Developmental Stimuli Research Articles

Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells

BackgroundEndothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood.ResultsTo characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We developed a robust procedure for comparative epigenome analysis that circumvents variations at the level of the individual and technical noise derived from sample preparation under various conditions. Through this approach, we identified 3765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. We found that the nine EC types can be divided into two subgroups, corresponding to those with upper-body origins and lower-body origins, based on their epigenomic landscape. Epigenomic variations were highly correlated with gene expression patterns, but also provided unique information. Most of the deferentially expressed genes and enhancers were cooperatively enriched in more than one EC type, suggesting that the distinct combinations of multiple genes play key roles in the diverse phenotypes across EC types. Notably, many homeobox genes were differentially expressed across EC types, and their expression was correlated with the relative position of each organ in the body. This reflects the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing.ConclusionsThis comprehensive analysis of epigenome characterization of EC types reveals diverse transcriptional regulation across human vascular systems. These datasets provide a valuable resource for understanding the vascular system and associated diseases.

Rab11 is required for neurite pruning and developmental membrane protein degradation in Drosophila sensory neurons

Neurons, with their distinct neurites, require elaborate membrane trafficking pathways and regulation to uphold neurite identity and to be able to respond to neuronal or developmental stimuli. In a survey of trafficking regulators required for developmental dendrite pruning in Drosophila sensory neurons, we identified the small GTPase Rab11, a regulator of recycling endosomes. Dendrite pruning requires the developmentally regulated degradation of the cell adhesion molecule Neuroglian, and loss of Rab11 causes defects in the developmental degradation of Neuroglian and another target, the ion channel Ppk26. Rab11 often links vesicles to molecular motors, and we find that loss of the microtubule motor dynein also leads to defective Neuroglian and Ppk26 degradation. Loss of Rab11 also leads to defects in larval dendrite elaboration, and Neuroglian and Ppk26 localization is already altered at this stage. Our data highlight the importance of membrane protein recycling during development.

Development of efficient protocol for rice transformation overexpressing MAP kinase and their effect on root phenotypic traits.

Exhaustive studies on mitogen-activated protein kinase (MAPK) have reported the importance in regulating a variety of responses during plant growth and development. In particular, the potential MAPK genes, MPK3 and MPK6, seem to regulate a plethora of responses, conferring tolerance to varied abiotic, biotic, and developmental stimuli. This makes both MPK3 and MPK6 potential targets for further studies. It would be an important concern to overexpress and knock out these pivotal proteins and then, in turn, to monitor the plant response which is expected to correlate action of a gene to a trait in cellular and organismal contexts. However, overexpression of MAPK genes has remained a puzzle in plants. In the present study, we report the generation of stable transgenic lines overexpressing OsMPK3 in indica and japonica cultivars and OsMPK6 in japonica cultivar under the control of an inducible promoter. We also establish the crucial steps and troubleshooting for each of the indicated rice transformation medium components. Later, we study the potential role of these MAPKs in high-throughput analysis of root system architectural (RSA) traits. It was observed that OsMPK6 overexpression lines had a more robust and spread out root architectural system while OsMPK3 overexpression lines had a typical bushy phenotype.

Oncogenic Hijacking of the PIN1 Signaling Network.

Cellular choices are determined by developmental and environmental stimuli through integrated signal transduction pathways. These critically depend on attainment of proper activation levels that in turn rely on post-translational modifications (PTMs) of single pathway members. Among these PTMs, post-phosphorylation prolyl-isomerization mediated by PIN1 represents a unique mechanism of spatial, temporal and quantitative control of signal transduction. Indeed PIN1 was shown to be crucial for determining activation levels of several pathways and biological outcomes downstream to a plethora of stimuli. Of note, studies performed in different model organisms and humans have shown that hormonal, nutrient, and oncogenic stimuli simultaneously affect both PIN1 activity and the pathways that depend on PIN1-mediated prolyl-isomerization, suggesting the existence of evolutionarily conserved molecular circuitries centered on this isomerase. This review focuses on molecular mechanisms and cellular processes like proliferation, metabolism, and stem cell fate, that are regulated by PIN1 in physiological conditions, discussing how these are subverted in and hijacked by cancer cells. Current status and open questions regarding the use of PIN1 as biomarker and target for cancer therapy as well as clinical development of PIN1 inhibitors are also addressed.

YC3.60-Based Imaging Analysis on Calcium Level in Cotton Cells.

