Chromatin Immunoprecipitation to Investigate H2A.Z Dynamics in Response to Environmental Changes.

DNA methylation and posttranslational modifications of histones instruct gene expression in eukaryotes. Besides canonical histones, histone variants also play a critical role in transcriptional regulation. One of the best studied histone variants in plants is H2A.Z whose removal from gene bodies correlates with increased transcriptional activity. The eviction of H2A.Z is regulated by environmental cues such as increased ambient temperatures, and current models suggest that H2A.Z functions as a transcriptional buffer preventing environmentally responsive genes from undesired activation. To monitor temperature-dependent H2A.Z dynamics, chromatin immunoprecipitation (ChIP) of H2A.Z-occupied DNA can be performed. The following protocol describes a quick and easy ChIP approach to study in vivo H2A.Z occupancy.

A Comparison of Proximal and Tracheal Airway Pressures During Pressure Controlled Ventilation.

BACKGROUND: Airway pressure is usually measured by sensors placed in the ventilator or on the ventilator side of the endotracheal tube (ETT), at the Y-piece. These remote measurements serve as a surrogate for the tracheal or alveolar pressure. Tracheal pressure can only be predicted correctly by using a model that incorporates the pressure at the remote location, the flow through the ETT, and the resistance of the ETT if the latter is a predictable function of Y-piece flow. However, this is not consistently appropriate, and accuracy of prediction is hampered. METHODS: This in vitro study systematically examined the ventilator pressure in dependence of compliance of the respiratory system (CRS), inspiratory time, and expiratory time during pressure-controlled ventilation by using a small intratracheal pressure sensor and a mechanical lung simulator. Pressures were measured simultaneously at the ventilator outlet, at the Y-piece, and in the trachea during pressure-controlled ventilation with a peak inspiratory pressure of 20 cm H2O and a PEEP of 5 cm H2O while changing CRS (10, 30, 60, 90, and 100 mL/cm H2O) and varying inspiratory time and expiratory time. RESULTS: Tracheal pressures were always lower (maximum 8 cm H2O during inspiration) or higher (maximum 4 cm H2O during expiration) than the pressures measured proximal to the ETT if zero-flow conditions were not achieved at the end of the breathing cycles. CONCLUSIONS: Dependent on CRS and the breathing cycle, tracheal pressures deviated from those measured proximal to the ETT under non-zero-flow conditions. Intratracheal pressure and pressure curve dynamics can differ greatly from the ventilator pressure, depending on the ventilator setting and the CRS. The small pressure sensor may be used as a measurement method of tracheal pressure via integration onto an ETT.

Transcription Factor Dynamics in Cross-Regulation of Plant Hormone Signaling Pathways.

Cross-regulation between hormone signaling pathways is indispensable for plant growth and development. However, the molecular mechanisms by which multiple hormones interact and co-ordinate activity need to be understood. Here, we generated a cross-regulation network explaining how hormone signals are integrated from multiple pathways in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. To do so we comprehensively characterized transcription factor activity during plant hormone responses and reconstructed dynamic transcriptional regulatory models for six hormones; abscisic acid, brassinosteroid, ethylene, jasmonic acid, salicylic acid and strigolactone/karrikin. These models incorporated target data for hundreds of transcription factors and thousands of protein-protein interactions. Each hormone recruited different combinations of transcription factors, a subset of which were shared between hormones. Hub target genes existed within hormone transcriptional networks, exhibiting transcription factor activity themselves. In addition, a group of MITOGEN-ACTIVATED PROTEIN KINASES (MPKs) were identified as potential key points of cross-regulation between multiple hormones. Accordingly, the loss of function of one of these (MPK6) disrupted the global proteome, phosphoproteome and transcriptome during hormone responses. Lastly, we determined that all hormones drive substantial alternative splicing that has distinct effects on the transcriptome compared with differential gene expression, acting in early hormone responses. These results provide a comprehensive understanding of the common features of plant transcriptional regulatory pathways and how cross-regulation between hormones acts upon gene expression.

Testing of pandemic ventilators under early and agile development.

