Chromosome conformation capture technologies as tools to detect structural variations and their repercussion in chromatin 3D configuration.

3D genome organization regulates gene expression in different physiological and pathological contexts. Characterization of chromatin structure at different scales has provided information about how the genome organizes in the nuclear space, from chromosome territories, compartments of euchromatin and heterochromatin, topologically associated domains to punctual chromatin loops between genomic regulatory elements and gene promoters. In recent years, chromosome conformation capture technologies have also been used to characterize structural variations (SVs) de novo in pathological conditions. The study of SVs in cancer, has brought information about transcriptional misregulation that relates directly to the incidence and prognosis of the disease. For example, gene fusions have been discovered arising from chromosomal translocations that upregulate oncogenes expression, and other types of SVs have been described that alter large genomic regions encompassing many genes. However, studying SVs in 2D cannot capture all their regulatory implications in the genome. Recently, several bioinformatic tools have been developed to identify and classify SVs from chromosome conformation capture data and clarify how they impact chromatin structure in 3D, resulting in transcriptional misregulation. Here, we review recent literature concerning bioinformatic tools to characterize SVs from chromosome conformation capture technologies and exemplify their vast potential to rebuild the 3D landscape of genomes in cancer. The study of SVs from the 3D perspective can produce essential information about drivers, molecular targets, and disease evolution.

Plant In Situ Hi-C Experimental Protocol and Bioinformatic Analysis.

Hi-C enables the characterization of the 0conformation of the genome in the three-dimensional nuclear space. This technique has revolutionized our ability to detect interactions between linearly distant genomic sites on a genome-wide scale. Here, we detail a protocol to carry out in situ Hi-C in plants and describe a straightforward bioinformatics pipeline for the analysis of such data, in particular for comparing samples from different organs or conditions.

Isolation of Boechera stricta Developing Embryos for Hi-C.

The possibility of analyzing chromatin topology in developing plant embryos is hampered by inaccessibility of the embryo sac, deeply embedded in the maternal seed tissue, following double fertilization. Here we describe a protocol to isolate, purify, and prepare developing Boechera stricta embryos for chromosome conformation capture-based methods as in situ Hi-C experiments. Early globular embryos can be isolated by air-pressure microaspiration, and subsequently washed to eliminate residual cells from the endosperm and maternal seed coat, allowing for pure sampling of selected stages of embryogenesis. This protocol allows for the possibility of comparing genome topology during plant embryonic differentiation since early until late embryo development stages.

A Shift in Paradigms: Spatial Genomics Approaches to Reveal Single-Cell Principles of Genome Organization.

The genome tridimensional (3D) organization and its role towards the regulation of key cell processes such as transcription is currently a main question in biology. Interphase chromosomes are spatially segregated into "territories," epigenetically-defined large domains of chromatin that interact to form "compartments" with common transcriptional status, and insulator-flanked domains called "topologically associating domains" (TADs). Moreover, chromatin organizes around nuclear structures such as lamina, speckles, or the nucleolus to acquire a higher-order genome organization. Due to recent technological advances, the different hierarchies are being solved. Particularly, advances in microscopy technologies are shedding light on the genome structure at multiple levels. Intriguingly, more and more reports point to high variability and stochasticity at the single-cell level. However, the functional consequences of such variability in genome conformation are still unsolved. Here, I will discuss the implication of the cell-to-cell heterogeneity at the different scales in the context of newly developed imaging approaches, particularly multiplexed Fluorescence in situ hybridization methods that enabled "chromatin tracing." Extensions of these methods are now combining spatial information of dozens to thousands of genomic loci with the localization of nuclear features such as the nucleolus, nuclear speckles, or even histone modifications, creating the fast-moving field of "spatial genomics." As our view of genome organization shifts the focus from ensemble to single-cell, new insights to fundamental questions begin to emerge.

Entering the Next Dimension: Plant Genomes in 3D.

After linear sequences of genomes and epigenomic landscape data, the 3D organization of chromatin in the nucleus is the next level to be explored. Different organisms present a general hierarchical organization, with chromosome territories at the top. Chromatin interaction maps, obtained by chromosome conformation capture (3C)-based methodologies, for eight plant species reveal commonalities, but also differences, among them and with animals. The smallest structures, found in high-resolution maps of the Arabidopsis genome, are single genes. Epigenetic marks (histone modification and DNA methylation), transcriptional activity, and chromatin interaction appear to be correlated, and whether structure is the cause or consequence of the function of interacting regions is being actively investigated.

Shaping Up the Embryo: The Role of Genome 3D Organization.

The spatial organization of the chromatinized genome inside the cell nucleus impacts genomic function. In transcription, the hierarchical genome structure creates spatial regulatory landscapes, in which modulating elements like enhancers can contact their target genes and activate their expression, as a result of restricting their exploration to a specific topological neighbourhood. Here we describe exciting recent findings obtained through "C" technologies in pluripotent cells and early embryogenesis and emphasize some of the key unanswered questions arising from them.