Identification of developmental functions for Arabidopsis thaliana genes by a reverse genetics approach based on analysis of H3K27me3 distribution

Polycomb Group (PcG) protein mediated gene repression is essential for normal development in both plants and animals, as demonstrated by severe developmental defects resulting from their loss-of-function. PcG proteins convey repression of target genes by tri-methylation of lysine 27 of histone 3 (H3K27me3). Many H3K27me3 decorated genes encode developmental regulators in Arabidopsis thaliana and developmental functions are particularly overrepresented in tissue specific sub sets of H3K27me3 targets. This study identified 105 genes specifically expressed in the shoot apex and floral organs by transcriptional clustering analysis, which are particularly enriched for shoot developmental functions according to Gene Ontology analysis. As half of the genes in this group were not characterised in detail, these were screened for a role in shoot development by analysing loss-of-function mutants and selected can- didate gene overexpessor plants. Fourteen putative Development related PcG Targets in the Apex (DPAs) were identified. For five DPA putants developmental abnormalities were confirmedly associated with the respective loci. Among them were genes related to flowering time, leaf size and leaf shape regulation. dpa4 loss-of-function plants display enhanced leaf serrations and enlarged petals, while leaf margins of 35S::DPA4 plants are smooth. DPA4 encodes for a putative RAV (Related to ABI3/VP1) transcriptional repressor and is expressed in the lateral organ boundary region and in leaf sinuses. Total leaf area and cell numbers are not altered in dpa4 plants, suggesting that DPA4 regulates leaf margin outgrowth by inhibiting growth towards leaf serrations. DPA4 expression domains widely overlap with those of CUP-SHAPED COTYLEDON 2, known to regulate leaf margin shape. Genome-wide transcriptional profiling in dpa4 apices revealed 77 differentially expressed genes. An overrepresentation of auxin-response elements in the promoters of these otherwise poorly characterised genes indicates a role for DPA4 in auxin- mediated signalling. This is further supported by an auxin-influx carrier mutant-like phenotype observed for 35S::DPA4 plants displaying left-hand twisted rosette leaves. Taken together, the data confirm that DPA4, which was identified as a candidate by this reverse genetics screen, is a newly identified player in the signalling network controlling leaf serrations in Arabidopsis thaliana

Meeting Summary of the 6th European Workshop on Plant Chromatin 2019 in Cologne, Germany

In June 2019, more than a hundred plant researchers met in Cologne, Germany, for the 6th European Workshop on Plant Chromatin (EWPC). This conference brought together a highly dynamic community of researchers with the common aim to understand how chromatin organization controls gene expression, development, and plant responses to the environment. New evidence showing how epigenetic states are set, perpetuated, and inherited were presented, and novel data related to the three-dimensional organization of chromatin within the nucleus were discussed. At the level of the nucleosome, its composition by different histone variants and their specialized histone deposition complexes were addressed as well as the mechanisms involved in histone post-translational modifications and their role in gene expression. The keynote lecture on plant DNA methylation by Julie Law (SALK Institute) and the tribute session to Lars Hennig, honoring the memory of one of the founders of the EWPC who contributed to promote the plant chromatin and epigenetic field in Europe, added a very special note to this gathering. In this perspective article we summarize some of the most outstanding data and advances on plant chromatin research presented at this workshop

Looking at the past and heading to the Future : Meeting summary of the 6th European Workshop on Plant Chromatin 2019 in Cologne, Germany

Altres ajuts: CERCA Programme/Generalitat de CatalunyaIn June 2019, more than a hundred plant researchers met in Cologne, Germany, for the 6th European Workshop on Plant Chromatin (EWPC). This conference brought together a highly dynamic community of researchers with the common aim to understand how chromatin organization controls gene expression, development, and plant responses to the environment. New evidence showing how epigenetic states are set, perpetuated, and inherited were presented, and novel data related to the three-dimensional organization of chromatin within the nucleus were discussed. At the level of the nucleosome, its composition by different histone variants and their specialized histone deposition complexes were addressed as well as the mechanisms involved in histone post-translational modifications and their role in gene expression. The keynote lecture on plant DNA methylation by Julie Law (SALK Institute) and the tribute session to Lars Hennig, honoring the memory of one of the founders of the EWPC who contributed to promote the plant chromatin and epigenetic field in Europe, added a very special note to this gathering. In this perspective article we summarize some of the most outstanding data and advances on plant chromatin research presented at this workshop

