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DOE OSTI2018

Secretome Analysis from the Ectomycorrhizal Ascomycete Cenococcum geophilum

Cenococcum geophilum is an ectomycorrhizal fungus with global distribution in numerous habitats and associates with a large range of host species including gymnosperm and angiosperm trees. Moreover, C. geophilum is the unique ectomycorrhizal species within the clade Dothideomycetes, the largest class of Ascomycetes containing predominantly saprotrophic and many devastating phytopathogenic fungi. Recent studies highlight that mycorrhizal fungi, as pathogenic ones, use effectors in form of Small Secreted Proteins (SSPs) as molecular keys to promote symbiosis. In order to better understand the biotic interaction of C. geophilum with its host plants, the goal of this work was to characterize mycorrhiza-induced small-secreted proteins (MiSSPs) that potentially play a role in the ectomycorrhiza formation and functioning of this ecologically very important species. We combined different approaches such as gene expression profiling, genome localization and conservation of MiSSP genes in different C. geophilum strains and closely related species as well as protein subcellular localization studies of potential targets of MiSSPs in interacting plants using in tobacco leaf cells. Gene expression analyses of C. geophilum interacting with Pinus sylvestris (pine) and Populus tremula × Populus alba (poplar) showed that similar sets of genes coding for secreted proteins were up-regulated and only few were specific to each host. Whereas pine induced more carbohydrate active enzymes (CAZymes), the interaction with poplar induced the expression of specific SSPs. We identified a set of 22 MiSSPs, which are located in both, gene-rich, repeat-poor or gene-sparse, repeat-rich regions of the C. geophilum genome, a genome showing a bipartite architecture as seen for some pathogens but not yet for an ectomycorrhizal fungus. Genome re-sequencing data of 15 C. geophilum strains and two close relatives Glonium stellatum and Lepidopterella palustris were used to study sequence conservation of MiSSP-encoding genes. The 22 MiSSPs showed a high presence-absence polymorphism among the studied C. geophilum strains suggesting an evolution through gene gain/gene loss. Finally, we showed that six CgMiSSPs target four distinct sub-cellular compartments such as endoplasmic reticulum, plasma membrane, cytosol and tonoplast. Overall, this work presents a comprehensive analysis of secreted proteins and MiSSPs in different genetic level of C. geophilum opening a valuable resource to future functional analysis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Genome resequencing reveals demographic history and genetic architecture of seed salinity tolerance in Populus euphratica

Populus euphratica is a dominant tree species in desert riparian forests and possesses extraordinary adaptation to salinity stress. Exploration of its genomic variation and molecular underpinning of salinity tolerance is important for elucidating population evolution and identifying stress-related genes. Here, we identify approximately 3.15 million single nucleotide polymorphisms using whole-genome resequencing. The natural populations of P. euphratica in northwest China are divided into four distinct clades that exhibit strong geographical distribution patterns. Pleistocene climatic fluctuations and tectonic deformation jointly shaped the extant genetic patterns. A seed germination rate-based salinity tolerance index was used to evaluate seed salinity tolerance of P. euphratica and a genome-wide association study was implemented. A total of 38 single nucleotide polymorphisms were associated with seed salinity tolerance and were located within or near 82 genes. Expression profiles showed that most of these genes were regulated under salt stress, revealing the genetic complexity of seed salinity tolerance. Furthermore, DEAD-box ATP-dependent RNA helicase 57 and one undescribed gene (CCG029559) were demonstrated to improve the seed salinity tolerance in transgenic Arabidopsis. These results provide new insights into the demographic history and genetic architecture of seed salinity tolerance in desert poplar.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2011

A Conserved Developmental Patterning Network Produces Quantitatively Different Output in Multiple Species of Drosophila

Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3–4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of equivalent cells.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRSDate not supplied

