Genome-wide Transcriptome Analysis Research Articles

The Ameliorating Effects of n-3 Polyunsaturated Fatty Acids on Liver Steatosis Induced by a High-Fat Methionine Choline-Deficient Diet in Mice.

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is associated with abnormalities of liver lipid metabolism. On the contrary, a diet enriched with n-3 polyunsaturated fatty acids (n-3-PUFAs) has been reported to ameliorate the progression of NAFLD. The aim of our study was to investigate the impact of dietary n-3-PUFA enrichment on the development of NAFLD and liver lipidome. Mice were fed for 6 weeks either a high-fat methionine choline-deficient diet (MCD) or standard chow with or without n-3-PUFAs. Liver histology, serum biochemistry, detailed plasma and liver lipidomic analyses, and genome-wide transcriptome analysis were performed. Mice fed an MCD developed histopathological changes characteristic of NAFLD, and these changes were ameliorated with n-3-PUFAs. Simultaneously, n-3-PUFAs decreased serum triacylglycerol and cholesterol concentrations as well as ALT and AST activities. N-3-PUFAs decreased serum concentrations of saturated and monounsaturated free fatty acids (FAs), while increasing serum concentrations of long-chain PUFAs. Furthermore, in the liver, the MCD significantly increased the hepatic triacylglycerol content, while the administration of n-3-PUFAs eliminated this effect. Administration of n-3-PUFAs led to significant beneficial differences in gene expression within biosynthetic pathways of cholesterol, FAs, and pro-inflammatory cytokines (IL-1 and TNF-α). To conclude, n-3-PUFA supplementation appears to represent a promising nutraceutical approach for the restoration of abnormalities in liver lipid metabolism and the prevention and treatment of NAFLD.

Genome-wide investigation of the DMRT gene family sheds new insight into the regulation of sex differentiation in spotted knifejaw (Oplegnathus punctatus) with fusion chromosomes (Y)

The role of the DMRT family in male sex determination and differentiation is significant, but its regulatory role in spotted knifejaw with Y fusion chromosomes remains unclear. Through genome-wide scanning, transcriptome analysis, qPCR, FISH, and RNA interference (RNAi), we investigated the DMRT family and the dmrt1-based sex regulation network. Seven DMRTs were identified (DMRT1/2 (2a,2b)/6, DMRT4/5, DMRT3), and dmrt gene dispersion among chromosomes is possibly driven by three whole-genome duplications. Transcriptome analysis enriched genes were associated with sex regulation and constructed a network associated with dmrt1. qPCR and FISH results showed the expression dimorphism of sex-related genes in dmrt-related regulatory networks. RNAi experiments indicated a distinct sex regulation mode in spotted knifejaw. Dmrt1 knockdown upregulated male-related genes (sox9a, sox9b, dmrt1, amh, amhr2) and hsd11b2 expression, which is critical for androgen synthesis. Amhr2 is located on the heterozygous chromosome (Y) and is specifically localized in primary spermatocytes, and is extremely upregulated after dmrt1 knockdown which suggested besides the important role of dmrt1 in male differentiation, the amhr2 along with amhr2/amh system, also play important regulatory roles in maintaining high expression of the hsd11b2 and male differentiation. This study aims to further investigate sex regulatory mechanisms in species with fusion chromosomes.

Mouse Trophoblast Cells Have Attenuated Responses to TNF-α and IFN-γ and Can Avoid Synergic Cytotoxicity of the Two Cytokines.

TNF-α and IFN-γ are two inflammatory cytokines that play critical roles in immune responses, but they can also negatively affect cell proliferation and viability. In particular, the combination of the two cytokines (TNF-α/IFN-γ) synergistically causes cytotoxicity in many cell types. We recently reported that mouse embryonic stem cells (ESCs) isolated from the blastocyst stage embryo do not respond to TNF-α and have limited response to IFN-γ, thereby avoiding TNF-α/IFN-γ cytotoxicity. The current study expanded our investigation to mouse trophoblast stem cells (TSCs) and their differentiated trophoblasts (TSC-TBs), the precursors and the differentiated cells of the placenta, respectively. In this study, we report that the combination of TNF-α/IFN-γ does not show the cytotoxicity to TSCs and TSC-TBs that otherwise effectively kills fibroblasts, similar to ESCs. Although ESCs, TSCs, and TSC-TBs are dramatically different in their growth rate, morphology, and physiological functions, they nevertheless share a similarity in being able to avoid TNF-α/IFN-γ cytotoxicity. We propose that this unique immune property may serve as a protective mechanism that limits cytokine cytotoxicity in the blastocyst. With molecular and cellular approaches and genome-wide transcriptomic analysis, we have demonstrated that the attenuated NF-κB and STAT1 transcription activation is a limiting factor that restricts the effect of TNF-α/IFN-γ on TSCs and TSC-TBs.