Automated Pupillometry Identifies Absence of Intracranial Pressure Elevation in Intracerebral Hemorrhage Patients

Abstract Introduction Although automated pupillometry is increasingly used in critical care settings, predictive value of automatically assessed pupillary parameters during different intracranial pressure (ICP) levels and possible clinical implications are unestablished. Methods This retrospective cohort study at the neurocritical care unit of the University of Erlangen-Nuremberg (2016–2018) included 23 nontraumatic supratentorial (intracerebral hemorrhage) ICH patients without signs of abnormal pupillary function by manual assessment, i.e., absent light reflex. We assessed ICP levels by an external ventricular drain simultaneously with parameters of pupillary reactivity [i.e., maximum and minimum apertures, light reflex latency (Lat), constriction and redilation velocities (CV, DV), and percentage change of apertures (per-change)] using a portable pupillometer (NeurOptics®). Computed tomography (CT) scans were analyzed to determine lesion location, size, intraventricular hemorrhage, hydrocephalus, midline shift, and compression or absence of the basal cisterns. We performed receiver operating characteristics analysis to investigate associations of ICP levels with pupillary parameters and to determine best cutoff values for prediction of ICP elevation. After dichotomization of assessments according to ICP values (normal: < 20 mmHg, elevated: ≥ 20 mmHg), prognostic performance of the determined cutoff parameters of pupillary function versus of CT-imaging findings was analyzed by calculating sensitivity, specificity, positive and negative predictive values (logistic regression, corresponding ORs with 95% CIs). Results In 23 patients (11 women, median age 59.0 (51.0–69.0) years), 1,934 assessments were available for analysis. A total of 74 ICP elevations ≥ 20 mmHg occurred in seven patients. Best discriminative thresholds for ICP elevation were: CV  0.3 s (AUC 0.616). Positive predictive value of all four parameters to indicate ICP elevation ranged between 7.2 and 8.3% only and was similarly low for CT abnormalities (9.1%). We found high negative predictive values of pupillary parameters [CV: 99.2% (95% CI 98.3–99.6), per-change: 98.7% (95% CI 97.8–99.2), DV: 98.0% (95% CI 97.0–98.7), Lat: 97.0% (95% CI 96.0–97.7)], and CT abnormalities [99.7% (95% CI 99.2–99.9)], providing evidence that both techniques adequately identified ICH patients without ICP elevation. Conclusions Our data suggest an association between noninvasively detected changes in pupillary reactivity and ICP levels in sedated ICH patients. Although automated pupillometry and neuroimaging seem not sufficient to noninvasively indicate ICP elevation, both techniques, however, adequately identified ICH patients without ICP elevation. This finding may facilitate routine management by saving invasive ICP monitoring or repeated CT controls in patients with specific automated pupillometry readings

ULTRAPETALA1 and LEAFY pathways function independently in specifying identity and determinacy at the Arabidopsis floral meristem

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Interactions between transcription factors and chromatin regulators in the control of flower development

International audienceChromatin modifiers and remodelers are involved in generating dynamic changes at the chromatin, which allow differential and specific readouts of the genome. While genetic evidence indicates that several chromatin factors play a key role in controlling basic developmental programs for inflorescence and flower morphogenesis, it remained unknown until recently how they exert their specificity toward gene expression, both temporally and spatially. An emerging topic is the recruitment or eviction of chromatin factors through the activity of sequence-specific DNA-binding domains, present in the chromatin factors themselves or in partnering transcription factors. Here we summarize recent progress that has been made in this regard in the model plant Arabidopsis thaliana. We further outline the different possible modes through which chromatin complexes specifically target genes involved in flower development

Gene activation and cell fate control in plants: a chromatin perspective.