Aiming to address clinical requirements subsequent to SARS-CoV-2-related pulmonary disease, multiple research groups and industry groups carried out intensive studies to develop pandemic ventilators (PDVs). In vitro testing to critically evaluate the specific performance of the developed apparatuses is an essential requirement. This study presents a test protocol which promotes a test-oriented, iterative, and agile assessment and consecutive development of such PDVs. It allows for fast identification of specific characteristics of each PDV in the individual test features. The test protocol includes an evaluation of the accuracy of control systems and instruments at changing parameters, the oxygen dynamics, and the response to trigger signals. The test environment is a mechanical lung, which allows reproducing various lung mechanics and to simulate active breathing cycles. A total of three PDVs that are under development were iteratively tested, with a Hamilton T1 as a reference. Continuous testing of the PDVs under development enables quick identification of critical application aspects that deserve further improved. Based on the present test protocol, the ventilators demonstrate a promising performance justifying continued development.

Dnmt3a knockout in excitatory neurons impairs postnatal synapse maturation and increases the repressive histone modification H3K27me3.

Two epigenetic pathways of transcriptional repression, DNA methylation and polycomb repressive complex 2 (PRC2), are known to regulate neuronal development and function. However, their respective contributions to brain maturation are unknown. We found that conditional loss of the de novo DNA methyltransferase Dnmt3a in mouse excitatory neurons altered expression of synapse-related genes, stunted synapse maturation, and impaired working memory and social interest. At the genomic level, loss of Dnmt3a abolished postnatal accumulation of CG and non-CG DNA methylation, leaving adult neurons with an unmethylated, fetal-like epigenomic pattern at ~222,000 genomic regions. The PRC2-associated histone modification, H3K27me3, increased at many of these sites. Our data support a dynamic interaction between two fundamental modes of epigenetic repression during postnatal maturation of excitatory neurons, which together confer robustness on neuronal regulation.

In-Vitro Visualization of Thrombus Growth in Artificial Lungs Using Real-Time X-Ray Imaging: A Feasibility Study.

PURPOSE: Extracorporeal membrane oxygenation has gained increasing attention in the treatment of patients with acute and chronic cardiopulmonary and respiratory failure. However, clotting within the oxygenators or other components of the extracorporeal circuit remains a major complication that necessitates at least a device exchange and bears risks of adverse events for the patients. In order to better predict thrombus growth within oxygenators, we present an approach for in-vitro visualization of thrombus growth using real-time X-ray imaging. METHODS: An in-vitro test setup was developed using low-dose anticoagulated ovine blood and allowing for thrombus growth within 4 h. The setup was installed in a custom-made X-ray setup that uses phase-contrast for imaging, thus providing enhanced soft-tissue contrast, which improves the differentiation between blood and potential thrombus growth. During experimentation, blood samples were drawn for the analysis of blood count, activated partial thromboplastin time and activated clotting time. Additionally, pressure and flow data was monitored and a full 360° X-ray scan was performed every 15 min. RESULTS: Thrombus formation indicated by a pressure drop and changing blood parameters was monitored in all three test devices. Red and white thrombi (higher/lower attenuation, respectively) were successfully segmented in one set of X-ray images. CONCLUSION: We showed the feasibility of a new in-vitro method for real-time thrombus growth visualization by means of phase contrast X-ray imaging. In addition, with more blood parameters that are clinically relevant, this approach might contribute to improved oxygenator exchange protocols in the clinical routine.

PHYTOCHROME-INTERACTING FACTORs trigger environmentally responsive chromatin dynamics in plants.

The interplay between light receptors and PHYTOCHROME-INTERACTING FACTORs (PIFs) serves as a regulatory hub that perceives and integrates environmental cues into transcriptional networks of plants1,2. Although occupancy of the histone variant H2A.Z and acetylation of histone H3 have emerged as regulators of environmentally responsive gene networks, how these epigenomic features interface with PIF activity is poorly understood3-7. By taking advantage of rapid and reversible light-mediated manipulation of PIF7 subnuclear localization and phosphorylation, we simultaneously assayed the DNA-binding properties of PIF7, as well as its impact on chromatin dynamics genome wide. We found that PIFs act rapidly to reshape the H2A.Z and H3K9ac epigenetic landscape in response to a change in light quality. Furthermore, we discovered that PIFs achieve H2A.Z removal through direct interaction with EIN6 ENHANCER (EEN), the Arabidopsis thaliana homolog of the chromatin remodeling complex subunit INO80 Subunit 6 (Ies6). Thus, we describe a PIF-INO80 regulatory module that is an intermediate step for allowing plants to change their growth trajectory in response to environmental changes.

