Biology subjects

Dvorackova, M.

Publications and source records attributed to Dvorackova, M..

5 recordsLinked to original sources

bioRxiv2023

In-section Click-iT detection and super-resolution CLEM: Shedding light on nucleolar ultrastructure and S-phase progression in plants.

Correlative light and electron microscopy (CLEM) is an essential tool that allows for localisation of a particular target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometer scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of serial sections by both, single-molecule localization microscopy and transmission electron microscopy for correlative analysis. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows a dramatic improvement in fluorophore blinking behavior relative to the whole-mount approaches. Here we used commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as FASCIATA1 and NUCLEOLIN1 mutants and applied our on-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

plant biology↗

bioRxiv2021

Loss of FAS1 function reveals rescue of an aberrant intra-S-phase checkpoint by the G2/M checkpoint regulator SOG1

The WEE1 and ATR kinases represent important regulators of the plant intra-S-phase checkpoint, as evidenced by the hypersensitivity of WEE1KO and ATRKO roots to replication inhibitory drugs. Here, we report on the identification of a defective allele of the FASCIATA1 (FAS1) subunit of the chromatin assembly factor 1 (CAF-1) complex as a suppressor of WEE1- or ATR-deficient plants. We demonstrate that lack of FAS1 activity results in the activation of an ATM- and SOG1-mediated G2/M-arrest that makes the ATR and WEE1 checkpoint regulators redundant. This ATM activation accounts for telomere erosion and loss of ribosomal DNA described for the fas1 plants. Knocking out SOG1 in the fas1 wee1 background restores replication stress sensitivity, demonstrating that SOG1 plays a prominent role as secondary checkpoint regulator in plants that fail to activate the intra-S-phase checkpoint. One-Sentence SummaryLack of the chromatin assembly factor-1 subunit FAS1 results in a DNA damage response that overrules the need for replication checkpoint activators.

plant biology↗

bioRxiv2020

Multiple epigenetic layers accompany the spatial distribution of ribosomal genes in Arabidopsis

45S ribosomal genes in A. thaliana (rDNA) are located in tandem arrays on termini of chromosomes 2 and 4 (NOR2 and NOR4) and encode rRNA, crucial structural elements of the ribosome. Inactive rDNA genes accumulate in the condensed chromocenters in the nucleus and at the nucleolar periphery, while nucleolus delimits the active genes. We show that a subset of nucleolar rDNA assembles into condensed foci marked by H3.1 and H3.3 histones and that progressive rDNA condensation is connected with rDNA transcriptional activity, cell ploidy and rDNA copy number. Interestingly, some nucleolar foci are reminiscent of perinucleolar chromocenters, containing the NOR4 region. We further demonstrate that rDNA promoter is a key regulatory region of the rDNA repeat and describe large involvement of repressive epigenetic mark H3K9me2 and H2A.W histone variant in rDNA activity regulation. In addition, we found euchromatic H3.3 histone enrichment at the rDNA transcription start site in actively dividing tissues, despite its accumulation in nucleolar foci containing condensed rDNA repeats.

cell biology↗

bioRxiv2020

Low ribosomal RNA genes copy number provoke genomic instability and chromosomal segment duplication events that modify global gene expression and plant-pathogen response

Among the hundreds of ribosomal RNA (rRNA) gene copies organized as tandem repeats in the nucleolus organizer regions (NORs), only a portion is usually actively expressed in the nucleolus and participate in the ribosome biogenesis process. The role of these extra-copies remains elusive, but previous studies suggested their importance in genome stability and global gene expression. Because the nucleolus is also a platform for nuclear organization, we tested the impact of a decreased amount of rRNA gene copies on the Arabidopsis thaliana 3D genome organization and stability, using an A. thaliana line only containing 20% of rRNA gene copies (20rDNA line). Compared to the wild-type Col-0, the 20rDNA line shows several signs of genomic instability, such as variations in 3D genome organization, spontaneous double-strand breaks accumulation, transcriptomic changes, and higher DNA methylation level. Strikingly, using genomic and microscopic approaches, we identified seven large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. As a consequence, more than 600 genes were duplicated, often associated with an increase in their expression level. Among them, we found several upregulated genes involved in plant-pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and we discuss their potential implications on plant genome evolution.

plant biology↗

bioRxiv2020

Disruption of NAP1 genes supresses the fas1 mutant phenotype and enhances genome stability

Histone chaperones mediate assembly and disassembly of nucleosomes and participate in essentially all DNA-dependent cellular processes. In Arabidopsis thaliana, loss-of-functions of FAS1 or FAS2 subunits of the H3-H4 histone chaperone complex CHROMATIN ASSEMBLY FACTOR 1(CAF-1) has a dramatic effect on plant morphology, growth and overall fitness. Altered chromatin compaction, systematic loss of repetitive elements or increased DNA damage clearly demonstrate the severity of CAF-1 dysfunction. How histone chaperone molecular networks change without a functional CAF-1 remains elusive. Here we present an intriguing observation that disruption of the H2A-H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1) supresses FAS1 loss-of function. The quadruple mutant fas1nap1;1-3 shows wild-type growth and decreased sensitivity to genotoxic stress. Chromatin of fas1nap1;1-3 plants is less accessible to micrococcal nuclease and progressive loss of telomeres and 45S rDNA is supressed. Interestingly, the strong genetic interaction between FAS1 and NAP1 does not occur via direct protein-protein interaction. We propose that NAP1;1-3 play an essential role in nucleosome assembly in fas1, thus their disruption abolishes fas1 defects. Our data altogether reveal a novel function of NAP1 proteins, unmasked by CAF-1 dysfunction. It emphasizes the importance of a balanced composition of chromatin and shed light on the histone chaperone molecular network.

molecular biology↗