SEARCH · Engineering Papers

Results for “gene expression profiling”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 667 records · Page 37

All sources NASA NTRS DOE OSTI

DOE OSTI2022

A Novel Trichothecene Toxin Phenotype Associated with Horizontal Gene Transfer and a Change in Gene Function in Fusarium

Fusarium trichothecenes are among the mycotoxins of most concern to food and feed safety. Production of these mycotoxins and presence of the trichothecene biosynthetic gene (TRI) cluster have been confirmed in only two multispecies lineages of Fusarium: the Fusarium incarnatum-equiseti (Incarnatum) and F. sambucinum (Sambucinum) species complexes. Here, we identified and characterized a TRI cluster in a species that has not been formally described and is represented by Fusarium sp. NRRL 66739. This fungus is reported to be a member of a third Fusarium lineage: the F. buharicum species complex. Cultures of NRRL 66739 accumulated only two trichothecenes, 7-hydroxyisotrichodermin and 7-hydroxyisotrichodermol. Although these are not novel trichothecenes, the production profile of NRRL 66739 is novel, because in previous reports 7-hydroxyisotrichodermin and 7-hydroxyisotrichodermol were components of mixtures of 6–8 trichothecenes produced by several Fusarium species in Sambucinum. Heterologous expression analysis indicated that the TRI13 gene in NRRL 66739 confers trichothecene 7-hydroxylation. This contrasts the trichothecene 4-hydroxylation function of TRI13 in other Fusarium species. Phylogenetic analyses suggest that NRRL 66739 acquired the TRI cluster via horizontal gene transfer from a close relative of Incarnatum and Sambucinum. These findings provide insights into evolutionary processes that have shaped the distribution of trichothecene production among Fusarium species and the structural diversity of the toxins.

Food Science & Technology↗

DOE OSTI2017

Interleukin-24 as a target cytokine of environmental aryl hydrocarbon receptor agonist exposure in the lung

Exposure to environmental aryl hydrocarbon receptor (AhR) agonists, such as halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs), has great impacts on the development of various lung diseases. As emerging molecular targets for AhR agonists, cytokines may contribute to the inflammatory or immunotoxic effects of environmental AhR agonists. However, general cytokine expression may not specifically indicate environmental AhR agonist exposure. By comparing cytokine and chemokine expression profiles in human lung adenocarcinoma cell line CL5 treated with AhR agonists and the non-AhR agonist polychlorinated biphenyl (PCB) 39, we identified a target cytokine of environmental AhR agonist exposure of in the lungs. Thirteen cytokine and chemokine genes were altered in the AhR agonists-treated cells, but none were altered in the PCB39-treated cells. Interleukin (IL)-24 was the most highly induced gene among AhR-modulated cytokines. Cotreatment with AhR antagonist completely prevented IL-24 induction by AhR agonists in the CL5 cells. Knockdown AhR expression with short-hairpin RNA (shRNA) significantly reduced benzo[a]pyrene (BaP)-induced IL-24 mRNA levels. We further confirmed that gene transcription, but not mRNA stability, was involved in IL-24 upregulation by BaP. Particulate matter (PM) in the ambient air contains some PAHs and is reported to activate AhR. Oropharyngeal aspiration of PM significantly increased IL-24 levels in lung epithelia and in bronchoalveolar lavage fluid of mice 4 weeks after treatment. Thus, our data suggests that IL-24 is a pulmonary exposure target cytokine of environmental AhR agonists. - Graphical abstract: (A) Cytokine and chemokine gene expressions were examined in CL5 cells treated with AhR and non-AhR agonists. Thirteen cytokines and chemokines genes were altered in the AhR agonist-treated cells, but not in the non-AhR agonist-treated cells. IL-24 was the most highly induced gene among the AhR-modulated cytokines. (B) Both AhR agonists and PM{sub 2.5} induced IL-24 production in the CL5 cells and macrophages. Cotreatment with an AhR antagonist (DMF) or transfections with shRNA for AhR abolished IL-24 induction by BaP in CL5 cells. Intratracheal instillation of PM activated AhR-mediated inflammatory responses and IL-24 expression in mouse lungs. Thus, our data suggests that IL-24 is a pulmonary exposure target cytokine of environmental AhR agonist. - Highlights: • IL-24 is identified as a target cytokine of environmental AhR agonist exposure. • AhR agonists increased IL-24 expression in an AhR-dependent manner in lung cells. • Ambient particulate matter induces IL-24 secretion in BALF in mice. • IL-24 can be used to evaluate environmental lung diseases.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2016