Efficacy of Dried Plum to Countermeasure Simulated Spaceflight Exposure

Human exploration of the space environment has been a topic of interest for decades; however, the threat to physiological health posed by ionizing radiation and microgravity effects remains a concern. Previous research utilizing combined hindlimb unloading and irradiation to simulate spaceflight effects found that a dried plum diet can mitigate adverse effects of simulated spaceflight on skeletal health. Though the mechanism is not fully understood we believe this protective effect is due to its high antioxidant capacity and polyphenolic content. This study seeks to extend these findings to investigate if dried plum is an effective countermeasure in other tissues relating to aging and neurotoxicity. Male C57BL/6 J mice were assigned to one of eight groups (n = 10/group) and were fed either a controlled diet or dried plum diet, underwent normal loading or hindlimb unloading as well as exposure 2 Gy gamma radiation or sham exposure. At the end of the experiment, mice were euthanized, and RNA was extracted from brain and bone marrow tissue and PCR was employed to analyze significant changes in gene expression profiles. Analysis of five house-keeping genes revealed genes B2M (neurotoxicity) and GUSB (aging) to be the most stable genes across all treatments and were used to find the fold change within our genes of interest. Through this, and analysis via ANOVA, we were able to identify genes with significant altered expression levels within brain and bone marrow tissues related to neurotoxicity and aging. Considering previous research, we hypothesize that the dried plum diet’s antioxidative properties ameliorated the oxidative stress from irradiation and microgravity via altered levels of gene expression. Identified expression changes will provide insights into molecular mechanisms behind the observed protective effects. These findings present potential avenues for future interventions to alleviate negative impacts of spaceflight on astronaut health.

Faisal Ahmad↗

NASA NTRSDate not supplied

The Efficacy of Dried Plum to Counter Spaceflight Microgravity and Radiation

Faisal Ahmad↗

DOE OSTI2016

Comprehensive Assessments of RNA-seq by the SEQC Consortium: FDA-Led Efforts Advance Precision Medicine

Studies on gene expression in response to therapy have led to the discovery of pharmacogenomics biomarkers and advances in precision medicine. Whole transcriptome sequencing (RNA-seq) is an emerging tool for profiling gene expression and has received wide adoption in the biomedical research community. However, its value in regulatory decision making requires rigorous assessment and consensus between various stakeholders, including the research community, regulatory agencies, and industry. The FDA-led SEquencing Quality Control (SEQC) consortium has made considerable progress in this direction, and is the subject of this review. Specifically, three RNA-seq platforms (Illumina HiSeq, Life Technologies SOLiD, and Roche 454) were extensively evaluated at multiple sites to assess cross-site and cross-platform reproducibility. The results demonstrated that relative gene expression measurements were consistently comparable across labs and platforms, but not so for the measurement of absolute expression levels. As part of the quality evaluation several studies were included to evaluate the utility of RNA-seq in clinical settings and safety assessment. The neuroblastoma study profiled tumor samples from 498 pediatric neuroblastoma patients by both microarray and RNA-seq. RNA-seq offers more utilities than microarray in determining the transcriptomic characteristics of cancer. However, RNA-seq and microarray-based models were comparable in clinical endpoint prediction, even when including additional features unique to RNA-seq beyond gene expression. The toxicogenomics study compared microarray and RNA-seq profiles of the liver samples from rats exposed to 27 different chemicals representing multiple toxicity modes of action. Cross-platform concordance was dependent on chemical treatment and transcript abundance. Though both RNA-seq and microarray are suitable for developing gene expression based predictive models with comparable prediction performance, RNA-seq offers advantages over microarray in profiling genes with low expression. The rat BodyMap study provided a comprehensive rat transcriptomic body map by performing RNA-Seq on 320 samples from 11 organs in either sex of juvenile, adolescent, adult and aged Fischer 344 rats. Lastly, the transferability study demonstrated that signature genes of predictive models are reciprocally transferable between microarray and RNA-seq data for model development using a comprehensive approach with two large clinical data sets. This result suggests continued usefulness of legacy microarray data in the coming RNA-seq era. In conclusion, the SEQC project enhances our understanding of RNA-seq and provides valuable guidelines for RNA-seq based clinical application and safety evaluation to advance precision medicine.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2014

A Sorghum bicolor expression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet and bioenergy sorghums

