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Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe the further development of the RNA-guided endonuclease-in situ labeling (RGEN-ISL) method CRISPR-FISH. Using soybean and mouse chromosomes, we demonstrate that the treatment of conventionally fixed chromosomes (in ethanol or methanol:acetic acid) with 40 mM Tris-HCl (pH 9.0) for 30 minutes at 37°C prior to CRISPR-FISH allows the application of this method for the detection of high-copy sequences. Wheat, rye, maize, and Nicotiana benthamiana were used to confirm the applicability of the identified CRISPR-FISH conditions also in other species.

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.

Based on the analysis of 20 different monocot and eudicot species, we propose that the centromeric distribution of the phosphorylated histone H2AThr120 is evolutionary highly conserved across species with mono- and holocentric chromosomes. Therefore, antibodies recognizing the phosphorylated threonine 120 of the histone H2A can serve as a universal marker for the cytological detection of centromeres of mono- and holokinetic plant species. In addition, super resolution microscopy of signals specific to the centromere-specific histone H3 variant CENH3 and to H2AThr120ph revealed that these histone variants are incorporated into different nucleosomes, which form distinct, partly intermingled chromatin domains. This specific arrangement of both histone variants suggests different centromeric functions during the cell cycle.

RNA polymerase II (RNAPII) is responsible for the transcription of most eukaryotic genes. In mammalian nuclei, RNAPII is mainly localized in relatively few distinct transcription factories. In this study - applying super-resolution microscopy - it is shown that in plants, inactive (non-phosphorylated) and active (phosphorylated) RNAPII modifications compose distinct ‘transcription networks' within the euchromatin. These reticulate structures sometimes attach to each other, but they are absent from heterochromatin and nucleoli. The global RNAPII distribution within nuclei is not influenced by interphase chromatin organization such as Rabl (rye) versus non-Rabl (Arabidopsis thaliana) orientation. Replication of sister chromatids without cell division causes endopolyploidy, a phenomenon widespread in plants and animals. Endopolyploidy raises the number of gene copies per nucleus. Here, it is shown that the amounts of active and inactive RNAPII enzymes in differentiated 2-32C leaf nuclei of A. thaliana proportionally increase with rising endopolyploidy. Thus, increasing the transcriptional activity of cells and tissues seems to be an important function of endopolyploidy.

Classical neocentromeres, proven mainly in Poaceae species, cause meiotic drive in higher plants. Here I present data indicating that the morphological stretching of a chromatin domain at the long arm of Aegilops markgrafii chromosome E during meiosis is not due to a supposed neocentric activity. The stretching is visible at the alien univalent in metaphase I and anaphase I pollen mother cells of Triticum aestivum – Ae. markgrafii monosomic addition line E and at one univalent of the 49-chromosome-containing plants containing the haploid Ae. markgrafii genome added to wheat. The absence of both centromeric histone variant CENH3 and attachment of spindle fibers, proven by immunostaining with antibodies against CENH3 and tubulin, respectively, revealed that the univalent elongation of chromosome E is not caused by a classical plant neocentromere.