Metatranscriptomics reveals temperature-driven functional changes in microbiome impacting cheese maturation rate

Traditional cheeses harbour complex microbial consortia that play an important role in shaping typical sensorial properties. However, the microbial metabolism is considered difficult to control. Microbial community succession and the related gene expression were analysed during ripening of a traditional Italian cheese, identifying parameters that could be modified to accelerate ripening. Afterwards, we modulated ripening conditions and observed consistent changes in microbial community structure and function. We provide concrete evidence of the essential contribution of non-starter lactic acid bacteria in ripening-related activities. An increase in the ripening temperature promoted the expression of genes related to proteolysis, lipolysis and amino acid/lipid catabolism and significantly increases the cheese maturation rate. Moreover, temperature-promoted microbial metabolisms were consistent with the metabolomic profiles of proteins and volatile organic compounds in the cheese. Finally, the results clearly indicate how processing-driven microbiome responses can be modulated in order to optimize production efficiency and product quality.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2014

Evolution of H3K27me3-marked chromatin is linked to gene expression evolution and to patterns of gene duplication and diversification

Histone modifications are critical for the regulation of gene expression, cell type specification, and differentiation. However, evolutionary patterns of key modifications that regulate gene expression in differentiating organisms have not been examined. Here we mapped the genomic locations of the repressive mark histone 3 lysine 27 trimethylation (H3K27me3) in four species of Drosophila, and compared these patterns to those in C. elegans. We found that patterns of H3K27me3 are highly conserved across species, but conservation is substantially weaker among duplicated genes. We further discovered that retropositions are associated with greater evolutionary changes in H3K27me3 and gene expression than tandem duplications, indicating that local chromatin constraints influence duplicated gene evolution. These changes are also associated with concomitant evolution of gene expression. Our findings reveal the strong conservation of genomic architecture governed by an epigenetic mark across distantly related species and the importance of gene duplication in generating novel H3K27me3 profiles.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Unbiased compound screening with a reporter gene assay highlights the role of p13 in the cardiac cellular stress response

Highlights: • Unbiased screening with 2320 known compounds identified 12 chemical probes for p13. • > 80% screened agents increased or decreased p13 mRNA and affected cardiac function. • Four cardiotonic agents decreased p13 levels and induced oxidative stress. • In vivo profile of p13 expression shows its importance in heart pathophysiology. • PGC1-alpha, a mitochondrial master regulator, markedly elevated p13 expression. We recently showed that a 13-kDa protein (p13), the homolog protein of formation of mitochondrial complex V assembly factor 1 in yeast, acts as a potential protective factor in pancreatic islets under diabetes. Here, we aimed to identify known compounds regulating p13 mRNA expression to obtain therapeutic insight into the cellular stress response. A luciferase reporter system was developed using the putative promoter region of the human p13 gene. Overexpression of peroxisome proliferator-activated receptor gamma coactivator 1α, a master player regulating mitochondrial metabolism, increased both reporter activity and p13 expression. Following unbiased screening with 2320 known compounds in HeLa cells, 12 pharmacological agents (including 8 cardiotonics and 2 anthracyclines) that elicited >2-fold changes in p13 mRNA expression were identified. Among them, four cardiac glycosides decreased p13 expression and concomitantly elevated cellular oxidative stress. Additional database analyses showed highest p13 expression in heart, with typically decreased expression in cardiac disease. Accordingly, our results illustrate the usefulness of unbiased compound screening as a method for identifying novel functional roles of unfamiliar genes. Our findings also highlight the importance of p13 in the cellular stress response in heart.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Dysregulation of long noncoding RNAs in mouse testes and spermatozoa after exposure to cadmium