Background: Effective improvement in sorghum crop development necessitates a genomics-based approach to identify functional genes and QTLs. Sequenced in 2009, a comprehensive annotation of the sorghum genome and the development of functional genomics resources is key to enable the discovery and deployment of regulatory and metabolic genes and gene networks for crop improvement. Results: This study utilizes the first commercially available whole-transcriptome sorghum microarray (Sorgh-WTa520972F) to identify tissue and genotype-specific expression patterns for all identified Sorghum bicolor exons and UTRs. The genechip contains 1,026,373 probes covering 149,182 exons (27,577 genes) across the Sorghum bicolor nuclear, chloroplast, and mitochondrial genomes. Specific probesets were also included for putative non-coding RNAs that may play a role in gene regulation (e. g., microRNAs), and confirmed functional small RNAs in related species (maize and sugarcane) were also included in our array design. We generated expression data for 78 samples with a combination of four different tissue types (shoot, root, leaf and stem), two dissected stem tissues (pith and rind) and six diverse genotypes, which included 6 public sorghum lines (R159, Atlas, Fremont, PI152611, AR2400 and PI455230) representing grain, sweet, forage, and high biomass ideotypes. Conclusions: Here we present a summary of the microarray dataset, including analysis of tissue-specific gene expression profiles and associated expression profiles of relevant metabolic pathways. With an aim to enable identification and functional characterization of genes in sorghum, this expression atlas presents a new and valuable resource to the research community.

Shakoor, N↗

DOE OSTI2019

Campylobacter jejuni Demonstrates Conserved Proteomic and Transcriptomic Responses When Co-cultured With Human INT 407 and Caco-2 Epithelial Cells

Major foodborne bacterial pathogens, such as Campylobacter jejuni , have devised complex strategies to establish and foster intestinal infections. For more than two decades, researchers have used immortalized cell lines derived from human intestinal tissue to dissect C. jejuni -host cell interactions. Known from these studies is that C. jejuni virulence is multifactorial, requiring a coordinated response to produce virulence factors that facilitate host cell interactions. This study was initiated to identify C. jejuni proteins that contribute to adaptation to the host cell environment and cellular invasion. We demonstrated that C. jejuni responds to INT 407 and Caco-2 cells in a similar fashion at the cellular and molecular levels. Active protein synthesis was found to be required for C. jejuni to maximally invade these host cells. Proteomic and transcriptomic approaches were then used to define the protein and gene expression profiles of C. jejuni co-cultured with cells. By focusing on those genes showing increased expression by C. jejuni when co-cultured with epithelial cells, we discovered that C. jejuni quickly adapts to co-culture with epithelial cells by synthesizing gene products that enable it to acquire specific amino acids for growth, scavenge for inorganic molecules including iron, resist reactive oxygen/nitrogen species, and promote host cell interactions. Based on these findings, we selected a subset of the genes involved in chemotaxis and the regulation of flagellar assembly and generated C. jejuni deletion mutants for phenotypic analysis. Binding and internalization assays revealed significant differences in the interaction of C. jejuni chemotaxis and flagellar regulatory mutants. The identification of genes involved in C. jejuni adaptation to culture with host cells provides new insights into the infection process.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2012

Global identification of genes regulated by estrogen signaling and demethylation in MCF-7 breast cancer cells

