A Compendium of Methods to Analyze the Spatial Organization of Plant Chromatin

Abstract

The long linear chromosomes of eukaryotic organisms are tightly packed into the nucleus of the cell. Beyond a first organization into nucleosomes and higher-order chromatin fibers, the positioning of nuclear DNA within the three-dimensional space of the nucleus plays a critical role in genome function and gene expression. Different techniques have been developed to assess nanoscale chromatin organization, nuclear position of genomic regions or specific chromatin features and binding proteins as well as higher-order chromatin organization. Here, I present an overview of imaging and molecular techniques applied to study nuclear architecture in plants, with special attention to the related protocols published in the "Plant Chromatin Dynamics" edition from Methods in Molecular Biology.

Plant 3D genomics: the exploration and application of chromatin organization

“…There are two main kinds of technical approaches for 3D genomic studies. The first approach is cytological evaluation and microscopy, the principle of which is to label DNA or chromatin and then use the microscope to observe the spatial organization of chromatin (Probst, 2018) (Fig. 1).…”

“…In the past, widefield microscopy (WF), confocal microscopy (CLSM) and light sheet fluorescence microscopy (LSFM) combined labeling techniques could only resolve structures > 200 nm, such as CTs and nuclear bodies in the nucleus (Schwarzacher et al ., 1992; Aragon‐Alcaide et al .,1997; Bass et al ., 2000; Baroux & Schubert, 2018). In recent years, new advances in labeling techniques include custom oligonucleotide arrays (such as Oligopaint; Beliveau et al ., 2012) and modified clustered regularly interspaced short palindromic repeats/CRISPR‐associated caspase 9 (CRISPR/Cas9) systems (Dreissig et al ., 2017; Hong et al ., 2018), and microscopic techniques including super resolution microscopy (SRM) (Probst, 2018) (such as stochastic optical reconstruction microscopy (STORM) (Rust et al ., 2006) and photoactivated localization microscopy (PALM) (Betzig et al ., 2006)) and electron microscopy imaging (ELMI) (Lobastov et al ., 2005). The resolution of the SRM can reach 20 nm, and the resolution of the ELMI is even higher (Baroux & Schubert, 2018).…”

Understanding 3D Genome Organization and Its Effect on Transcriptional Gene Regulation Under Environmental Stress in Plant: A Chromatin Perspective

“…The 3D genome mapping approaches can be broadly divided into two categories. The first category of approaches is based on cytology/microscopy, which utilizes fluorescent dye to label DNA/chromatin and/or visualization of the spatial chromatin organization using a microscope ( Probst, 2018 ). Combining microscopy with fluorescent in situ hybridization (FISH) boosted the progress in understanding how the spatial organization of CTs affects gene expression within the nucleus ( Zhang and Wang, 2021 ).…”

Rapid in-solution preparation of somatic and meiotic plant cell nuclei for high-quality 3D immunoFISH and immunoFISH-GISH

“…The compartmentalisation of individual genomic sequences or the characteristic shaping of chromosome regions through different stages of the plant cell cycle (telomer/leptotene bouquet [ 2 ], Rabl configuration [ 3 ]), the spatial distribution of genes and transposable elements [ 4 ], and specific epigenetic states (DNA methylation, histone modifications) are all indispensable factors for the chromatin to express its complex functions. It is therefore essential that a complementary array of methods is available for the detection and dynamic monitoring of these factors [ 5 ].…”