The absence of the arabidopsis chaperone complex CAF-1 produces mitotic chromosome abnormalities and changes in the expression profiles of genes involved in DNA repair

Chromatin Assembly Factor 1 (CAF-1) is an evolutionary conserved heterotrimeric chaperone complex that facilitates the incorporation of histones H3 and H4 onto newly synthesized DNA. We demonstrate here that the mutant deficient for the large subunit of the complex, fas1-4, and in minor extent, the mutant deficient for the middle subunit, fas2-1, display chromosome abnormalities throughout Arabidopsis mitosis. Among them, we observed multicentromeric chromosomes at metaphase, and chromatid bridges and acentric fragments at anaphase-telophase. 45S rDNA and telomeric sequences were frequently involved in bridges and fragments. Gene expression analysis by real-time qPCR has revealed that several genes related to homologous recombination (HR) and alternative non-homologous end-joining (aNHEJ) are overexpressed in fas1-4. These results concur with previous studies which have indicated that HR may be involved in the progressive loss of 45S rDNA and telomeres displayed by fas mutants. However, increased expression of PARP1, PARP2, and LIG6 in fas1-4, and the phenotype shown by the double mutant fas1 rad51 suggest that aNHEJ should also be responsible for the chromosomal aberrations observed. The activity of different DNA repair pathways in absence of CAF-1 is discussed

Latent Tree Learning with Differentiable Parsers: Shift-Reduce Parsing and Chart Parsing

Latent tree learning models represent sentences by composing their words according to an induced parse tree, all based on a downstream task. These models often outperform baselines which use (externally provided) syntax trees to drive the composition order. This work contributes (a) a new latent tree learning model based on shift-reduce parsing, with competitive downstream performance and non-trivial induced trees, and (b) an analysis of the trees learned by our shift-reduce model and by a chart-based model.Comment: ACL 2018 workshop on Relevance of Linguistic Structure in Neural Architectures for NL

Analysis of the Relationships between DNA Double-Strand Breaks, Synaptonemal Complex and Crossovers Using the Atfas1-4 Mutant

Chromatin Assembly Factor 1 (CAF-1) is a histone chaperone that assembles acetylated histones H3/H4 onto newly synthesized DNA, allowing the de novo assembly of nucleosomes during replication. CAF-1 is an evolutionary conserved heterotrimeric protein complex. In Arabidopsis, the three CAF-1 subunits are encoded by FAS1, FAS2 and MSI1. Atfas1-4 mutants have reduced fertility due to a decrease in the number of cells that enter meiosis. Interestingly, the number of DNA double-strand breaks (DSBs), measured by scoring the presence of γH2AX, AtRAD51 and AtDMC1 foci, is higher than in wild-type (WT) plants, and meiotic recombination genes such AtCOM1/SAE2, AtBRCA1, AtRAD51 and AtDMC1 are overexpressed. An increase in DSBs in this mutant does not have a significant effect in the mean chiasma frequency at metaphase I, nor a different number of AtMLH1 nor AtMUS81 foci per cell compared to WT at pachytene. Nevertheless, this mutant does show a higher gene conversion (GC) frequency. To examine how an increase in DSBs influences meiotic recombination and synaptonemal complex (SC) formation, we analyzed double mutants defective for AtFAS1 and different homologous recombination (HR) proteins. Most showed significant increases in both the mean number of synapsis initiation points (SIPs) and the total length of AtZYP1 stretches in comparison with the corresponding single mutants. These experiments also provide new insight into the relationships between the recombinases in Arabidopsis, suggesting a prominent role for AtDMC1 versus AtRAD51 in establishing interhomolog interactions. In Arabidopsis an increase in the number of DSBs does not translate to an increase in the number of crossovers (COs) but instead in a higher GC frequency. We discuss different mechanisms to explain these results including the possible existence of CO homeostasis in plants

Untangling chromatin interactions

International audienceUntangling 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

Synthetically induced Arabidopsis thaliana autotetraploids provide insights into the analysis of meiotic mutants with altered crossover frequency

