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International audience; Untangling chromatin interactions The plant nucleus contains myriad subdomains that define and are defined by the composition of their chromatin. On a whole nucleus scale, this includes regions of active euchromatin and inactive heterochromatin, although the boundaries between these states are blurred. Different loci constantly enter and exit nuclear structures including the nucleolus and transient regulatory nuclear bodies. Chromatin is defined by its complex combinations of epigenetic modifications and variation in its constituent histone proteins. Here we preview articles that describe many of the mechanisms that regulate this dynamic chromatin.

Background The three-dimensional nuclear arrangement of chromatin impacts many cellular processes operating at the DNA level in animal and plant systems. Chromatin organization is a dynamic process that can be affected by biotic and abiotic stresses. Three-dimensional imaging technology allows to follow these dynamic changes, but only a few semi-automated processing methods currently exist for quantitative analysis of the 3D chromatin organization. Results We present an automated method, Nuclear Object DetectionJ (NODeJ), developed as an imageJ plugin. This program segments and analyzes high intensity domains in nuclei from 3D images. NODeJ performs a Laplacian convolution on the mask of a nucleus to enhance the contrast of intra-nuclear objects and allow their detection. We reanalyzed public datasets and determined that NODeJ is able to accurately identify heterochromatin domains from a diverse set of Arabidopsis thaliana nuclei stained with DAPI or Hoechst. NODeJ is also able to detect signals in nuclei from DNA FISH experiments, allowing for the analysis of specific targets of interest. Conclusion and availability NODeJ allows for efficient automated analysis of subnuclear structures by avoiding the semi-automated steps, resulting in reduced processing time and analytical bias. NODeJ is written in Java and provided as an ImageJ plugin with a command line option to perform more high-throughput analyses. NODeJ can be downloaded from https://gitlab.com/axpoulet/image2danalysis/-/releases with source code, documentation and further information avaliable at https://gitlab.com/axpoulet/image2danalysis. The images used in this study are publicly available at https://www.brookes.ac.uk/indepth/images/ and https://doi.org/10.15454/1HSOIE.

The nucleoskeleton forms a filamentous meshwork under the nuclear envelope and contributes to the regulation of nuclear morphology and gene expression. To understand how the Arabidopsis nucleoskeleton physically connects to the nuclear periphery, we investigated the nucleoskeleton protein KAKU4 and sought for functional regions responsible for its localization at the nuclear periphery. Computational predictions identified three evolutionary conserved peptide motifs within the N-terminal region of KAKU4. Functional analysis revealed that these motifs are required for homomerization of KAKU4, interaction with the nucleoskeleton proteins CROWDED NUCLEI (CRWN) and localization at the nuclear periphery. We find that similar protein motifs are present in NUP82 and NUP136, two plant specific nucleoporins from the Nuclear Pore Complex (NPC) basket. These conserved motifs allow the two nucleoporins to bind CRWN proteins, thus revealing a physical link between the nucleoskeleton and nuclear pores in plants. Finally, whilst NUP82, NUP136 and KAKU4 have a common evolutionary history predating non-vascular land plants, KAKU4 mainly localizes outside the NPC suggesting neofunctionalization of an ancient nucleoporin into a new nucleoskeleton component.

Centromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed CenH3. As for other histones, escort and deposition of CenH3 must be ensured by histone chaperones, which handle the non-nucleosomal CenH3 pool and replenish CenH3 chromatin in dividing cells. Here, we show that the Arabidopsis orthologue of the mammalian NUCLEAR AUTOANTIGENIC SPERM PROTEIN (NASP) and Schizosaccharomyces pombe histone chaperone Sim3 is a soluble nuclear protein that binds the histone variant CenH3 and affects its abundance at the centromeres. NASP

Identification of membrane protein interactomes is a key issue to better understand how these molecules carry out their functions. However, protein-protein interactions using conventional interaction assays are particularly challenging for integral membrane proteins, because of their hydrophobic nature. Here we describe the membrane yeast two-hybrid (MbY2H) system, a powerful tool for identifying the interactors of membrane and membrane-associated proteins.