Highlights: Black-Right-Pointing-Pointer Estrogen signaling and demethylation can both control gene expression in breast cancers. Black-Right-Pointing-Pointer Cross-talk between these mechanisms is investigated in human MCF-7 breast cancer cells. Black-Right-Pointing-Pointer 137 genes are influenced by both 17{beta}-estradiol and demethylating agent 5-aza-2 Prime -deoxycytidine. Black-Right-Pointing-Pointer A set of genes is identified as targets of both estrogen signaling and demethylation. Black-Right-Pointing-Pointer There is no direct molecular interplay of mediators of estrogen and epigenetic signaling. -- Abstract: Estrogen signaling and epigenetic modifications, in particular DNA methylation, are involved in regulation of gene expression in breast cancers. Here we investigated a potential regulatory cross-talk between these two pathways by identifying their common target genes and exploring underlying molecular mechanisms in human MCF-7 breast cancer cells. Gene expression profiling revealed that the expression of approximately 140 genes was influenced by both 17{beta}-estradiol (E2) and a demethylating agent 5-aza-2 Prime -deoxycytidine (DAC). Gene ontology (GO) analysis suggests that these genes are involved in intracellular signaling cascades, regulation of cell proliferation and apoptosis. Based on previously reported association with breast cancer, estrogen signaling and/or DNA methylation, CpG island prediction and GO analysis, we selected six genes (BTG3, FHL2, PMAIP1, BTG2, CDKN1A and TGFB2) for further analysis. Tamoxifen reverses the effect of E2 on the expression of all selected genes, suggesting that they are direct targets of estrogen receptor. Furthermore, DAC treatment reactivates the expression of all selected genes in a dose-dependent manner. Promoter CpG island methylation status analysis revealed that only the promoters of BTG3 and FHL2 genes are methylated, with DAC inducing demethylation, suggesting DNA methylation directs repression of these genes in MCF-7 cells. In a further analysis of the potential interplay between estrogen signaling and DNA methylation, E2 treatment showed no effect on the methylation status of these promoters. Additionally, we show that the ER{alpha} recruitment occurs at the FHL2 promoter in an E2- and DAC-independent fashion. In conclusion, we identified a set of genes regulated by both estrogen signaling and DNA methylation. However, our data does not support a direct molecular interplay of mediators of estrogen and epigenetic signaling at promoters of regulated genes.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Analysis of individual cells identifies cell-to-cell variability following induction of cellular senescence

Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single-cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell-to-cell variability resulted in a loss of correlation among the expression of several senescence associated genes. Many genes encoding senescence-associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.

transcriptomics↗

DOE OSTI2012

Development of Thermoanaerobacterium saccharolyticum for the conversion of lignocellulose to ethanol (Final Technical Report)

This project addressed the need for economical technology for the conversion of lignocellulosic biomass to fuels, specifically the conversion of pretreated hardwood to ethanol. The technology developed is a set of strains of the bacterium Thermoanaerobacterium saccharolyticum and an associated fermentation process for pretreated hardwood. Tools for genetic engineering and analysis of the organism were developed, including a markerless mutation method, a complete genome sequence and a set of gene expression profiles that show the activity of its genes under a variety of conditions relevant to lignocellulose conversion. Improved strains were generated by selection and genetic engineering to be able to produce higher amounts of ethanol (up to 70 g/L) and to be able to better tolerate inhibitory compounds from pretreated hardwood. Analysis of these strains has generated useful insight into the genetic basis for desired properties of biofuel producing organisms. Fermentation conditions were tested and optimized to achieve ethanol production targets established in the original project proposal. The approach proposed was to add cellulase enzymes to the fermentation, a method called Simultaneous Saccharification and Fermentation (SSF). We had reason to think SSF would be an efficient approach because the optimal temperature and pH for the enzymes and bacterium are very close. Unfortunately, we discovered that commercially available cellulases are inactivated in thermophilic SSF by a combination of low redox potential and ethanol. Despite this, progress was made against the fermentation targets using bacterial cellulases. Thermoanaerobacterium saccharolyticum may still prove to be a commercially viable technology should cellulase enzyme issues be addressed. Moreover, the organism was demonstrated to produce ethanol at approximately theoretical yield from oligomeric hemicellulose extracts, an ability that may prove to be uniquely valuable in pretreatment configurations in which cellulose and hemicellulose are separated.