Calcium ion (Ca2+) is a core regulator of cell functions in response to many developmental and environmental stimuli. A hallmark for Ca2+ signaling is the change of this ion in cells. Fluorescent resonance energy transfer (FRET)-based Ca2+ sensors provide a powerful tool for qualitatively and quantitatively measuring cytosolic Ca2+ level. Using YC3.60, one of those sensors, we have imaged cytosolic changes of Ca2+ in cotton fibers during the initiation stage. In this chapter, the imaging method is described in detail. The description is not limited to fiber cells but also examples leaf trichomes and protoplasts of cotton.

Autophagy in crop plants: what's new beyond Arabidopsis?

Autophagy is a major degradation and recycling pathway in plants. It functions to maintain cellular homeostasis and is induced by environmental cues and developmental stimuli. Over the past decade, the study of autophagy has expanded from model plants to crop species. Many features of the core machinery and physiological functions of autophagy are conserved among diverse organisms. However, several novel functions and regulators of autophagy have been characterized in individual plant species. In light of its critical role in development and stress responses, a better understanding of autophagy in crop plants may eventually lead to beneficial agricultural applications. Here, we review recent progress on understanding autophagy in crops and discuss potential future research directions.

H2S is involved in ABA-mediated stomatal movement through MPK4 to alleviate drought stress in Arabidopsis thaliana

Hydrogen sulfide (H2S) is a gaseous signaling molecule that participates in multiple physiological processes in both animals and plants. Mitogen-activated protein kinase (MAPK) is important signaling molecule that links the growth and developmental signals and environment stimuli to cellular responses. In the current study we explored the relationship between H2S and MAPK in drought stress resistance in Arabidopsis. The quantitative real-time (qRT)-PCR, root tip bending experiment and stomatal aperture assay were used in this paper. Drought stress activated both H2S biosynthesis and gene expression of MAPKs. The increase in MAPK expression was depressed in lcd/des1, a double mutant of H2S synthesis. Then we selected MPK4 as our target and used mpk4 mutants for further studies. H2S was able to alleviate the drought stress in wild-type (WT) Arabidopsis but not in mpk4 mutants. Meanwhile, H2S-induced stomatal movement was impaired in mpk4 mutants. We then examined the role of H2S and MPK4 in stomatal movements in response to abscisic acid (ABA) and hydrogen peroxide (H2O2). ABA- and H2O2- mediated stomatal movements were impaired in lcd/des1 and mpk4 mutants, and H2S-induced stomatal closure was impaired in slac1–3 mutants. Our results suggested that MPK4 is important downstream of H2S in the drought stress response and in stomatal movement, and that the H2S-MPK4 cascade is involved in ABA-mediated stomatal movement to regulate the drought stress.

Arabidopsis Group IIId ERF proteins positively regulate primary cell wall-type CESA genes

The cell wall determines morphology and the environmental responses of plant cells. The primary cell wall (PCW) is produced during cell division and expansion, determining the cell shape and volume. After cell expansion, specific types of plant cells produce a lignified wall, known as a secondary cell wall (SCW). We functionally analyzed Group IIId Arabidopsis AP2/EREBP genes, namely ERF34, ERF35, ERF38, and ERF39, which are homologs of a rice ERF gene previously proposed to be related to SCW biosynthesis. Expression analysis revealed that these four genes are expressed in regions related to cell division and/or cell differentiation in seedlings (i.e., shoot apical meristems, the primordia of leaves and lateral roots, trichomes, and central cylinder of primary roots) and flowers (i.e., vascular tissues of floral organs and replums and/or valve margins of pistils). Overexpression of ERF genes significantly upregulated PCW-type, but not SCW-type, CESA genes encoding cellulose synthase catalytic subunits in Arabidopsis seedlings. Transient co-expression reporter analysis indicated that ERF35, ERF38, and ERF39 possess transcriptional activator activity, and that ERF34, ERF35, ERF38, and ERF39 upregulated the promoter activity of CESA1, a PCW-type CESA gene, through the DRECRTCOREAT elements, the core cis-acting elements known to be recognized by AP2/ERF proteins. Together, our findings show that Group IIId ERF genes are positive transcriptional regulators of PCW-type CESA genes in Arabidopsis and are possibly involved in modulating cellulose biosynthesis in response to developmental requirements and environmental stimuli.

Microtubule-associated protein IQ67 DOMAIN5 regulates morphogenesis of leaf pavement cells in Arabidopsis thaliana.

Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation, and cell morphology. Their organization and dynamics are co-ordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization, and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced anisotropic expansion. Lastly, we demonstrate IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays and provide first evidence for important roles for calcium in regulation of PC morphogenesis. Our work identifies IQD5 as a novel player in PC shape regulation and, for the first time, links calcium signaling to developmental processes that regulate anisotropic growth in PCs.