The analysis of nuclear envelope components and their function has recently been progressed by the use of computational methods of analysis. The methods in this chapter provided by members of the International Plant Nucleus Consortium address the identification of novel nuclear envelope proteins and the study of structure and mobility of the nucleus. DORY2 is an upgrade of the KASH-finder DORY, and NucleusJ is used to characterize the three-dimensional structure of the nucleus in light microscope images. Finally, a method is provided for analysis of the migration of the nucleus, a key technique for exploring the function of plant nuclear proteins.

Image analysis is a classical way to study nuclear organization. While nuclear organization used to be investigated by colorimetric or fluorescent labeling of DNA or specific nuclear compartments, new methods in microscopy imaging now enable qualitative and quantitative analyses of chromatin pattern, and nuclear size and shape. Several procedures have been developed to prepare samples in order to collect 3D images for the analysis of spatial chromatin organization, but only few preserve the positional information of the cell within its tissue context. Here, we describe a whole mount tissue preparation procedure coupled to DNA staining using the PicoGreen® intercalating agent suitable for image analysis of the nucleus in living and fixed tissues. 3D Image analysis is then performed using NucleusJ, an open source ImageJ plugin, which allows for quantifying variations in nuclear morphology such as nuclear volume, sphericity, elongation, and flatness as well as in heterochromatin content and position in respect to the nuclear periphery.

International audience; The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its involvement in chromatin organisation has not been studied in plants. Here, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana in which heterochromatin clusters in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of the 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally, we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences.

International audience; In this study, we explore the plasticity during evolution of proteins of the higher plant nuclear envelope (NE) from the most ancestral plant species to advanced angiosperms. The higher plant NE contains a functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex based on conserved Sad1-Unc84 (SUN) domain proteins and plant specific Klarsicht/Anc1/Syne homology (KASH) domain proteins. Recent evidence suggests the presence of a plant lamina underneath the inner membrane and various coiled-coil proteins have been hypothesized to be associated with it including Crowded Nuclei (CRWN; also termed LINC and NMCP), Nuclear Envelope Associated Protein (NEAP) protein families as well as the CRWN binding protein KAKU4. SUN domain proteins appear throughout with a key role for mid-SUN proteins suggested. Evolution of KASH domain proteins has resulted in increasing complexity, with some appearing in all species considered, while other KASH proteins are progressively gained during evolution. Failure to identify CRWN homologs in unicellular organisms included in the study and their presence in plants leads us to speculate that convergent evolution may have occurred in the formation of the lamina with each kingdom having new proteins such as the Lamin B receptor (LBR) and Lamin-Emerin-Man1 (LEM) domain proteins (animals) or NEAPs and KAKU4 (plants). Our data support a model in which increasing complexity at the nuclear envelope occurred through the plant lineage and suggest a key role for mid-SUN proteins as an early and essential component of the nuclear envelope.

The precise location of chromatin domains within the cell nucleus has\ud seen growing recognition in the past decade as an additional\ud mechanism of controlling gene expression in both plants and\ud animals (Dekker et al., 2017). Consequently, international efforts are\ud devoted to understanding the organising principle of this organelle in\ud plants, and notably the nature and the role of functional compartments\ud on gene expression (Graumann et al., 2013; Sotelo-Silveira et al.,\ud 2018). The European cooperation ‘Impact of Nuclear Domains on\ud Gene Expression and Plant Traits’ (INDEPTH) brings together\ud molecular cell biologists, plant physiologists, bioinformaticians, image\ud analysts and computer scientists. They aim to address the question of\ud how nuclear architecture, chromatin organisation and gene expression\ud are connected in plants, particularly in relation to traits of interest such\ud as biomass, reproduction and resistance to pathogens (https://\ud www.brookes.ac.uk/indepth/). The kick-off meeting of the INDEPTH\ud consortium took place in Clermont-Ferrand, France, on 12–14thMarch\ud 2018, where more than 80 researchers set the agenda for the coming\ud four years of research and collaboration.