Publisher Correction: Integrated multi-omics framework of the plant response to jasmonic acid.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Integrated multi-omics framework of the plant response to jasmonic acid.

Understanding the systems-level actions of transcriptional responses to hormones provides insight into how the genome is reprogrammed in response to environmental stimuli. Here, we investigated the signalling pathway of the hormone jasmonic acid (JA), which controls a plethora of critically important processes in plants and is orchestrated by the transcription factor MYC2 and its closest relatives in Arabidopsis thaliana. We generated an integrated framework of the response to JA, which spans from the activity of master and secondary regulatory transcription factors, through gene expression outputs and alternative splicing, to protein abundance changes, protein phosphorylation and chromatin remodelling. We integrated time-series transcriptome analysis with (phospho)proteomic data to reconstruct gene regulatory network models. These enabled us to predict previously unknown points of crosstalk of JA to other signalling pathways and to identify new components of the JA regulatory mechanism, which we validated through targeted mutant analysis. These results provide a comprehensive understanding of how a plant hormone remodels cellular functions and plant behaviour, the general principles of which provide a framework for analyses of cross-regulation between other hormone and stress signalling pathways.

Chimeric Activators and Repressors Define HY5 Activity and Reveal a Light-Regulated Feedback Mechanism.

The first exposure to light marks a crucial transition in plant development. This transition relies on the transcription factor HY5 controlling a complex downstream growth program. Despite its importance, its function in transcription remains unclear. Previous studies have generated lists of thousands of potential target genes and competing models of HY5 transcription regulation. In this work, we carry out detailed phenotypic and molecular analysis of constitutive activator and repressor HY5 fusion proteins. Using this strategy, we were able to filter out large numbers of genes that are unlikely to be direct targets, allowing us to eliminate several proposed models of HY5's mechanism of action. We demonstrate that the primary activity of HY5 is promoting transcription and that this function relies on other, likely light-regulated, factors. In addition, this approach reveals a molecular feedback loop via the COP1/SPA E3 ubiquitin ligase complex, suggesting a mechanism that maintains low HY5 in the dark, primed for rapid accumulation to reprogram growth upon light exposure. Our strategy is broadly adaptable to the study of transcription factor activity. Lastly, we show that modulating this feedback loop can generate significant phenotypic diversity in both Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum).

The JA-pathway MYC transcription factors regulate photomorphogenic responses by targeting HY5 gene expression.

Jasmonates are key regulators of the balance between defence and growth in plants. However, the molecular mechanisms by which activation of defence reduces growth are not yet fully understood. Here, we analyze the role of MYC transcription factors (TFs) and jasmonic acid (JA) in photomorphogenic growth. We found that multiple myc mutants share light-associated phenotypes with mutants of the phytochrome B photoreceptor, such as delayed seed germination in the dark and long hypocotyl growth. Overexpression of MYC2 in a phyB background partially suppressed its long hypocotyl phenotype. Transcriptomic analysis of multiple myc mutants confirmed that MYCs are required for full expression of red (R) light-regulated genes, including the master regulator HY5. ChIP-seq analyses revealed that MYC2 and MYC3 bind directly to the promoter of HY5 and that HY5 gene expression and protein levels are compromised in multiple myc mutants. Altogether, our results pinpoint MYCs as photomorphogenic TFs that control phytochrome responses by activating HY5 expression. This has important implications in understanding the trade-off between growth and defence as the same TFs that activate defence responses are photomorphogenic growth regulators.

A MYC2/MYC3/MYC4-dependent transcription factor network regulates water spray-responsive gene expression and jasmonate levels.

Mechanical stimuli, such as wind, rain, and touch affect plant development, growth, pest resistance, and ultimately reproductive success. Using water spray to simulate rain, we demonstrate that jasmonic acid (JA) signaling plays a key role in early gene-expression changes, well before it leads to developmental changes in flowering and plant architecture. The JA-activated transcription factors MYC2/MYC3/MYC4 modulate transiently induced expression of 266 genes, most of which peak within 30 min, and control 52% of genes induced >100-fold. Chromatin immunoprecipitation-sequencing analysis indicates that MYC2 dynamically binds >1,300 promoters and trans-activation assays show that MYC2 activates these promoters. By mining our multiomic datasets, we identified a core MYC2/MYC3/MYC4-dependent "regulon" of 82 genes containing many previously unknown MYC2 targets, including transcription factors bHLH19 and ERF109 bHLH19 can in turn directly activate the ORA47 promoter, indicating that MYC2/MYC3/MYC4 initiate a hierarchical network of downstream transcription factors. Finally, we also reveal that rapid water spray-induced accumulation of JA and JA-isoleucine is directly controlled by MYC2/MYC3/MYC4 through a positive amplification loop that regulates JA-biosynthesis genes.