There is increasing evidence that cadmium (Cd) exposure can cause male subfertility and even complete infertility in mammals. Long noncoding (lnc) RNAs are critical for spermatogenesis, and their dysregulation might lead to male infertility. However, whether they are involved in Cd-induced subfertility is unknown. Here we found that intraperitoneal exposure to Cd in mice led to male subfertility indicated by reductions in testicular sperm production and motility, and by abnormal morphology. Testicular and sperm RNAs were used to investigate lncRNA expression profiles by strand-specific RNA sequencing at the transcriptome level to help determine any RNA-related mechanisms in Cd-induced subfertility. The Cd-treated testes and spermatozoa exhibited aberrant expression profiles for lncRNAs and mRNAs. Of the lncRNAs, there were 139 with upregulated expression and 174 with downregulated expression in testes; in contrast, 685 were upregulated and 375 were downregulated in spermatozoa. For mRNA expression, 214 were upregulated and 226 were downregulated in testes; 272 were upregulated and 111 were downregulated in spermatozoa. Gene ontology and pathway analyses showed that the functions of differentially expressed lncRNA targets and mRNAs were closely linked with many processes involved in spermatogenesis. Additionally, many newly identified lncRNAs showed inducible expression, suggesting that they might be good candidate markers for Cd-induced male reproductive toxicity. This study provides a preliminary database for further exploring lncRNA-related mechnisms in male infertility induced by Cd. - Highlights: • LncRNA profiles were changed by Cd exposure in mouse testes and spermatozoa. • Differentially expressed lncRNA targets and mRNAs were linked with spermatogenesis. • Providing a database for exploring lncRNA-related mechnisms in male infertility. • LncRNA might be candidate markers for Cd-induced male reproductive toxicity.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2015

Identification and proteomic analysis of osteoblast-derived exosomes

Exosomes are nanometer-sized vesicles with the function of intercellular communication, and they are released by various cell types. To reveal the knowledge about the exosomes from osteoblast, and explore the potential functions of osteogenesis, we isolated microvesicles from supernatants of mouse Mc3t3 by ultracentrifugation, characterized exosomes by electron microscopy and immunoblotting and presented the protein profile by proteomic analysis. The result demonstrated that microvesicles were between 30 and 100 nm in diameter, round shape with cup-like concavity and expressed exosomal marker tumor susceptibility gene (TSG) 101 and flotillin (Flot) 1. We identified a total number of 1069 proteins among which 786 proteins overlap with ExoCarta database. Gene Oncology analysis indicated that exosomes mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The Ingenuity Pathway Analysis showed pathways are mostly involved in exosome biogenesis, formation, uptake and osteogenesis. Among the pathways, eukaryotic initiation factor 2 pathways played an important role in osteogenesis. Our study identified osteoblast-derived exosomes, unveiled the content of them, presented potential osteogenesis-related proteins and pathways and provided a rich proteomics data resource that will be valuable for further studies of the functions of individual proteins in bone diseases. - Highlights: • We for the first time identified exosomes from mouse osteoblast. • Osteoblasts-derived exosomes contain osteoblast peculiar proteins. • Proteins from osteoblasts-derived exosomes are intently involved in EIF2 pathway. • EIF2α from the EIF2 pathway plays an important role in osteogenesis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2014

miR-34a expands myeloid-derived suppressor cells via apoptosis inhibition

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population and show significant expansion under pathological conditions. microRNA plays important roles in many biological processes, whether microRNAs have a function in the expansion of MDSCs is still not very clear. In this study, miR-34a overexpression can induce the expansion of MDSCs in bone marrow chimera and transgenic mice model. The experimental results suggest that miR-34a inhibited the apoptosis of MDSCs but did not affect the proliferation of MDSCs. The distinct mRNA microarray profiles of MDSCs of wild type and miR-34a over-expressing MDSCs combined with the target prediction of miR-34a suggest that miR-34a may target genes such as p2rx7, Tia1, and plekhf1 to inhibit the apoptosis of MDSCs. Taken together, miR-34a contributes to the expansion of MDSCs by inhibiting the apoptosis of MDSCs. - Highlights: • Over-expression of miR-34a increases the number of MDSCs. • miR-34a inhibits the apoptosis of MDSCs, but does not affects their proliferation. • miR-34a may inhibit the apoptosis of MDSCs via targeting the p2rx7, Tia1 and plekhf1.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