International audience; The centromere is a specific chromosomal region where the kinetochore assembles to ensure the faithful segregation of sister chromatids during mitosis and meiosis. Centromeres are defined by a local enrichment of the specific histone variant CenH3 mostly at repetitive satellite sequences. A larger pericentromeric region containing repetitive sequences and transposable elements surrounds the centromere that adopts a particular chromatin state characterized by specific histone variants and post-translational modifications and forms a transcriptionally repressive chromosomal environment. In the model organism Arabidopsis thaliana centromeric and pericentromeric domains form conspicuous heterochromatin clusters called chromocenters in interphase. Here we discuss, using Arabidopsis as example, recent insight into mechanisms involved in maintenance and establishment of centromeric and pericentromeric chromatin signatures as well as in chromocenter formation.

International audience; SUN-domain proteins belong to a gene family including classical Cter-SUN and mid-SUN subfamilies differentiated by the position of the SUN domain within the protein. Although present in animal and plant species, mid-SUN proteins have so far remained poorly described. Here, we used a combination of genetics, yeast two-hybrid and in planta transient expression methods to better characterize the SUN family in Arabidopsis thaliana. First, we validated the mid-SUN protein subfamily as a monophyletic group conserved from yeast to plant. Arabidopsis Cter-SUN (AtSUN1 and AtSUN2) and mid-SUN (AtSUN3 and AtSUN4) proteins expressed as fluorescent protein fusions are membrane-associated and localize to the nuclear envelope (NE) and endoplasmic reticulum. However, only the Cter-SUN subfamily is enriched at the NE. We investigated interactions in and between members of the two subfamilies and identified the coiled-coil domain as necessary for mediating interactions. The functional significance of the mid-SUN subfamily was further confirmed in mutant plants as essential for early seed development and involved in nuclear morphology. Finally, we demonstrated that both subfamilies interact with the KASH domain of AtWIP1 and identified a new root-specific KASH-domain protein, AtTIK. AtTIK localizes to the NE and affects nuclear morphology. Our study indicates that Arabidopsis Cter-SUN and mid-SUN proteins are involved in a complex protein network at the nuclear membranes, reminiscent of the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex found in other kingdoms.

International audience; Significant advances in understanding the plant nuclear envelope have been made over the past few years; indeed, knowledge of the protein network at the nuclear envelope is rapidly growing. One such network, the linker of nucleoskeleton and cytoskeleton (LINC) complex, is known in animals to connect chromatin to the cytoskeleton through the nuclear envelope. The LINC complex is made of Sad1/Unc84 (SUN) and Klarsicht/Anc1/Syne1 homology (KASH) proteins which have been recently characterized in plants. SUN proteins are located within the inner nuclear membrane, while the KASH proteins are included into the outer nuclear membrane. SUN and KASH domains interact and bridge the two nuclear membranes. In Arabidopsis, KASH proteins also interact with the tryptophan-proline-proline (WPP) domain-interacting tail-anchored protein 1 (WIT1), associated with the nuclear pore complex and with myosin XI-i which directly interacts with the actin cytoskeleton. Although evidence for a plant LINC complex connecting the nucleus to the cytoskeleton is growing, its interaction with chromatin is still unknown, but knowledge gained from animal models strongly suggests its existence in plants. Possible functions of the plant LINC complex in cell division, nuclear shape, and chromatin organization are discussed.

Short interspersed nuclear elements (SINEs) are ubiquitous components of complex animal and plant genomes. SINEs are believed to be important players in eukaryotic genome evolution. Studies on SINE integration sites have revealed non-random integration without strict nucleotide sequence requirements for the integration target, suggesting that the targeted DNA might assume specific secondary structures or protein associations. Here, we report that S1 SINE elements in the genomes of Brassica show an interesting preference for matrix attachment regions (MARs). Ten cloned genomic regions were tested for their ability to bind the nuclear matrix both before and after a SINE integration event. Eight of the genomic regions targeted by S1 display strong affinity for the nuclear matrix, while two show weaker binding. The SINE S1 did not display any matrix-binding capacity on its own in either non-methylated or methylated forms. In vivo, an integrated S1 is methylated while the surrounding genomic regions may remain undermethylated or undergo methylation. However, tested genomic regions containing methylated'S1, with or without methylated flanking genomic sequences, were found to vary in their ability to bind the matrix in vitro. These results suggest a possible molecular basis for a preferential targeting of SINEs to MARs and a possible impact of the integration events upon gene and genome function.