09 BIOMASS FUELS↗

NASA NTRS2007

Expression of Genes Associated with DNA Damage Sensing in Human Fibroblasts Exposed to Low-dose-rate Gamma Rays

Understanding of the molecular response to low-dose and low-dose-rate radiation exposure is essential for the extrapolation of high-dose radiation risks to those at dose levels relevant to space and other environmental concerns. Most of the reported studies of gene expressions induced by low-dose or low-dose-rate radiation were carried out on exponentially growing cells. In the present study, we analyzed expressions of 84 genes associated with DNA damage sensing using real time PCR in human fibroblasts in mostly G1 phase of the cell cycle. The cells were exposed continuously to gamma rays at a dose rate of 0.8 cGy/hr for 1, 2, 6 or 24 hrs at 37 C throughout the exposure. The total RNA was isolated immediately after the exposure was terminated. Of the 84 genes, only a few showed significant changes of the expression level. Some of the genes (e.g. DDit3 and BTG2) were found to be up or down regulated only after a short period of exposure, while other genes (e.g. PRKDC) displayed a highest expression level at the 24 hr time point. The expression profiles for the exposed cells which had a smaller portion of G1 cells indicated more cell cycle signaling and DNA repair genes either up or down regulated. Interestingly, the panel of genes changed from radiation exposure in G1 cells is different from the panel in cells having less G1 arrest cells. The gene expression profile of the cells responding to low-dose-radiation insult apparently depends on the cell growth stage. The response pathway in G1 cells may differ from that in exponentially growing cells.

Zhang, Ye↗

DOE OSTI2014

Development of a Low Input and sustainable Switchgrass Feedstock Production System Utilizing Beneficial Bacterial Endophytes

Switchgrass represents a promising feedstock crop for US energy sustainability. However, its broad utilization for bioenergy requires improvements of biomass yields and stress tolerance. In this DOE funded project, we have been working on harnessing beneficial bacterial endophytes to enhance switchgrass performance and to develop a low input feedstock production system for marginal lands that do not compete with the production of food crops. We have demonstrated that one of most promising plant growth-promoting bacterial endophytes, Burkholderia phytofirmans strain PsJN, is able to colonize roots and significantly promote growth of switchgrass cv. Alamo under in vitro, growth chamber, greenhouse, as well as field conditions. Furthermore, PsJN bacterization improved growth and development of switchgrass seedlings, significantly stimulated plant root and shoot growth, and tiller number in the field, and enhanced biomass accumulation on both poor (p<0.001) and rich (p<0.05) soils, with more effective stimulation of plant growth in low fertility soil. Plant physiology measurements showed that PsJN inoculated Alamo had consistently lower transpiration, lower stomatal conductance, and higher water use efficiency in greenhouse conditions. These physiological changes may significantly contribute to the recorded growth enhancement. PsJN inoculation rapidly results in an increase in photosynthetic rates which contributes to the advanced growth and development. Some evidence suggests that this initial growth advantage decreases with time when resources are not limited such as in greenhouse studies. Additionally, better drought resistance and drought hardening were observed in PsJN inoculated switchgrass. Using the DOE-funded switchgrass EST microarray, in a collaboration with the Genomics Core Facility at the Noble Foundation, we have determined gene expression profile changes in both responsive switchgrass cv. Alamo and non-responsive cv. Cave-in-Rock (CR) following PsJN bacterization. With the MapMan software to analyze microarray data, the number of up- and down-regulated probes was calculated. The number of up-regulated probes in Alamo was 26, 14, 14, and 12% at 0.5, 2, 4 and 8 days after inoculation (DAI) with PsJN, respectively while the corresponding number in CR was 24, 22, 21, and 19%, respectively. In both cultivars, the largest number of up-regulated probes occurred at 0.5 DAI. Noticeable differences throughout the timeframe between Alamo and CR were that the number was dramatically decreased to half (12%) in Alamo but remained high in CR (approximately 20%). The number of down regulated genes demonstrated different trends in Alamo and CR. Alamo had an increasing trend from 9% at 0.5 DAI to 11, 17, and 28% at 2, 4, and 8 DAI, respectively. However, CR had 13% at 0.5 and 2 DAI, and declined to 10% at 4 and 8 DAI. With the aid of MapMan and PageMan, we mapped the response of the ID probes to the observed major gene regulatory network and major biosynthetic pathway changes associated with the beneficial bacterial endophyte infection, colonization, and early growth promotion process. We found significant differences in gene expression patterns between responsive and non-responsive cultivars in many pathways, including redox state regulation, signaling, proteolysis, transcription factors, as well as hormone (SA and JA in particular)-associated pathways. Form microarray data, a total of 50 key genes have been verified using qPCR. Ten of these genes were chosen for further functional study via either overexpression and/or RNAi knockout technologies. These genes were calmodulin-related calcium sensor protein (CAM), glutathione S-transferase (GST), histidine-containing phosphotransfer protein (H-221), 3 different zinc finger proteins (ZF-371, ZF131 and ZF242), EF hand transcription factor (EF-622), peroxidase, cellulose synthase catalytic submit A2 (CESA2), and Aux/IAA family. A total of 8 overexpression and 5 RNAi transgenic plants have been regenerated, and their gene expression levels determined using qPCR. Consequently high, medium and low expression lines were propagated in vitro for gene function study. When adequate numbers of individual transgenic lines were obtained, they were challenged with PsJN to see if PsJN promotes or inhibits growth of transgenic plants. Our results demonstrated that EF-622 overexpression, ZF-371, GST, H-221 and CAM RNAi transgenic lines lost responses to PsJN, i.e. PsJN had no growth promotive effects on these transgenic plants. Further study needs to be done to characterize this loss of responsiveness to PsJN. During this funding period, we have done more work related to this funded project and established collaborations with other institutions and obtained some interesting results, building a foundation for further research projects. For example, we isolated a naturally-occurring bacterium from surface-sterilized switchgrass seeds, identified as a unique Panteoa agglomerans species, and named strain PaKM. PaKM has been proved to be an efficient growth promoter of switchgrass over a broad spectrum of genotypes and has potential in applications with low input and sustainable production systems on marginal lands. In collaboration with Dr. Shuijin Hu (North Carolina State University), we conducted experiments on how endophyte-inoculated switchgrass affects soil N and P availability and the number of AMF in roots. Our preliminary results showed that PsJN increased AMF infection of switchgrass roots, and enhanced soil N availability and soil N mineralization on a low nutrient field. Further study of this phenomenon on different soils, over longer time periods, is needed to assess its potential impact on the productivity and longevity of switchgrass stands.