Molecular evolution of SUN-domain containing proteins in diverse plant species and their expression profiling in response to developmental and perturbation stimuli

SUN (Sad1/UNC-84) domain-containing proteins are highly conserved throughout evolution. They are localized to the inner membrane of the nuclear envelope and are involved in nuclear migration and nucleoskeleton formation. In the present study, a genome-wide investigation was performed in three dicotyledonous (Arabidopsis thaliana, Glycine max and Medicago truncatula) and three monocotyledonous (Oryza sativa, Zea mays and Sorghum bicolor) plants. A total of 56 SUN proteins encoded by 30 genes were identified. Based on their length, transmembrane topology, conserved domains and phylogenetic relationships, they could be divided into two previously defined groups- Cter-SUN and mid-SUN proteins. Expression of these genes was analyzed in different developmental stages, tissues and various unfavorable conditions such as salinity, drought, and hormonal treatment. Analyses indicated that the expression of SUN1/2 transcripts are ubiquitous; that of SUN3/4 are development/tissue regulated, and SUN5 are inflorescence stage-specific. This study provides an initial framework for the characterization and functional validation of the plant SUN family.

Phosphoregulation of the Plant Cellulose Synthase Complex and Cellulose Synthase-Like Proteins.

Cellulose, the most abundant biopolymer on the planet, is synthesized at the plasma membrane of plant cells by the cellulose synthase complex (CSC). Cellulose is the primary load-bearing polysaccharide of plant cell walls and enables cell walls to maintain cellular shape and rigidity. The CSC is comprised of functionally distinct cellulose synthase A (CESA) proteins, which are responsible for synthesizing cellulose, and additional accessory proteins. Moreover, CESA-like (CSL) proteins are proposed to synthesize other essential non-cellulosic polysaccharides that comprise plant cell walls. The deposition of cell-wall polysaccharides is dynamically regulated in response to a variety of developmental and environmental stimuli, and post-translational phosphorylation has been proposed as one mechanism to mediate this dynamic regulation. In this review, we discuss CSC composition, the dynamics of CSCs in vivo, critical studies that highlight the post-translational control of CESAs and CSLs, and the receptor kinases implicated in plant cell-wall biosynthesis. Furthermore, we highlight the emerging importance of post-translational phosphorylation-based regulation of CSCs on the basis of current knowledge in the field.

The Arabidopsis Calcium-Dependent Protein Kinases (CDPKs) and Their Roles in Plant Growth Regulation and Abiotic Stress Responses.

As a ubiquitous secondary messenger in plant signaling systems, calcium ions (Ca2+) play essential roles in plant growth and development. Within the cellular signaling network, the accurate decoding of diverse Ca2+ signal is a fundamental molecular event. Calcium-dependent protein kinases (CDPKs), identified commonly in plants, are a kind of vital regulatory protein deciphering calcium signals triggered by various developmental and environmental stimuli. This review chiefly introduces Ca2+ distribution in plant cells, the classification of Arabidopsis thaliana CDPKs (AtCDPKs), the identification of the Ca2+-AtCDPK signal transduction mechanism and AtCDPKs’ functions involved in plant growth regulation and abiotic stress responses. The review presents a comprehensive overview of AtCDPKs and may contribute to the research of CDPKs in other plants.

Autophagy-related approaches for improving nutrient use efficiency and crop yield protection.

Autophagy is a eukaryotic catabolic pathway essential for growth and development. In plants, it is activated in response to environmental cues or developmental stimuli. However, in contrast to other eukaryotic systems, we know relatively little regarding the molecular players involved in autophagy and the regulation of this complex pathway. In the framework of the COST (European Cooperation in Science and Technology) action TRANSAUTOPHAGY (2016-2020), we decided to review our current knowledge of autophagy responses in higher plants, with emphasis on knowledge gaps. We also assess here the potential of translating the acquired knowledge to improve crop plant growth and development in a context of growing social and environmental challenges for agriculture in the near future.

M-AAA Complexes Are Not Crucial for the Survival of Arabidopsis Under Optimal Growth Conditions Despite Their Importance for Mitochondrial Translation.