Abstract A166: CRISPR-Cas9 genome editing in iPSCs for functional genetic screening

Abstract Induced pluripotent stem cells (iPSCs) are widely used for disease modeling, drug discovery, and cell therapy development. However, iPSCs are difficult to engineer with an efficient CRISPR/Cas9 system for functional genetic screens. We used WTC11 human iPS cells to generate functionally validated, lentiviral-transduced Cas9 lines, characterized by high gene-editing activity and sustained potential to differentiate. Flow cytometry was used to check for the expression of pluripotency markers Oct3/4, TRA1-60, and SSEA-4 in the iPS-Cas9 cells, and their actual pluripotency was then confirmed by testing the ability to differentiate into the three germ layers: ectoderm, mesoderm, and endoderm. Furthermore, genome-wide transcriptome analysis was performed to confirm the expression of germ layer-specific markers in the differentiated cells. Sustained Cas9 activity was confirmed in the differentiated cells. This study provides proof-of-principle that patient-derived iPSCs can be used to enable CRISPR/Cas9 functional genetic screening technology in reconstituted patient-specific tissues or disease models. Citation Format: Nadja Isachenko, Dongfang Hu, Alex Chenchik, Paul Diehl, Donato Tedesco. CRISPR-Cas9 genome editing in iPSCs for functional genetic screening [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A166.

The bZIP transcription factor ATF1 regulates blue light and oxidative stress responses in Trichoderma guizhouense.

In several filamentous fungi, incident light and environmental stress signaling share the mitogen-activated protein kinase (MAPK) HOG (SAK) pathway. It has been revealed that short-term illumination with blue light triggers the activation of the HOG pathway in Trichoderma spp. In this study, we demonstrate the crucial role of the basic leucine zipper transcription factor ATF1 in blue light responses and signaling downstream of the MAPK HOG1 in Trichoderma guizhouense. The lack of ATF1 severely impaired photoconidiation and delayed vegetative growth and conidial germination. Upon blue light or H2O2 stimuli, HOG1 interacted with ATF1 in the nucleus. Genome-wide transcriptome analyses revealed that 61.8% (509 out of 824) and 85.2% (702 out of 824) of blue light-regulated genes depended on ATF1 and HOG1, respectively, of which 58.4% (481 out of 824) were regulated by both of them. Our results also show that blue light promoted conidial germination and HOG1 and ATF1 played opposite roles in controlling conidial germination in the dark. Additionally, the lack of ATF1 led to reduced oxidative stress resistance, probably because of the downregulation of catalase-encoding genes. Overall, our results demonstrate that ATF1 is the downstream component of HOG1 and is responsible for blue light responses, conidial germination, vegetative growth, and oxidative stress resistance in T. guizhouense.

Abstract LB_A24: Baicalein suppresses tumorigenesis of the ER𝛼 positive of breast cancer cells by targeting multiple signaling pathways

Abstract The anti-tumor properties of baicalein, a flavonoid compound, have been extensively documented. However, the specific effects of baicalein on ER𝛼 positive breast cancer cell lines, namely T47-D and MCF-7, are less well-known. In this study, we conducted comprehensive in vitro experiments to elucidate the comparative impacts of baicalein on these two cell lines. Our results demonstrate that baicalein exerts robust anti-cancer effects, inducing morphological changes, reducing colony formation, and inhibiting cell proliferation in both T47-D and MCF-7 cells. These effects were more pronounced at higher concentrations. Additionally, baicalein treatment impeded the migratory capabilities of both cell lines, as shown by wound healing assays. Furthermore, our data show that baicalein triggers apoptosis, leading to an increase in early apoptotic, late apoptotic, and dead cells. This apoptotic process appears to be caspase 3/7-dependent. Interestingly, the pro-apoptotic effects were more potent in T47-D cells, accompanied by significant upregulation of pro-apoptotic genes and downregulation of anti-apoptotic markers. Baicalein treatment also induced oxidative stress and DNA damage, evidenced by an increase in reactive oxygen species (ROS) and phosphorylation of ATM and H2A.X. Importantly, baicalein inhibited the PI3K signaling pathway, a key mediator of cancer progression. Genome-wide transcriptomic analysis revealed significant changes in gene expression patterns, with baicalein treatment causing both upregulation and downregulation of several genes. Pathway analysis revealed that cell cycle and ferroptosis pathways were activated in both cell lines upon baicalein treatment. In conclusion, our findings highlight the potential of baicalein as a therapeutic agent against breast cancer, while also revealing nuanced differences in response between the T47-D and MCF-7 breast cancer cell lines. Citation Format: Besa Xhabija, Aidan J. McLaughlin, Ibiere Lovelyn Epelle, Tristan H. Nielson, Elisabeta Ujeniuc, Mariam I. Duhaini. Baicalein suppresses tumorigenesis of the ER𝛼 positive of breast cancer cells by targeting multiple signaling pathways [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A24.