Epigenetic silencing of a multifunctional plant stress regulator.

The central regulator of the ethylene (ET) signaling pathway, which controls a plethora of developmental programs and responses to environmental cues in plants, is ETHYLENE-INSENSITIVE2 (EIN2). Here we identify a chromatin-dependent regulatory mechanism at EIN2 requiring two genes: ETHYLENE-INSENSITIVE6 (EIN6), which is a H3K27me3 demethylase also known as RELATIVE OF EARLY FLOWERING6 (REF6), and EIN6 ENHANCER (EEN), the Arabidopsis homolog of the yeast INO80 chromatin remodeling complex subunit IES6 (INO EIGHTY SUBUNIT). Strikingly, EIN6 (REF6) and the INO80 complex redundantly control the level and the localization of the repressive histone modification H3K27me3 and the histone variant H2A.Z at the 5' untranslated region (5'UTR) intron of EIN2. Concomitant loss of EIN6 (REF6) and the INO80 complex shifts the chromatin landscape at EIN2 to a repressive state causing a dramatic reduction of EIN2 expression. These results uncover a unique type of chromatin regulation which safeguards the expression of an essential multifunctional plant stress regulator.

The complex architecture and epigenomic impact of plant T-DNA insertions.

The bacterium Agrobacterium tumefaciens has been the workhorse in plant genome engineering. Customized replacement of native tumor-inducing (Ti) plasmid elements enabled insertion of a sequence of interest called Transfer-DNA (T-DNA) into any plant genome. Although these transfer mechanisms are well understood, detailed understanding of structure and epigenomic status of insertion events was limited by current technologies. Here we applied two single-molecule technologies and analyzed Arabidopsis thaliana lines from three widely used T-DNA insertion collections (SALK, SAIL and WISC). Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements, and the length of individual insertions from 27 to 236 kilobases. De novo nanopore sequencing-based assemblies for two segregating lines partially resolved T-DNA structures and revealed multiple translocations and exchange of chromosome arm ends. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. The unprecedented contiguous nucleotide-level resolution enabled an in-depth study of the epigenome at T-DNA insertion sites. SALK_059379 line T-DNA insertions were enriched for 24nt small interfering RNAs (siRNA) and dense cytosine DNA methylation, resulting in transgene silencing via the RNA-directed DNA methylation pathway. In contrast, SAIL_232 line T-DNA insertions are predominantly targeted by 21/22nt siRNAs, with DNA methylation and silencing limited to a reporter, but not the resistance gene. Additionally, we profiled the H3K4me3, H3K27me3 and H2A.Z chromatin environments around T-DNA insertions using ChIP-seq in SALK_059379, SAIL_232 and five additional T-DNA lines. We discovered various effect s ranging from complete loss of chromatin marks to the de novo incorporation of H2A.Z and trimethylation of H3K4 and H3K27 around the T-DNA integration sites. This study provides new insights into the structural impact of inserting foreign fragments into plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes.

Technical Indicators to Evaluate the Degree of Large Clot Formation Inside the Membrane Fiber Bundle of an Oxygenator in an In Vitro Setup.

