Biology subjects

Houben, A.

Publications and source records attributed to Houben, A..

24 records · Page 2Linked to original sources

bioRxiv2020

Application of aptamers improves CRISPR-based live imaging of plant telomeres

Development of live imaging techniques for providing information how chromatin is organized in living cells is pivotal to decipher the regulation of biological processes. Here, we demonstrate the improvement of a live imaging technique based on CRISPR/Cas9. In this approach, the sgRNA scaffold is fused to RNA aptamers including MS2 and PP7. When the dead Cas9 (dCas9) is co-expressed with chimeric sgRNA, the aptamer-binding proteins fused to fluorescent protein (MCP-FP and PCP-FP) are recruited to the targeted sequence. Compared to previous work with dCas9:GFP, we show that the quality of telomere labelling was improved in transiently transformed Nicotiana benthamiana using aptamer-based CRISPR-imaging constructs. Labelling is influenced by the copy number of aptamers and less by the promoter types. The same constructs were not applicable for labelling of repeats in stably transformed plants and roots. The constant interaction of the RNP complex with its target DNA might interfere with cellular processes. HighlightAptamer-based CRISPR imaging: an opportunity for improving live-cell imaging in plants

cell biology↗

bioRxiv2020

Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

Expansion Microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to [~]4.2-times physically expanded nuclei corresponding to a maximal resolution of [~]50-60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution the chromatin structures were observed at a resolution of [~]25-35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM we observed that the preservation of the chromatin ultrastructure after expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei we detected successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunostaining. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation) their ultrastructural arrangement was impaired.

cell biology↗

bioRxiv2020

Unequal contribution of two paralogous centromeric histones to function the cowpea centromere

The legume cowpea (Vigna unguiculata, 2n=2x=22) has significant tolerance to drought and heat stress. Here we analysed and manipulated cowpea centromere-specific histone H3 (CENH3) genes, aiming to establish a centromere-based doubled-haploid method for use in genetic improvement of this dryland crop in future. Cowpea encodes two functional CENH3 variants (CENH3.1 and CENH3.2) and two CENH3 pseudogenes. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of V. unguiculata and the related V. mungo without a genome duplication event. Both functional cowpea CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the outer kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. Differences between both CENH3 paralogs are restricted to the N-terminus. The complete CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development, suggesting that the gene is an early stage of subfunctionalization or pseudogenization. One-sentence summaryThe two paralogous centromeric histones (CENH3) of cowpea contribute unequal to the function of the centromere.

genetics↗

bioRxiv2019

Chromosome-scale genome assembly provides insights into rye biology, evolution, and agronomic potential

We present a chromosome-scale annotated assembly of the rye (Secale cereale L. inbred line Lo7) genome, which we use to explore Triticeae genomic evolution, and ryes superior disease and stress tolerance. The rye genome shares chromosome-level organization with other Triticeae cereals, but exhibits unique retrotransposon dynamics and structural features. Crop improvement in rye, as well as in wheat and triticale, will profit from investigations of rye gene families implicated in pathogen resistance, low temperature tolerance, and fertility control systems for hybrid breeding. We show that rye introgressions in wheat breeding panels can be characterised in high-throughput to predict the yield effects and trade-offs of rye chromatin.

genomics↗

bioRxiv2019

The Arabidopsis condensin CAP-D subunits arrange interphase chromatin

Condensins are best known for their role in shaping chromosomes. However, other functions as organizing interphase chromatin and transcriptional control have been reported in yeasts and animals. Yeasts encode one condensin complex, while higher eukaryotes have two of them (condensin I and II). Both, condensin I and II, are conserved in Arabidopsis thaliana, but so far little is known about their function. Here we show that the A. thaliana CAP-D2 (condensin I) and CAP-D3 (condensin II) subunits are highly expressed in mitotically active tissues. In silico and pull-down experiments indicate that both CAP-D proteins interact with the other condensin I and II subunits. Our data suggest that the expression, localization and composition of the condensin complexes in A. thaliana are similar as in other higher eukaryotes. Previous experiments showed that the lack of A. thaliana CAP-D3 leads to centromere association during interphase. To study the function of CAP-D3 in chromatin organization more in detail we compared the nuclear distribution of rDNA, of centromeric chromocenters and of different epigenetic marks, as well as the nuclear size between wild-type and cap-d3 mutants. In these mutants an association of heterochromatic sequences occurs, but nuclear size and the general methylation and acetylation patterns remain unchanged. In addition, transcriptome analyses revealed a moderate influence of CAP-D3 on general transcription, but a stronger one on transcription of stress-related genes. We propose a model for the CAP-D3 function during interphase, where CAP-D3 localizes in euchromatin loops to stiff them, and consequently separates centromeric regions and 45S rDNA repeats.

cell biology↗

bioRxiv2019

Nondisjunction and unequal spindle organization accompany the drive of Aegilops speltoides B chromosomes

Supernumerary B chromosomes (Bs), which are often preferentially inherited, deviating from usual Mendelian segregation. This chromosome drive is one of the most important features of Bs. Here we analyzed the drive mechanism of Aegilops speltoides Bs and provide direct insight into its cellular mechanism. Comparative genomics resulted in the identification of the tandem repeat AesTR-183 of Ae. speltoides Bs, which also can be found on the Bs of Ae. mutica and rye, was used to track Bs during microgametogenesis. Nondisjunction of CENH3-positive, tubulin interacting B sister chromatids and an asymmetric spindle during first pollen grain mitosis are likely components of the accumulation process. A quantitative flow cytometric approach revealed, that independent on the number of Bs present in the mother plant Bs accumulate in the generative nuclei with more than 93%. Nine of eleven tested (peri)centromeric repeats were shared by A and B chromosomes. A common origin of the drive process in Poaceae is likely.

genetics↗