Transcriptomic and proteomic signatures of stemness and differentiation in the colon crypt

The intestinal epithelium is replaced weekly by non-quiescent stem cells with kinetics that rely on a rapid loss of stemness and choice for secretory or absorptive lineage differentiation. To determine how the cellular transcriptome and proteome changes during these transitions, we developed a new cell sorting method to purify stem cells, secretory and absorptive progenitor cells, and mature, differentiated cells. Transcriptome analyses revealed that as stem cells transition to the progenitor stage, alternative mRNA splicing and polyadenylation dominate changes in the transcriptome. In contrast, as progenitors differentiate into mature cell types, alterations in gene expression and mRNA levels drive the changes. RNA processing targets mRNAs encoding regulators of cell cycle, RNA regulators, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, global proteome profiling of sorted cell populations detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

TransPlatformer

We propose TransPlatformer for translating toxicogenomics from one platform to another. Transcriptomic profiling has evolved through multiple generations of technology, from microarrays (e.g., Affymetrix, CodeLink) to more recent high-throughput sequencing and targeted panels such as S1500+. Microarrays, which dominated gene expression studies in the early 2000s, provided affordable and high-throughput transcript quantification but suffered from cross-hybridization issues and limited dynamic range . RNA-Seq, introduced in the late 2000s, revolutionized transcriptomics by enabling unbiased and comprehensive gene expression analysis, albeit at higher costs and computational demands . Despite advances, many studies rely on historical microarray data, necessitating the translation of legacy data into modern platforms to ensure continuity and comparability. This translation is complicated by factors such as platform-specific probe design, differences in transcript coverage, and batch effects . Existing methods for cross-platform mapping include statistical normalization, machine learning models, and biological anchoring approaches. The ability to translate transcriptomic data between platforms has broad implications, including enhanced meta-analyses, improved toxicological modeling, and better integration of historical datasets with contemporary research. TransPlatformer seeks to contribute to this effort by evaluating translation methodologies and proposing novel strategies to improve cross-platform gene expression harmonization. In this repository there are code examples for TransPlatformer implementation

Cong, Guojing↗

DOE OSTI2014

Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons

Highlights: • Identification of dystrophin (Dp) shortest isoform, Dp40, is a neuron-type Dp. • Dp40 expression is temporally and differentially regulated in comparison to Dp71. • Somatodendritic and nuclear localization of Dp40. • Dp40 is localized to excitatory postsynapses. • Dp40 might play roles in dendritic and synaptic functions. - Abstract: The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH{sub 2}-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2011

Histone demethylase JMJD2B is required for tumor cell proliferation and survival and is overexpressed in gastric cancer

Highlights: Black-Right-Pointing-Pointer JMJD2B is required for cell proliferation and in vivo tumorigenesis. Black-Right-Pointing-Pointer JMJD2B depletion induces apoptosis and/or cell cycle arrest. Black-Right-Pointing-Pointer JMJD2B depletion activates DNA damage response and enhances p53 stabilization. Black-Right-Pointing-Pointer JMJD2B is overexpressed in human primary gastric cancer. -- Abstract: Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Jumonji domain containing 2B (JMJD2B) is a newly identified histone demethylase that regulates chromatin structure or gene expression by removing methyl residues from trimethylated lysine 9 on histone H3. Recent observations have shown oncogenic activity of JMJD2B. We explored the functional role of JMJD2B in cancer cell proliferation, survival and tumorigenesis, and determined its expression profile in gastric cancer. Knocking down JMJD2B expression by small interfering RNA (siRNA) in gastric and other cancer cells inhibited cell proliferation and/or induced apoptosis and elevated the expression of p53 and p21{sup CIP1} proteins. The enhanced p53 expression resulted from activation of the DNA damage response pathway. JMJD2B knockdown markedly suppressed xenograft tumor growth in vivo in mice. Moreover, JMJD2B expression was increased in primary gastric-cancer tissues of humans. Thus, JMJD2B is required for sustained proliferation and survival of tumor cells in vitro and in vivo, and its aberrant expression may contribute to the pathogenesis of gastric cancer.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2016