The most common technical complication during ECMO is clot formation. A large clot inside a membrane oxygenator reduces effective membrane surface area and therefore gas transfer capabilities, and restricts blood flow through the device, resulting in an increased membrane oxygenator pressure drop (dpMO). The reasons for thrombotic events are manifold and highly patient specific. Thrombus formation inside the oxygenator during ECMO is usually unpredictable and remains an unsolved problem. Clot sizes and positions are well documented in literature for the Maquet Quadrox-i Adult oxygenator based on CT data extracted from devices after patient treatment. Based on this data, the present study was designed to investigate the effects of large clots on purely technical parameters, for example, dpMO and gas transfer. Therefore, medical grade silicone was injected into the fiber bundle of the devices to replicate large clot positions and sizes. A total of six devices were tested in vitro with silicone clot volumes of 0, 30, 40, 50, 65, and 85 mL in accordance with ISO 7199. Gas transfer was measured by sampling blood pre and post device, as well as by sampling the exhaust gas at the devices' outlet at blood flow rates of 0.5, 2.5, and 5.0 L/min. Pre and post device pressure was monitored to calculate the dpMO at the different blood flow rates. The dpMO was found to be a reliable parameter to indicate a large clot only in already advanced "clotting stages." The CO2 concentration in the exhaust gas, however, was found to be sensitive to even small clot sizes and at low blood flows. Exhaust gas CO2 concentration can be monitored continuously and without any risks for the patient during ECMO therapy to provide additional information on the endurance of the oxygenator. This may help detect a clot formation and growth inside a membrane oxygenator during ECMO even if the increase in dpMO remains moderate.

TGA2 signaling in response to reactive electrophile species is not dependent on cysteine modification of TGA2.

Reactive electrophile species (RES), including prostaglandins, phytoprostanes and 12-oxo phytodienoic acid (OPDA), activate detoxification responses in plants and animals. However, the pathways leading to the activation of defense reactions related to abiotic or biotic stress as a function of RES formation, accumulation or treatment are poorly understood in plants. Here, the thiol-modification of proteins, including the RES-activated basic region/leucine zipper transcription factor TGA2, was studied. TGA2 contains a single cysteine residue (Cys186) that was covalently modified by reactive cyclopentenones but not required for induction of detoxification genes in response to OPDA or prostaglandin A1. Activation of the glutathione-S-transferase 6 (GST6) promoter was responsive to cyclopentenones but not to unreactive cyclopentanones, including jasmonic acid suggesting that thiol reactivity of RES is important to activate the TGA2-dependent signaling pathway resulting in GST6 activation We show that RES modify thiols in numerous proteins in vivo, however, thiol reactivity alone appears not to be sufficient for biological activity as demonstrated by the failure of several membrane permeable thiol reactive reagents to activate the GST6 promoter.

Cryptochromes Interact Directly with PIFs to Control Plant Growth in Limiting Blue Light.

Sun-loving plants have the ability to detect and avoid shading through sensing of both blue and red light wavelengths. Higher plant cryptochromes (CRYs) control how plants modulate growth in response to changes in blue light. For growth under a canopy, where blue light is diminished, CRY1 and CRY2 perceive this change and respond by directly contacting two bHLH transcription factors, PIF4 and PIF5. These factors are also known to be controlled by phytochromes, the red/far-red photoreceptors; however, transcriptome analyses indicate that the gene regulatory programs induced by the different light wavelengths are distinct. Our results indicate that CRYs signal by modulating PIF activity genome wide and that these factors integrate binding of different plant photoreceptors to facilitate growth changes under different light conditions.

A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis.

Here, we have asked about post-transcriptional mechanisms regulating murine developmental neurogenesis, focusing upon the RNA-binding proteins Smaug2 and Nanos1. We identify, in embryonic neural precursors of the murine cortex, a Smaug2 protein/nanos1 mRNA complex that is present in cytoplasmic granules with the translational repression proteins Dcp1 and 4E-T. We show that Smaug2 inhibits and Nanos1 promotes neurogenesis, with Smaug2 knockdown enhancing neurogenesis and depleting precursors, and Nanos1 knockdown inhibiting neurogenesis and maintaining precursors. Moreover, we show that Smaug2 likely regulates neurogenesis by silencing nanos1 mRNA. Specifically, Smaug2 knockdown inappropriately increases Nanos1 protein, and the Smaug2 knockdown-mediated neurogenesis is rescued by preventing this increase. Thus, Smaug2 and Nanos1 function as a bimodal translational repression switch to control neurogenesis, with Smaug2 acting in transcriptionally primed precursors to silence mRNAs important for neurogenesis, including nanos1 mRNA, and Nanos1 acting during the transition to neurons to repress the precursor state. SIGNIFICANCE STATEMENT: The mechanisms instructing neural stem cells to generate the appropriate progeny are still poorly understood. Here, we show that the RNA-binding proteins Smaug2 and Nanos1 are critical regulators of this balance and provide evidence supporting the idea that neural precursors are transcriptionally primed to generate neurons but translational regulation maintains these precursors in a stem cell state until the appropriate developmental time.