Mutations affecting crossover (CO) frequency and distribution lead to the presence of univalents during meiosis, giving rise to aneuploid gametes and sterility. These mutations may have a different effect after chromosome doubling. The combination of altered ploidy and mutations could be potentially useful to gain new insights into the mechanisms and regulation of meiotic recombination; however, studies using autopolyploid meiotic mutants are scarce. Here, we have analyzed the cytogenetic consequences in colchicine-induced autotetraploids (colchiploids) from different Arabidopsis mutants with an altered CO frequency. We have found that there are three types of mutants: mutants in which chiasma frequency is doubled after chromosome duplication (zip4, mus81), as in the control; mutants in which polyploidy leads to a higher-than-expected increase in chiasma frequency (asy1, mer3, hei10, and mlh3); and mutants in which the rise in chiasma frequency produced by the presence of two extrachromosomal sets is less than doubled (msh5, fancm). In addition, the proportion of class I/class II COs varies after chromosome duplication in the control. The results obtained reveal the potential of colchiploid meiotic mutants for better understanding of the function of key proteins during plant meiosis. This is especially relevant considering that most crops are polyploids.Depto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEpu

Loss of function of Arabidopsis microRNA-machinery genes impairs fertility, and has effects on homologous recombination and meiotic chromatin dynamics

MicroRNAs (miRNAs) are ~22-nt single-stranded noncoding RNAs with regulatory roles in a wide range of cellular functions by repressing eukaryotic gene expression at a post-transcriptional level. Here, we analyzed the effects on meiosis and fertility of hypomorphic or null alleles of the HYL1, HEN1, DCL1, HST and AGO1 genes, which encode miRNA-machinery components in Arabidopsis. Reduced pollen and megaspore mother cell number and fertility were shown by the mutants analyzed. These mutants also exhibited a relaxed chromatin conformation in male meiocytes at the first meiotic division, and increased chiasma frequency, which is likely to be due to increased levels of mRNAs from key genes involved in homologous recombination. The hen1-13 mutant was found to be hypersensitive to gamma irradiation, which mainly causes double-strand breaks susceptible to be repaired by homologous recombination. Our findings uncover a role for miRNA-machinery components in Arabidopsis meiosis, as well as in the repression of key genes required for homologous recombination. These genes seem to be indirect miRNA targets

Involvement of the Cohesin Cofactor PDS5 (SPO76) During Meiosis and DNA Repair in Arabidopsis thaliana

Maintenance and precise regulation of sister chromatid cohesion is essential for faithful chromosome segregation during mitosis and meiosis. Cohesin cofactors contribute to cohesin dynamics and interact with cohesin complexes during cell cycle. One of these, PDS5, also known as SPO76, is essential during mitosis and meiosis in several organisms and also plays a role in DNA repair. In yeast, the complex Wapl-Pds5 controls cohesion maintenance and colocalizes with cohesin complexes into chromosomes. In Arabidopsis, AtWAPL proteins are essential during meiosis, however, the role of AtPDS5 remains to be ascertained. Here we have isolated mutants for each of the five AtPDS5 genes (A–E) and obtained, after different crosses between them, double, triple, and even quadruple mutants (Atpds5a Atpds5b Atpds5c Atpds5e). Depletion of AtPDS5 proteins has a weak impact on meiosis, but leads to severe effects on development, fertility, somatic homologous recombination (HR) and DNA repair. Furthermore, this cohesin cofactor could be important for the function of the AtSMC5/AtSMC6 complex. Contrarily to its function in other species, our results suggest that AtPDS5 is dispensable during the meiotic division of Arabidopsis, although it plays an important role in DNA repair by HR

Absence of SUN1 and SUN2 proteins in Arabidopsis thaliana leads to a delay in meiotic progression and defects in synapsis and recombination

The movement of chromosomes during meiosis involves the location of their telomeres at the inner surface of the nuclear envelope (NE). Sad1/UNC-84 (SUN)-domain proteins are inner NE proteins that are part of complexes linking cytoskeletal elements with the nucleo skeleton, connecting telomeres to the force-generating mechanism in the cytoplasm. These proteins play a conserved role in chromosome dynamics in eukaryotes. Homologues of SUN-domain proteins have been identified in several plant species. In Arabidopsis thaliana two proteins which interact with each other, named AtSUN1 and AtSUN2, have been identified. Immuno localisation with antibodies to AtSUN1 and AtSUN2 proteins revealed that they were associated with the nuclear envelope during meiotic prophase I. Analysis of the double mutant Atsun1-1 Atsun2-2 has revealed severe meiotic defects, namely, a delay in the progression of meiosis, an absence of full synapsis, unresolved interlock-like structures and a reduction in the mean cell chiasma frequency. We propose that in Arabidopsis thaliana, overlapping functions of SUN1 and SUN2 ensure normal meiotic recombination and synapsis