For optimal mitochondrial activity, the mitochondrial proteome must be properly maintained or altered in response to developmental and environmental stimuli. Based on studies of yeast and humans, one of the key players in this control are m-AAA proteases, mitochondrial inner membrane-bound ATP-dependent metalloenzymes. This study focuses on the importance of m-AAA proteases in plant mitochondria, providing their first experimentally proven physiological substrate. We found that the Arabidopsis m- AAA complexes composed of AtFTSH3 and/or AtFTSH10 are involved in the proteolytic maturation of ribosomal subunit L32. Consequently, in the double Arabidopsis ftsh3/10 mutant, mitoribosome biogenesis, mitochondrial translation and functionality of OXPHOS (oxidative phosphorylation) complexes are impaired. However, in contrast to their mammalian or yeast counterparts, plant m-AAA complexes are not critical for the survival of Arabidopsis under optimal conditions; ftsh3/10 plants are only slightly smaller in size at the early developmental stage compared with plants containing m-AAA complexes. Our data suggest that a lack of significant visible morphological alterations under optimal growth conditions involves mechanisms which rely on existing functional redundancy and induced functional compensation in Arabidopsis mitochondria.

Translational Implications of Platelets as Vascular First Responders.

Platelets play a vital role in normal hemostasis to stem blood loss at sites of vascular injury by tethering and adhering to sites of injury, recruiting other platelets and blood cells to the developing clot, releasing vasoactive small molecules and proteins, and assembling and activating plasma coagulation proteins in a tightly regulated temporal and spatial manner. In synchrony with specific end products of coagulation, primarily cross-linked fibrin, a stable thrombus quickly forms. Far beyond physiological hemostasis and pathological thrombosis, emerging evidence supports platelets playing a pivotal role in vascular homeostasis, inflammation, cellular repair, regeneration, and wide range of autocrine and paracrine functions. In essence, platelets play both structural and functional roles as reporters, messengers, and active transporters surveying the vasculature for cues of environmental or developmental stimuli and participating as first responders.1 In this review, we will provide a contemporary perspective of platelet physiology, including fundamental, translational, and clinical constructs that apply directly to human health and disease.

UTR-Dependent Control of Gene Expression in Plants.

Throughout their lives, plants sense many developmental and environmental stimuli, and activation of optimal responses against these stimuli requires extensive transcriptional reprogramming. To facilitate this activation, plant mRNA contains untranslated regions (UTRs) that significantly increase the coding capacity of the genome by producing multiple mRNA variants from the same gene. In this review we compare UTRs of arabidopsis (Arabidopsis thaliana) and rice (Oryza sativum) at the genome scale to highlight their complexity in crop plants. We discuss different modes of UTR-based regulation with emphasis on genes that regulate multiple plant processes, including flowering, stress responses, and nutrient homeostasis. We demonstrate functional specificity in genes with variable UTR length and propose future research directions.

The Blockade of Glutamate N-methyl-D-aspartate Receptors into the Prelimbic of Prefrontal Cortex Decreases Morphine-induced Conditioned Place Preference in Rat

BACKGROUND: The prelimbic area (PL) of the prefrontal cortex is susceptible to abnormal developmental stimuli that raises the risk of addiction. Glutamate receptors play a key role in opiate reinforcement and reward functions in this area. Therefore, we examined the effect of the DL-2-amino-5-phosphonopentanoic acid (AP5), as N-methyl-D-aspartate (NMDA) receptor antagonist into the PL on the phases of conditioned place preference (CPP) induced by morphine.METHODS: Male Wistar rats were divided into 12 groups (3 surgical groups for each dose of morphine in any phase of CPP) and anaesthetized with chloral hydrate. Cannula was implanted into the PL and the AP5 was injected into this area and morphine-induced CPP was investigated. Data were processed with the commercially available SPSS 22 software using one-way ANOVA and Tukey's test. p<0.05 were considered statistically significant.RESULTS: Our findings indicated, morphine in doses of 2.5 to 10 mg/kg induced CPP. Microinjection of various doses of the AP5 into the PL before the administration of the effective dose of morphine significantly reduced place preference in the acquisition and the expression phases of the CPP test compared to the sham group (p<0.001). In another set of our experiments was seen that, different doses of the AP5 with the ineffective dose of morphine only reduced the expression phase of the CPP (p<0.001) while, produced neither preference nor aversion effect on the acquisition phase (p=0.147).CONCLUSION: It seems that the glutamate NMDA receptors in the PL through memory formation and morphine-related reward signals play a critical role in addiction process during morphine-induced CPP.KEYWORDS: N-methyl-aspartate, morphine, glutamate receptor, prefrontal cortex, reward

Plant Cell Cultures as Model Systems to Study Programmed Cell Death.