Short interspersed nuclear elements (SINEs) are small noncoding transposable elements that are widespread in most eukaryotic genomes. Plant S1 SINEs are present in crucifers, especially in species of the Brassiceae tribe, and were generated by several waves of amplification of different evolutionary ages, suggesting that S1 insertional variability (presence/absence of an S1 at a given genomic site) could be used as a classification criterion to evaluate phylogenetic relationships. We applied this strategy on closely related species from the Brassica oleracea species complex using 21 independent S1 genomic sites. The microsatellite-like variation of S1 39 poly(A) tails was also used as a complementary classification criterion to obtain internal resolution in two different clades. The phylogenetic tree obtained by this approach is in general agreement with the classification made from chloroplast DNA restriction site polymorphisms and differs significantly from two other (nonequivalent) classifications made using nuclear restriction fragment length polymorphisms. Brassica incana, Brassica montana, and Brassica hilarionis are confirmed as the closest relatives of B. oleracea. From our data, we suggest that Brassica drepanensis emerged recently from a B. incana/Brassica villosa hybrid (with B. villosa as the maternal parent) following backcrosses to B. incana. We also detected several introgressions, confirming that these highly related species are capable of genetic exchange in their natural habitat. S1 markers are therefore very useful in understanding the detailed evolutionary history of wild Brassica species and could also be used to identify potential gene flow between cultivated (including transgenic) Brassica and their wild relatives.

DNA methylation has been often proposed to operate as a genome defence system against parasitic mobile elements. To test this possibility, the methylation status of a class of plant mobile elements, the S1Bn SINEs, was analysed in detail using the bisulfite modification method. We observed that S1Bn SINE retroposons are methylated at symmetrical and asymmetrical positions. Methylated cytosines are not limited to transcriptionally important regions but are well distributed along the sequence. S1Bn SINE retroposons are two-fold more methylated than the average methylation level of the Brassica napus nuclear DNA. By in situ hybridization, we showed that this high level of methylation does not result from the association of S1Bn elements to genomic regions known to be highly methylated suggesting that S1Bn elements were specifically methylated. A detailed analysis of the methylation context showed that S1Bn cytosines in symmetrical CpG and CpNpG sites are methylated at a level of 87% and 44% respectively. We observed that 5.3% of S1Bn cytosines in non- symmetrical positions were also methylated. Of this asymmetrical methylation, 57% occurred at a precise motif (Cp(A/T)pA) that only represented 12% of the asymmetrical sites in S1Bn sequences suggesting that it represents a preferred asymmetrical methylation site. This motif is methylated in S1Bnelements at only half the level observed for the Cp(A/T)pG sites. We show that non-S1Bn CpTpA sites can also be methylated in DNA from B. napus and from other plant species.

The characterization of a simple bacterial system using commercially available plasmids and strains, developed to assess the effectiveness of trans-acting (antisense RNA and ribozymes) RNA in Escherichia coli, is reported. This system was used to test various trans-acting RNA molecules against the expression of the I factor, a functional transposable element from Drosophila melanogaster. For this target, results indicate that antisense RNA efficiency depends on the hybridization length between sense and antisense RNA. The introduction of a single hammerhead ribozyme within the antisense RNAs does not increase its inhibitory activity. These predictions were subsequently confirmed in Drosophila melanogaster.