Metabolomic and Genomic Markers of Atherosclerosis as Related to Oxidative Stress, Inflammation, and Vascular Function in Twin Astronauts

Background: Future human space travel will consist primarily of long-duration missions onboard the International Space Station (ISS) or exploration-class missions to Mars, its moons, or nearby asteroids. Astronauts participating in long-duration missions may be at an increased risk of oxidative stress and inflammatory damage due to radiation, psychological stress, altered physical activity, nutritional insufficiency, and hyperoxia during extravehicular activity. By studying one identical twin during his 1-year ISS mission and one ground-based twin, this work extends a current NASA-funded investigation to determine whether these spaceflight factors contribute to an accelerated progression of atherosclerosis. This study of twins affords a unique opportunity to examine the spaceflight-related atherosclerosis risk independent of the confounding factors associated with different genotypes. Purpose: The purpose of this investigation is to determine whether biomarkers of oxidative and inflammatory stress are elevated during and after long-duration spaceflight and determine if a relation exists between levels of these biomarkers and structural and functional indices of atherosclerotic risk measured in the carotid and brachial arteries. These physiological and biochemical data will be extended by using an exploratory approach to investigate the relationship between intermediate phenotypes and risk factors for atherosclerosis and the metabolomic signature from plasma and urine samples. Since metabolites are often the indirect products of gene expression, we will simultaneously assess gene expression and DNA methylation in leukocytes. Hypothesis: We predict that the space-flown twin will experience elevated biomarkers of oxidative stress and inflammatory damage, altered arterial structure and function, accelerated telomere shortening, dysregulation of genes associated with oxidative stress and inflammation, and a metabolic profile shift that is associated with elevated atherosclerosis risk factors. Conversely, these will not be observed in the ground-based twin. Methods: We will measure blood and urine biomarkers of oxidative stress and inflammation as well as arterial structure and function (carotid intima-medial thickness and brachial artery flow-mediated dilation) in one twin astronaut before, during, and after long-duration spaceflight and in his twin serving as a ground-based control. Furthermore, we will measure metabolomics (targeted and untargeted approaches) and genomic markers (DNA methylation, mRNA gene expression, telomere length) to elucidate the molecular mechanisms involved. A panel of biomarkers of oxidative and inflammatory stress will be measured in venous blood samples and 24-hour (in-flight) and 48-hour (pre- and post-flight) urine pools twice before flight, early (flight days 15 and 60) and late (2 weeks before landing) during the mission, and early in the post-flight recovery phase (approximately 3-5 days after landing). Arterial structure, assessed from measures of intima-media thickness, will be measured at the same times. Arterial function will be assessed using brachial flow-mediated dilation, a well-validated measure used to assess endothelium-dependent vasodilation and a sensitive predictor of atherosclerotic risk, only before and after spaceflight. Discussion: Pre- and in-flight data collection is in progress for the space-flown twin, and similar data have been obtained from the ground-based twin. Blood and urine samples will be batch processed when received from ISS after the conclusion of the 1-year mission. Results from these individual subjects will be compared to the larger complement of subjects participating in the companion study currently ongoing in ISS astronauts.