09 BIOMASS FUELS↗

DOE OSTI2012

Autoimmune regulator is acetylated by transcription coactivator CBP/p300

The Autoimmune Regulator (AIRE) is a regulator of transcription in the thymic medulla, where it controls the expression of a large set of peripheral-tissue specific genes. AIRE interacts with the transcriptional coactivator and acetyltransferase CBP and synergistically cooperates with it in transcriptional activation. Here, we aimed to study a possible role of AIRE acetylation in the modulation of its activity. We found that AIRE is acetylated in tissue culture cells and this acetylation is enhanced by overexpression of CBP and the CBP paralog p300. The acetylated lysines were located within nuclear localization signal and SAND domain. AIRE with mutations that mimicked acetylated K243 and K253 in the SAND domain had reduced transactivation activity and accumulated into fewer and larger nuclear bodies, whereas mutations that mimicked the unacetylated lysines were functionally similar to wild-type AIRE. Analogously to CBP, p300 localized to AIRE-containing nuclear bodies, however, the overexpression of p300 did not enhance the transcriptional activation of AIRE-regulated genes. Further studies showed that overexpression of p300 stabilized the AIRE protein. Interestingly, gene expression profiling revealed that AIRE, with mutations mimicking K243/K253 acetylation in SAND, was able to activate gene expression, although the affected genes were different and the activation level was lower from those regulated by wild-type AIRE. Our results suggest that the AIRE acetylation can influence the selection of AIRE activated genes. -- Highlights: Black-Right-Pointing-Pointer AIRE is acetylated by the acetyltransferases p300 and CBP. Black-Right-Pointing-Pointer Acetylation occurs between CARD and SAND domains and within the SAND domain. Black-Right-Pointing-Pointer Acetylation increases the size of AIRE nuclear dots. Black-Right-Pointing-Pointer Acetylation increases AIRE protein stability. Black-Right-Pointing-Pointer AIRE acetylation mimic regulates a different set of AIRE target genes.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2013

