Use of Fluorescence-Activated Nuclear Sorting (FANS) from Rare Plant Cell Types for analysis of gene expression and chromatin interactions

Analysis of individual cell types can add significant understanding to our knowledge the processes that drive the formation of complex tissues. However one major challenge in this area involves the difficulty in separating cells with different identifies that may be buried deep within tissues. Recently cell-type specific expression of fluorescent reporter proteins has facilitated the isolation of different cell types; initially by isolation of protoplasts and now through the use of flow cytometry and cell sorting. This latter technique allows the more rapid separation of the nuclei from specific cell files to ensure that a more realistic in-planta situation is revealed upon subsequent downstream analysis. This article provides a consensus methodology for the isolation of labelled nuclei and for the processing of these samples for analysis of gene expression, methylation state and of chromatin interactions

Protocol for Assay for Transposase-Accessible Chromatin (ATAC-seq)

The Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a powerful technique that enables the genome-wide analysis of chromatin accessibility. At the level of individual loci this allows an understanding of the positional relationship between chromatin, transcriptional regulators and gene expression whilst on a global level can provide an understanding of how large areas of the genome are regulated. ATAC-seq relies upon the engineered activity of the hyperactive transposase Tn5, which both cleaves accessible DNA and anneals specific adaptors to either end of the resulting fragments. These DNA fragments can be isolated, amplified and analysed by NGS. ATAC-seq requires a clean nuclear extraction that has limited contamination from, in particular, chloroplast DNA. This article outlines a consensus methodology for ATAC-seq that includes descriptions of a crude nuclear extraction, cleavage by Tn5 and amplification of DNA fragments in preparation for NGS

Single Molecule Fluorescence in situ Hybridization in Whole Mount Tissue

Traditional in situ hybridization is a powerful tool for the localization of RNA molecules within plant tissues. However the sensitivity of this technique is limited to single cell resolution and is unable to discriminate between sub-cellular expression domains. Single molecule fluorescent in situ hybridization (smFISH) is an evolution of this technique that allows for the resolution of expression to the level of a single messenger or non-coding RNA. This technique relies on the use of multiple fluorescent probes targeted to a single RNA sequence, which amplifies the signal such that is it visible using epi-fluorescent imaging. This allows both the localization and the quantification of single RNA molecules thus providing an increased level of precision in the study of gene expression

Membrane Yeast Two-Hybrid (MYTH) for analysis of targeted interactions or identification of novel interactors

Membrane Yeast Two Hybrid (MYTH) is a variation of standard yeast two hybrid that uses the split-ubiquitin system to allow the interrogation of interactions involving membrane bound proteins. This protocol has been used to study targeted interactions between pre- identified bait and prey proteins. In addition MYTH can use a known bait protein to screen a selection of unknown prey-proteins that have been generated from a cDNA library. This consensus methodology is for use of MYTH for both targeted screening as well as the identification of novel interactors

Isolation of the Plant Nucleolus

This article describes a protocol for Fluorescence-Activated Nucleolar Sorting (FANoS), which allows for clean extraction of the nucleolus from a range of plant tissues. This generates a nucleolar extract that is appropriate for use for downstream multi-‘omic characterisation of this important nuclear sub-domain

Hi-C protocol for analysis of plant nuclear chromatin interactions

The nuclear space is a dynamic environment in which DNA molecules interact across time, space and scale. Within the nucleus different yet adjacent chromosomes are co- regulated through shared chromatin modifications or the influence of global enhancer or repressor units. The interactions between adjacent chromosomes can be analysed though Hi-C, a technique that takes a whole genome view on samples obtained by chromosome confirmation capture. This technique has been used in many different plant species and this article provides a consensus protocol that is merged from those used in two expert labs

Localising Total mRNA in Plant Cells

Visualising the location of the total cellular mRNA pool can be important to understanding how different genes effect cellular physiology. Over the past decade researchers investigating RNA processing, nuclear transport and the function of the nuclear pore complex have used this situ hybridization protocol to visualize and quantify the accumulation of the total mRNA pool within the plant cell nucleus

Lect2 deficiency is characterised by altered cytokine levels and promotion of intestinal tumourigenesis

Leukocyte cell-derived chemotaxin 2 (Lect2) is a chemokine-like chemotactic factor that has been identified as a downstream target of the Wnt signalling pathway. Whilst the primary function of Lect2 is thought to be in modulating the inflammatory process, it has recently been implicated as a potential inhibitor of the Wnt pathway. Deregulation of the Wnt pathway, often due to loss of the negative regulator APC, is found in ~80% of colorectal cancer (CRC). Here we have used the ApcMin/+Lect2−/− mouse model to characterise the role of Lect2 in Wnt-driven intestinal tumourigenesis. Histopathological, immunohistochemical, PCR and flow cytometry analysis were employed to identify the role of Lect2 in the intestine. The ApcMin/+Lect2−/− mice had a reduced mean survival and a significantly increased number of adenomas in the small intestine with increased severity. Analysis of Lect2 loss indicated it had no effect on the Wnt pathway in the intestine but significant differences were observed in circulating inflammatory markers, CD4+ T cells, and T cell lineage-specification factors. In summary, in the murine intestine loss of Lect2 promotes the initiation and progression of Wnt-driven colorectal cancer. This protection is performed independently of the Wnt signalling pathway and is associated with an altered inflammatory environment during Wnt-driven tumorigenesis

Components of the Arabidopsis nuclear pore complex play multiple diverse roles in control of plant growth

Abstract The nuclear pore complex (NPC) is a multisubunit protein conglomerate that facilitates movement of RNA and protein between the nucleus and cytoplasm. Relatively little is known regarding the influence of the Arabidopsis NPC on growth and development. Seedling development, flowering time, nuclear morphology, mRNA accumulation, and gene expression changes in Arabidopsis nucleoporin mutants were investigated. Nuclear export of mRNA is differentially affected in plants with defects in nucleoporins that lie in different NPC subcomplexes. This study reveals differences in the manner by which nucleoporins alter molecular and plant growth phenotypes, suggesting that nuclear pore subcomplexes play distinct roles in nuclear transport and reveal a possible feedback relationship between the expression of genes involved in nuclear transport

Components of the Arabidopsis nuclear pore complex play multiple diverse roles in control of plant growth

The nuclear pore complex (NPC) is a multisubunit protein conglomerate that facilitates movement of RNA and protein between the nucleus and cytoplasm. Relatively little is known regarding the influence of the Arabidopsis NPC on growth and development. Seedling development, flowering time, nuclear morphology, mRNA accumulation, and gene expression changes in Arabidopsis nucleoporin mutants were investigated. Nuclear export of mRNA is differentially affected in plants with defects in nucleoporins that lie in different NPC subcomplexes. This study reveals differences in the manner by which nucleoporins alter molecular and plant growth phenotypes, suggesting that nuclear pore subcomplexes play distinct roles in nuclear transport and reveal a possible feedback relationship between the expression of genes involved in nuclear transport