International audience; The reverse transcription of RNA in DNA is responsible for the generation of large families of repetitive sequences called retroposons or non-LTR retrotransposons. Recent reports established that the integration of mammalian SINE and LINE retroposons occurs at nonrandom staggered breaks, probably resulting from the action of a LINE-encoded endonuclease (Feng et al. 1996; Jurka 1997; Jurka et al. 1998). We report here that plant SINE S1 retroposons also integrate at nonrandom staggered breaks. One of the two nicks involved in S1 integration is associated mainly with the 5'-Y/AAANNNG-3' motif. The other nick at opposite DNA strand occurs preferably within 14-16 bp, a situation also observed for mammalian retroposons, but is not associated with any specific motif. Further studies on the distribution of dinucleotides surrounding the two nicking sites showed that, as for mammalian retroposons, S1 retroposons integrate at sites rich in TA, CA, and TG dinucleotides. These dinucleotides were reported as specific DNA sites where special DNA structures called "kinks" may occur under bending constraints. Nicking sites are preceded by peaks in frequency of di-pyrimidine followed by peaks of di-purine. These results suggest that the general A/T richness of a given DNA region and the presence of short runs of pyrimidines followed by short runs of purines could represent a favorable context for the integration of retroposons. In such a context, an endonuclease upon fixation could be able to generate the kink at the pyrimidine/purine transition and to nick the DNA. The similarities in target site selection observed for plant and mammalian retroposons suggest that retroposition is a surprisingly well conserved process.

International audience; The current model of short interspersed nuclear element (SINE) mobility suggests that these non-coding retroposons are able to recruit for their own benefits the enzymatic machinery encoded by autonomous long interspersed nuclear elements (LINEs). The recent characterization of potential SINE-LINE partner pairs that share common 3' end sequences concurs with this model and has led to a potent picture of tRNA-derived SINEs consisting of a tripartite functional structure (Mol. Cell. Biol. 16 (1996) 3756; Mol. Biol. Evol. 16 (1999) 1238; Proc. Natl. Acad. Sci. USA 96 (1999) 2869). This structure consist of a 5' polIII tRNA-related promoter region, a central conserved domain and a variable 3' region with homology to the 3' end of LINEs, believed to be essential to direct recognition by the LINE proteins. To test this model in vivo, we have designed synthetic SINEs possessing this 'canonical' structure, including 3' homology to the 3' UTR of the LINE I factor from Drosophila. These synthetic elements were introduced in a Drosophila reactive strain, and SINE retroposition was assessed following dysgenic crosses that are known to induce high levels of I factor germinal transposition. In the progeny from the dysgenic crosses 3400-4000 flies were analyzed but no retroposed copy of the chimeric SINEs was detected, indicating that what is assumed to be a typical SINE structure is not sufficient per se to allow efficient trans-mobilization of our synthetic SINEs by an actively amplifying partner LINE. Alternatively, the apparent absence of natural fly SINEs may underline intrinsic properties of fly biology that are incompatible with the genesis and/or propagation of SINE-like elements.

The Polycomb Response Element (PRE) is the nucleation site for the Polycomb silencing complexes. The sequences responsible for the recruitment of the components of the Polycomb complex are not well understood. A comparison of the bxd PRE sequences from several different Drosophila species shows that some changes have occurred during phylogeny but large blocks of sequence are conserved after a divergence of some 60 million years. We compare the PRE sequences, the sites of some known PRE binding proteins, the conservation of DNasel hypersensitive sites and relate them to the sequence of the Ultrabithorax promoter which these PREs regulate.

The I factor (IF) is a LINE-like transposable element from Drosophila melanogaster. IF is silenced in most strains, but under special circumstances its transposition can be induced and correlates with the appearance of a syndrome of female sterility called hybrid dysgenesis. To elucidate the relationship between IF expression and female sterility, different transgenic antisense and/or sense RNAs homologous to the IF ORF1 have been expressed. Increasing the transgene copy number decreases both the expression of an IF-lacZ fusion and the intensity of the female sterile phenotype, demonstrating that IF expression is correlated with sterility. Some transgenes, however, exert their repressive abilities not only through a copy number-dependent zygotic effect, but also through additional maternal and paternal effects that may be induced at the DNA and/or RNA level. Properties of the maternal effect have been detailed: (1) it represses hybrid dysgenesis more efficiently than does the paternal effect; (2) its efficacy increases with both the transgene copy number and the aging of sterile females; (3) it accumulates slowly over generations after the transgene has been established; and (4) it is maintained for at least two generations after transgene removal. Conversely, the paternal effect increases only with female aging. The last two properties of the maternal effect and the genuine existence of a paternal effect argue for the occurrence, in the IF regulation pathway, of a cellular memory transmitted through mitosis, as well as through male and female meiosis, and akin to epigenetic phenomena.