International audienceIn plants, environment-adaptable organogenesis extends throughout the lifespan, and iterative development requires repetitive rounds of activation and repression of several sets of genes. Eukaryotic genome compaction into chromatin forms a physical barrier for transcription; therefore, induction of gene expression requires alteration in chromatin structure. One of the present great challenges in molecular and developmental biology is to understand how chromatin is brought from a repressive to permissive state on specific loci and in a very specific cluster of cells, as well as how this state is further maintained and propagated through time and cell division in a cell lineage. In this review, we report recent discoveries implementing our knowledge on chromatin dynamics that modulate developmental gene expression. We also discuss how new data sets highlight plant specificities, likely reflecting requirement for a highly dynamic chromatin

Profiling histone modifications in synchronized floral tissues for quantitative resolution of chromatin and transcriptome dynamics

Covalent histone modifications and their effects on chromatin state and accessibility play a key role in the regulation of gene expression in eukaryotes. To gain insights into their functions during plant growth and development, the distribution of histone modifications can be analyzed at a genome-wide scale through chromatin immunoprecipitation assays followed by sequencing of the isolated genomic DNA. Here, we present a protocol for systematic analysis of the distribution and dynamic changes of selected histone modifications, during flower development in the model plant Arabidopsis thaliana. This protocol utilizes a previously established floral induction system to synchronize flower development, which allows the collection of sufficient plant material for analysis by genomic technologies. In this chapter, we describe how to use this system to study, from the same set of samples, chromatin and transcriptome dynamics during early stages of flower formation

ULTRAPETALA1 and LEAFY pathways function independently in specifying identity and determinacy at the Arabidopsis floral meristem.

International audienceBackground and Aims The morphological variability of the flower in angiosperms, combined with its relatively simple structure, makes it an excellent model to study cell specification and the establishment of morphogenetic patterns. Flowers are the products of floral meristems, which are determinate structures that generate four different types of floral organs before terminating. The precise organization of the flower in whorls, each defined by the identity and number of organs it contains, is controlled by a multi-layered network involving numerous transcriptional regulators. In particular, the AGAMOUS (AG) MADS domain-containing transcription factor plays a major role in controlling floral determinacy in Arabidopsis thaliana in addition to specifying reproductive organ identity. This study aims to characterize the genetic interactions between the ULTRAPETALA1 (ULT1) and LEAFY (LFY) transcriptional regulators during flower morphogenesis, with a focus on AG regulation. Methods Genetic and molecular approaches were used to address the question of redundancy and reciprocal interdependency for the establishment of flower meristem initiation, identity and termination. In particular, the effects of loss of both ULT1 and LFY function were determined by analysing flower developmental phenotypes of double-mutant plants. The dependency of each factor on the other for activating developmental genes was also investigated in gain-of-function experiments. Key Results The ULT1 and LFY pathways, while both activating AG expression in the centre of the flower meristem, functioned independently in floral meristem determinacy. Ectopic transcriptional activation by ULT1 of AG and AP3, another gene encoding a MADS domain-containing flower architect, did not depend on LFY function. Similarly, LFY did not require ULT1 function to ectopically determine floral fate. Conclusions The results indicate that the ULT1 and LFY pathways act separately in regulating identity and determinacy at the floral meristem. In particular, they independently induce AG expression in the centre of the flower to terminate meristem activity. A model is proposed whereby these independent contributions bring about a switch at the AG locus from an inactive to an active transcriptional state at the correct time and place during flower development

Dynamic control of enhancer activity drives stage-specific gene expression during flower morphogenesis

Enhancers are critical for developmental stage-specific gene expression, but their dynamic regulation in plants remains poorly understood. Here we compare genome-wide localization of H3K27ac, chromatin accessibility and transcriptomic changes during flower development in Arabidopsis. H3K27ac prevalently marks promoter-proximal regions, suggesting that H3K27ac is not a hallmark for enhancers in Arabidopsis. We provide computational and experimental evidence to confirm that distal DNase. hypersensitive sites are predictive of enhancers. The predicted enhancers are highly stage-specific across flower development, significantly associated with SNPs for flowering-related phenotypes, and conserved across crucifer species. Through the integration of genome-wide transcription factor (TF) binding datasets, we find that floral master regulators and stage-specific TFs are largely enriched at developmentally dynamic enhancers. Finally, we show that enhancer clusters and intronic enhancers significantly associate with stage-specific gene regulation by floral master TFs. Our study provides insights into the functional flexibility of enhancers during plant development, as well as hints to annotate plant enhancers