A Genome-Wide Association Study Suggests Novel Loci Associated with a Schizophrenia-Related Brain-Based Phenotype

Patients with schizophrenia and their siblings typically show subtle changes of brain structures, such as a reduction of hippocampal volume. Hippocampal volume is heritable, may explain a variety of cognitive symptoms of schizophrenia and is thus considered an intermediate phenotype for this mental illness. The aim of our analyses was to identify singlenucleotide polymorphisms (SNP) related to hippocampal volume without making prior assumptions about possible candidate genes. In this study, we combined genetics, imaging and neuropsychological data obtained from the Mind Clinical Imaging Consortium study of schizophrenia (n = 328). A total of 743,591 SNPs were tested for association with hippocampal volume in a genome-wide association study. Gene expression profiles of human hippocampal tissue were investigated for gene regions of significantly associated SNPs. None of the genetic markers reached genome-wide significance. However, six highly correlated SNPs (rs4808611, rs35686037, rs12982178, rs1042178, rs10406920, rs8170) on chromosome 19p13.11, located within or in close proximity to the genes NR2F6, USHBP1, and BABAM1, as well as four SNPs in three other genomic regions (chromosome 1, 2 and 10) had p-values between 6.75610 -6 and 8.3610 -7 . Using existing data of a very recently published GWAS of hippocampal volume and additional data of a multicentre study in a large cohort of adolescents of European ancestry, we found supporting evidence for our results. Furthermore, allelic differences in rs4808611 and rs8170 were highly associated with differential mRNA expression in the cis-acting region. Associations with memory functioning indicate a possible functional importance of the identified risk variants. Our findings provide new insights into the genetic architecture of a brain structure closely linked to schizophrenia. In silico replication, mRNA expression and cognitive data provide additional support for the relevance of our findings. Identification of causal variants and their functional effects may unveil yet unknown players in the neurodevelopment and the pathogenesis of neuropsychiatric disorders.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Maternal exposure to di(2-ethylhexyl)phthalate (DEHP) promotes the transgenerational inheritance of adult-onset reproductive dysfunctions through the female germline in mice

Endocrine disruptors (EDs) are compounds known to promote transgenerational inheritance of adult-onset disease in subsequent generations after maternal exposure during fetal gonadal development. This study was designed to establish whether gestational and lactational exposure to the plasticizer di(2-ethylhexyl)phthalate (DEHP) at environmental doses promotes transgenerational effects on reproductive health in female offspring, as adults, over three generations in the mouse. Gestating F0 mouse dams were exposed to 0, 0.05, 5 mg/kg/day DEHP in the diet from gestational day 0.5 until the end of lactation. The incidence of adult-onset disease in reproductive function was recorded in F1, F2 and F3 female offspring. In adult F1 females, DEHP exposure induced reproductive adverse effects with: i) altered ovarian follicular dynamics with reduced primordial follicular reserve and a larger growing pre-antral follicle population, suggesting accelerated follicular recruitment; ii) reduced oocyte quality and embryonic developmental competence; iii) dysregulation of the expression profile of a panel of selected ovarian and pre-implantation embryonic genes. F2 and F3 female offspring displayed the same altered reproductive morphological phenotype and gene expression profiles as F1, thus showing transgenerational transmission of reproductive adverse effects along the female lineage. These findings indicate that in mice exposure to DEHP at doses relevant to human exposure during gonadal sex determination significantly perturbs the reproductive indices of female adult offspring and subsequent generations. Evidence of transgenerational transmission has important implications for the reproductive health and fertility of animals and humans, significantly increasing the potential biohazards of this toxicant. - Highlights: • Maternal exposure to DEHP transgenerationally affects female reproductive health. • DEHP reduced ovarian follicular reserve up to the third generation. • DEHP reduced oocyte and blastocyst developmental competence up to F3. • DEHP altered expression levels for key genes in ovary and blastocysts up to F4. • DEHP's adverse effects were observed at doses relevant for human exposure.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2014

