserved. Introduction The genetic information of an organism is represented by thousands of DNA elements that are linearly arranged along the chromosome. These include genes, regulatory elements, and other sequences such as transposable ele- ments (TEs) and telomeric repeats. For optimal func- tionality and stability of the genome, the DNA elements are under the control of various protein complexes and epigenetic modifications. Together these factors establish the 3-dimensional organization (3D) of the genome that facilitates an efficient regulation of the genome, including interactions between distant DNA elements. Under- standing the molecular mechanisms driving the 3D orga- nization of the genome is a major challenge. From microscopic and biochemical analyses, we can distinguish different organization levels of the genome, the nucleosome fiber, chromatin loops, chromatin domains and chromosome territories. Each of these may feature specific mechanisms regulating genome activity. The nucleosome consists of DNA wrapped around a multimer of histones and forms the basic unit of chro- matin; covalent modifications of the DNA and histones, and associated proteins affect the accessibility of the DNA. Microscopic studies showed that individual chro- mosomes occupy distinct domains, chromosome territories, inside the nucleus, each containing separate domains for the chromosome arms [1,2]. Fluorescence in situ hybrid- ization (FISH) and Chromosome Conformation Capture (3C) technology indicated that within territories, the chromatin fiber is folded in loops spanning tens of kb up to megabases, in mammalian cells [3–5], Drosophila [6,7] and plant cells [8,9]. How the chromatin fiber is organized inside chromosome territories is now being uncovered via large-scale genome-wide analysis, mainly by using 3C-based technologies. 3C technologies, such as Hi-C, are based on proximity ligation and determine the relative frequency at which genomic regions physically interact [10  ]. Hi-C, but espe- cially its derivatives (e.g. in situ Hi-C and Capture-C), generate high-resolution contact maps that provide detailed insights into the 3D genome organization, from individual locus up to the entire genome [11–15]. In this review, we discuss recent findings and novel insights into the 3D organization of genomes in plants in comparison to animals, with a focus on the underlying mechanisms of chromosome folding. Loops, TADs, and compartments in plants and animals 3C technology has demonstrated that chromosome terri- tories can be hierarchically subdivided into different types of chromatin domains: compartments and smaller domains called topologically associated domains (TADS; Figure 1), the latter contain chromatin loops. Similar hierarchical 3D structures have been observed in animals and plants [16,17  ,18  ,19,20  ,21  ,22  ,23–25]. The nomenclature used for the different types of chromatin domains in these organisms is, unfortunately, far from consistent. For clarity we apply the terms ‘compartments’ and ‘TADs’. It should be noted that, due to the develop- ment of high-resolution Hi-C techniques, better analyses methods, increased sequencing depth and sample quality, Available online at www.sciencedirect.com ScienceDirect Current Opinion in Plant Biology 2019, 48:36–46 www.sciencedirect.com