Lee, Stuart M. C.↗

DOE OSTI2019

Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer’s disease model

Many risk genes for the development of Alzheimer’s disease (AD) are exclusively or highly expressed in myeloid cells. Microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for their survival. We designed and synthesized a highly selective brain-penetrant CSF1R inhibitor (PLX5622) allowing for extended and specific microglial elimination, preceding and during pathology development. We find that in the 5xFAD mouse model of AD, plaques fail to form in the parenchymal space following microglial depletion, except in areas containing surviving microglia. Instead, Aβ deposits in cortical blood vessels reminiscent of cerebral amyloid angiopathy. Altered gene expression in the 5xFAD hippocampus is also reversed by the absence of microglia. Transcriptional analyses of the residual plaque-forming microglia show they exhibit a disease-associated microglia profile. Collectively, we describe the structure, formulation, and efficacy of PLX5622, which allows for sustained microglial depletion and identify roles of microglia in initiating plaque pathogenesis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2014

Distinct Strains of Toxoplasma gondii Feature Divergent Transcriptomes Regardless of Developmental Stage

Using high through-put RNA sequencing, we assayed the transcriptomes of three different strains of Toxoplasma gondii representing three common genotypes under both in vitro tachyzoite and in vitro bradyzoite-inducing alkaline stress culture conditions. Strikingly, the differences in transcriptional profiles between the strains, RH, PLK, and CTG, is much greater than differences between tachyzoites and alkaline stressed in vitro bradyzoites. With an FDR of 10%, we identify 241 genes differentially expressed between CTG tachyzoites and in vitro bradyzoites, including 5 putative AP2 transcription factors. We also observe close association between cell cycle regulated genes and differentiation. By Gene Set Enrichment Analysis (GSEA), there are a number of KEGG pathways associated with the in vitro bradyzoite transcriptomes of PLK and CTG, including pyrimidine metabolism and DNA replication. These functions are likely associated with cell-cycle arrest. When comparing mRNA levels between strains, we identify 1,526 genes that are differentially expressed regardless of culture-condition as well as 846 differentially expressed only in bradyzoites and 542 differentially expressed only in tachyzoites between at least two strains. Using GSEA, we identify ribosomal proteins as being expressed at significantly higher levels in the CTG strain than in either the RH or PLK strains. This association holds true regardless of life cycle stage.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors including histone PTMs are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for engineering solutions to increase the resilience of crops to climate change. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed as their intact forms by top-down mass spectrometry (MS) coupled to online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow thus yielding the global PTM profile of core histones (H4, H2A, H2B, H3). By comparing the PTM profile among samples corresponding to different conditions (e.g. drought vs. control), potential epigenetic marks can be discovered as targets for further characterization using approaches such as chromatin immunoprecipitation – sequencing (ChIP-seq).

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Characterization of terpene biosynthesis in Melaleuca quinquenervia and ecological consequences of terpene accumulation during myrtle rust infection

Abstract Plants use a wide array of secondary metabolites including terpenes as defense against herbivore and pathogen attack, which can be constitutively expressed or induced. Here, we investigated aspects of the chemical and molecular basis of resistance against the exotic rust fungus Austropuccinia psidii in Melaleuca quinquenervia , with a focus on terpenes. Foliar terpenes of resistant and susceptible plants were quantified, and we assessed whether chemotypic variation contributed to resistance to infection by A. psidii . We found that chemotypes did not contribute to the resistance and susceptibility of M. quinquenervia . However, in one of the chemotypes (Chemotype 2), susceptible plants showed higher concentrations of several terpenes including α‐pinene, limonene, 1,8‐cineole, and viridiflorol compared with resistant plants. Transcriptome profiling of these plants showed that several TPS genes were strongly induced in response to infection by A. psidii . Functional characterization of these TPS showed them to be mono‐ and sesquiterpene synthases producing compounds including 1,8‐cineole, β‐caryophyllene, viridiflorol and nerolidol. The expression of these TPS genes correlated with metabolite data in a susceptible plant. These results suggest the complexity of resistance mechanism regulated by M . quinquenervia and that modulation of terpenes may be one of the components that contribute to resistance against A. psidii .