Characterization of nitrogen use efficiency in sweet sorghum

Sweet sorghum (Sorghum bicolor L. Moench) has the potential to augment the increasing demand for alternative fuels and for the production of input efficient, environmentally friendly bioenergy crops. Nitrogen (N) and water availability are considered two of the major limiting factors in crop growth. Nitrogen fertilization accounts for about 40% of the total production cost in sorghum. In cereals, including sorghum, the nitrogen use efficiency (NUE) from fertilizer is approximately 33% of the amount applied. There is therefore extensive concern in relation to the N that is not used by the plant, which is lost by leaching of nitrate, denitrification from the soil, and loss of ammonia to the atmosphere, all of which can have deleterious environmental effects. To improve the potential of sweet sorghum as a leading and cost effective bioenergy crop, the enhancement of NUE must be addressed. To this end, we have identified a sorghum line (SanChi San) that displays about 25% increase in NUE over other sorghum lines. As such, the overarching goal of this project is to employ three complementary strategies to enhance the ability of sweet sorghum to become an efficient nitrogen user. To achieve the project goal, we will pursue the following specific objectives: Objective 1: Phenotypic characterization of SanChi San/Ck60 RILs under low and moderate N-availability including biochemical profiles, vegetative growth and seed yield Objective 2: Conduct quantitative trait loci (QTL) analysis and marker identification for nitrogen use efficiency (NUE) in a grain sorghum RIL population. Objective 3: Identify novel candidate genes for NUE using proteomic and gene expression profiling comparisons of high- and low-NUE RILs. Candidate genes will be brought into the pipeline for transgenic manipulation of NUE This project will apply the latest genomics resources to discover genes controlling NUE, one of the most complex and economically important traits in cereal crops. As a result of the completion of the proposed work, we will have: 1) identified novel alleles in wild sorghum germplasm that is useful to improve both cultivated grain and sweet sorghum; 2) been able to select individuals plants that exhibit high NUE within a breeding population on the basis of these markers; 3) acquired essential information necessary to examine the roles of GS and GOGAT, AlaT, along with impact of transcription factor Dof1, on N assimilation in sweet sorghum; and 4) The information learned will provide new opportunities for improving NUE in sorghum and other cereals.

09 BIOMASS FUELS↗

DOE OSTI2016

Nrf2-dependent protection against acute sodium arsenite toxicity in zebrafish

Transcription factor Nrf2 induces a number of detoxifying enzymes and antioxidant proteins to confer protection against the toxic effects of a diverse range of chemicals including inorganic arsenicals. Although a number of studies using cultured cells have demonstrated that Nrf2 has a cell-protective function against acute and high-dose arsenic toxicity, there is no clear in vivo evidence of this effect. In the present study, we genetically investigated the protective role of Nrf2 against acute sodium arsenite toxicity using the zebrafish Nrf2 mutant, nrf2a{sup fh318}. After treatment with 1 mM sodium arsenite, the survival of nrf2a{sup fh318} larvae was significantly shorter than that of wild-type siblings, suggesting that Nrf2 protected the zebrafish larvae against high-dose arsenite exposure. To understand the molecular basis of the Nrf2-dependent protection, we analyzed the gene expression profiles after arsenite exposure, and found that the genes involved in the antioxidative function (prdx1 and gclc), arsenic metabolism (gstp1) and xenobiotic elimination (abcc2) were induced in an Nrf2-dependent manner. Furthermore, pre-treatment with sulforaphane, a well-known Nrf2 activator improved the survival of zebrafish larvae after arsenic exposure. Based on these results, we concluded that Nrf2 plays a fundamental and conserved role in protection against acute sodium arsenite toxicity. - Highlights: • The role of Nrf2 under arsenite exposure was valuated using zebrafish. • Nrf2 mutant zebrafish was highly sensitive to acute arsenic toxicity. • Nrf2 induced anti-arsenic genes in response to arsenite. • Sulforaphane attenuated arsenic toxicity through Nrf2 activation. • Nrf2 system plays an important role in the defense against acute arsenic toxicity.

60 APPLIED LIFE SCIENCES↗