The study of programmed cell death (PCD) activated in a certain group of cells is complex when analyzed in the whole plant. Plant cell suspension cultures are useful when investigating PCD triggered by environmental and developmental stimuli. Due to their homogeneity and the possibility to synchronize their responses induced by external stimuli, these cultures are used for studying the signaling pathways leading to PCD. The first problem in the analysis of PCD in cell cultures is the quantification of cell viability/death over time. Cultured cells from different plant species may have specific mitotic patterns leading to calli or cell chains mixed to single cell suspensions. For this reason, not all cell cultures allow morphological parameters to be investigated using microscopy analysis, and adapted or ad hoc methods are needed to test cell viability.Here we report on some accurate methods to establish and propagate cell cultures from different plant species, including crops, as well as to determine cell viability and PCD morphological and genetic markers. In particular, we describe a protocol for extracting nucleic acids required for real-time PCR analysis which has been optimized for those cell cultures that do not allow the use of commercial kits.

Genetic Dissection of Morphometric Traits Reveals That Phytochrome B Affects Nucleus Size and Heterochromatin Organization in Arabidopsis thaliana.

Microscopically visible chromatin is partitioned into two major components in Arabidopsis thaliana nuclei. On one hand, chromocenters are conspicuous foci of highly condensed “heterochromatic” domains that contain mostly repeated sequences. On the other hand, less condensed and gene-rich “euchromatin” emanates from these chromocenters. This differentiation, together with the dynamic nature of chromatin compaction in response to developmental and environmental stimuli, makes Arabidopsis a powerful system for studying chromatin organization and dynamics. Heterochromatin dynamics can be monitored by measuring the Heterochromatin Index, i.e., the proportion of nuclei displaying well-defined chromocenters, or the DNA fraction of chromocenters (relative heterochromatin fraction). Both measures are composite traits, thus their values represent the sum of effects of various underlying morphometric properties. We exploited genetic variation between natural occurring accessions to determine the genetic basis of individual nucleus and chromocenter morphometric parameters (area, perimeter, density, roundness, and heterogeneity) that together determine chromatin compaction. Our novel reductionist genetic approach revealed quantitative trait loci (QTL) for all measured traits. Genomic colocalization among QTL was limited, which suggests a complex genetic regulation of chromatin compaction. Yet genomic intervals of QTL for nucleus size (area and perimeter) both overlap with a known QTL for heterochromatin compaction that is explained by natural polymorphism in the red/far-red light and temperature receptor Phytochrome B. Mutant analyses and genetic complementation assays show that Phytochrome B is a negative regulator of nucleus size, revealing that perception of climatic conditions by a Phytochrome-mediated hub is a major determinant for coordinating nucleus size and heterochromatin compaction.

Abstract 3578: Methyl2Acetyl: predicting epigenetic and transcriptomic activity from DNA methylation

Abstract DNA methylation and histone modifications are interconnected and interdependent epigenetic mechanisms that regulate chromatin accessibility in cells. They establish cell’s developmental identity and modulate individual response to endogenous developmental stimuli and environmental changes. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a popular assay to study genomewide histone modification profiles. However, ChIP-seq assays require relatively large amounts of starting materials (ranging from ~105 cells using a low cell number protocol to 107 cells in a standard protocol), which remains as a major obstacle for precious biological samples including primary tumor specimens. On the other hand, tagmentation-based whole genome bisulfite sequencing (TWGBS) protocol enables unbiased assessment of the genomewide methylation pattern using ~3,000 cells. Therefore, a quantitative relationship between DNA methylation and epigenetic/transcriptomic activity is in great demand. In the current study, we developed Methyl2Acetyl, a machine learning framework to infer promoter epigenetic activities (e.g. H3K27Ac enrichment) using TWGBS data as input. The proposed model utilizes random forest learners, which allow automatic extraction of complex interactions among DNA methylation features surrounding transcription start sites. We applied Methyl2Acetyl to a set of pediatric solid tumor samples with high quality H3K27Ac ChIPseq data to evaluate its performance. The model accurately predicted promoter H3K27Ac enrichment in independent test samples (Pearson correlation against measured enrichment: 0.81 ± 0.02). To further test the robustness of our model, we trained a Methyl2Acetyl model on solid tumors and applied it to a set of 10 pediatric leukemia samples. Despite dramatic global methylome differences between solid tumors and leukemia, Methyl2Acetyl predicted promoter H3K27Ac enrichment showed strong concordance with target gene expression level in the same sample (Pearson correlation: 0.75 ± 0.02, classification AUC: 0.89 ± 0.01). Collectively, our data suggested that DNA methylation is predictive of promoter active histone modification enrichment and gene expression activity. Epigenetically active promoters can be imputed from TWGBS data in samples with limiting starting materials. Citation Format: Xiang Chen. Methyl2Acetyl: predicting epigenetic and transcriptomic activity from DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3578. doi:10.1158/1538-7445.AM2017-3578