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Asparagine synthetase gene regulatory network and plant nitrogen metabolism

This DOE project concerns the molecular mechanisms by which plants coordinate carbon (C) and nitrogen (N) metabolism. Specifically, it focuses on a histone methyltransferase protein that targets C/N-metabolism genes for chromatin modifications. How the dynamic changes of chromatin regulate energy metabolism in response to developmental and environmental cues is a new area of study. We have filled this knowledge-gap with our studies of SDG8, a histone methyltransferase, that targets light, C and N metabolism genes. We aimed to understand how plants allocate C resources to assimilate inorganic N onto C-skeletons -- the most energy-intensive pathway in plants – in order to balance energy production and consumption in a changing environment. In order to achieve more C-efficient N-storage under low energy conditions, plants convert assimilated N from “reactive” Gln (C5:N2) into “inert” Asn (C4:N2) through the enzyme ASN1. To identify regulatory factors in this pathway, we used the ASN1 promoter in a genetic selection and discovered SDG8, a histone methyltransferase. We discovered that histone modification by SDG8 coordinates the expression of genes involved in energy generation (light-harvesting and C fixation) with energy consumption (N assimilation and sulfur metabolism). We used this opportunity to explore how histone modifications control energy metabolism genome-wide. Our aims focus on various levels of the regulation of energy related genes at the level of: 1) Chromatin; 2) Transcription; and 3) Metabolites of the C/N regulatory network. Specifically, we asked: 1) How does histone methylation regulate energy metabolism during the N response? 2) How does histone methylation control transcription of the energy metabolism genes? And 3) how does the histone methyltransferase SDG8 and other TF regulators affect C/N metabolism and N use efficiency? Our study indicates that N supply triggers genome-wide changes in chromatin modification via the histone methyltransferase SDG8 which impacts on gene expression, RNA processing, chlorophyll synthesis, N uptake and metabolism. Our study thus revealed a previously overlooked layer of chromatin regulation during nutrient signaling. In broad terms, our study provides a framework to understand how chromatin modifications influence responses to environmental changes and provides unique opportunities for breeding and engineering resilient crops with better performance in the current climate model. Accomplishments AIM 1 CHROMATIN: Role of histone methylation in the ASN1 energy metabolism network. This aim focuses on understanding the role of chromatin modification in response to the environment. We studied the targeting mechanism of histone methylation by SDG8, in particular the role of the CW domain in reading histone methylation status. We also began to study the effect of nitrogen-treatment on genome-wide chromatin modifications in WT and sdg8 mutant plants. AIM 2 TRANSCRIPTION: Transcription control of the ASN1 energy metabolism network. This aim investigated whether SDG8 and the histone modification it mediates affects mRNA processing. We performed genome-wide profiling that uncovered that histone methylation by SDG8 affects the co-transcriptional RNA processing of downstream genes involved in energy and photosynthesis. AIM 3 METABOLISM: A gene regulatory network controlling energy, C/N metabolism and N use efficiency. In Aim 3A, we showed that SDG8 affects the chromatin modification and expression of a transcription factor, CCT, which in turn affects the regulation of carbon responsive genes. In Aim 3B we showed that C and N metabolism is perturbed in the sdg8 mutant through differences in the chlorophyll content, free amino acids, and N uptake. Project activities In Aim 1, we uncovered that the histone methyltransferase SDG8 affects chromatin modification in response to N treatments. The N treatments causes changes in the H3K36me3 distribution off ~200 genes in WT plants, while the N-triggered changes in H3K36me3 of these genes is abrogated in the sdg8-5 mutant. The functional annotation of these 200 genes shows significant enrichment of N-related pathways. Overall, our study suggests that SDG8 mediates chromatin-level adaption of functionally relevant genes in response to changes in N. In Aim 2, we tested if SDG8 plays a role in transcriptional elongation and/or RNA processing. We detected more N-triggered isoform shifting events in the sdg8-5 mutant than in WT, which provides supports for SDG8 being required for proper RNA processing in response to N signaling. In Aim 3, we identified the gene network controlled by a CCT transcription factor under the epigenetic control of SDG8. Isoform abundance of the CCT protein is regulated by SDG8, which leads to an altered response of energy related genes to N level changes. Using physiological, metabolic and genetic approaches, we found that responses to N, including chlorophyll accumulation, N uptake and free amino acids, are altered in sdg8 mutants. Finally, this project provided training to a high school student who performed experiments on the complex interaction of photosynthesis and N availability. The student was named a semi-finalist in both the Regeneron and Siemens National Science competitions for her work.

59 BASIC BIOLOGICAL SCIENCES↗