A dual role of RBM42 in modulating splicing and translation of CDKN1A/p21 during DNA damage response

p53-mediated cell cycle arrest during DNA damage is dependent on the induction of p21 protein, encoded by the CDKN1A gene. p21 inhibits cyclin-dependent kinases required for cell cycle progression to guarantee accurate repair of DNA lesions. Hence, fine-tuning of p21 levels is crucial to preserve genomic stability. Currently, the multilayered regulation of p21 levels during DNA damage is not fully understood. Herein, we identify the human RNA binding motif protein 42 (RBM42) as a regulator of p21 levels during DNA damage. Genome-wide transcriptome and interactome analysis reveals that RBM42 alters the expression of p53-regulated genes during DNA damage. Specifically, we demonstrate that RBM42 facilitates CDKN1A splicing by counteracting the splicing inhibitory effect of RBM4 protein. Unexpectedly, we also show that RBM42, underpins translation of various splicing targets, including CDKN1A. Concordantly, transcriptome-wide mapping of RBM42-RNA interactions using eCLIP further substantiates the dual function of RBM42 in regulating splicing and translation of its target genes, including CDKN1A. Collectively, our data show that RBM42 couples splicing and translation machineries to fine-tune gene expression during DNA damage response.

Integrating genome-wide association and transcriptome analysis to provide molecular insights into heterophylly and eco-adaptability in woody plants.

Heterophylly is regard as an important adaptive mechanism in response to different environments within plants. However, the genetic mechanisms responsible for heterophylly in woody plants are still poorly understood. Herein, the divergence of heterophyllous leaves was investigated at morphogenesis and using microdissection and physiological indexes in paper mulberry, and the genetic basis of heterophylly was further revealed combined with genome-wide association study (GWAS), transcriptome analysis and weighted gene coexpression network analysis (WGCNA). Our results revealed that the flavonoid content and antioxidant activity increased gradually from the entire leaf to the palmatisect leaf, while the hormone content and net photosynthetic rate decreased. Through GWAS and transcriptome analysis, a total of 98 candidate genes and 2338 differentially expressed genes associated with heterophylly were identified. Importantly, we uncovered critical variations in the candidate genes Bp07g0981 (WOX) and Bp07g0920 (HHO), along with significant differences in haplotypes and expression levels among heterophyllous leaves. Our results also suggested that the genes involved in hormone signaling pathways, antioxidant activity, and flavonoid metabolism might be closely related to the heterophylly of paper mulberry, which could account for the physiological data. Indeed, CR-wox mutant lines showed significant changes in leaf phenotypes, and differential expression profile analysis also highlighted the expression of genes related to phytohormones and transcription factors. Together, the genetic variations and candidate genes detected in this study provide novel insights into the genetic mechanism of heterophylly, and would improve the understanding of eco-adaptability in heterophyllous woody plants.

Whole-genome microRNA sequencing analysis in patients with pulmonary hypertension.

Pulmonary hypertension (PH) is a pathological disorder with multiple clinical manifestations that lead to cardiovascular and respiratory diseases in most patients. Recent studies have revealed that microRNAs (miRNAs) play important roles as upstream signaling molecules in several diseases, including PH. However, miRNAs that can be used as diagnostic or prognostic biomarkers for PH have not been identified. Thus, in this study, peripheral blood samples obtained from patients with PH and healthy individuals were subjected to genome-wide miRNA sequencing and transcriptome analysis. We screened 136 differentially expressed miRNAs in patients with PH and verified that four differentially expressed miRNAs, namely, hsa-miR-1304-3p, hsa-miR-490-3p, hsa-miR-11400, and hsa-miR-31-5p, could be used as clinical diagnostic biomarkers for pulmonary arterial hypertension. Our findings provide a basis for further in-depth investigations of the specific mechanisms of miRNAs in PH.

Genome-wide transcriptome analysis of toxigenic Fusarium verticillioides in response to variation of temperature and water activity on maize kernels

Fusarium verticillioides is one of the important mycotoxigenic pathogens of maize since it causes severe yield losses and produces fumonisins (FBs) to threaten human and animal health. Previous studies showed that temperature and water activity (aw) are two pivotal environmental factors affecting F. verticillioides growth and FBs production during maize storage. However, the genome-wide transcriptome analysis of differentially expressed genes (DEGs) in F. verticillioides under the stress combinations of temperature and aw has not been studied in detail. In this study, DEGs of F. verticillioides and their related regulatory pathways were analyzed in response to the stress of temperature and aw combinations using RNA-Seq. The results showed that the optimal growth conditions for F. verticillioides were 0.98 aw and 25 °C, whereas the highest per-unit yield of the fumonisin B1 (FB1) was observed at 0.98 aw and 15 °C. The RNA-seq analysis showed that 9648 DEGs were affected by temperature regardless of aw levels, whereas only 218 DEGs were affected by aw regardless of temperature variations. Gene Ontology (GO) analysis revealed that a decrease in temperature at both aw levels led to a significant upregulation of genes associated with 24 biological processes, while three biological processes were downregulated. Furthermore, when aw was decreased at both temperatures, seven biological processes were significantly upregulated and four were downregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that the genes, whose expression was upregulated when the temperature decreased, were predominantly associated with the proteasome pathway, whereas the genes, whose expression was downregulated when the aw decreased, were mainly linked to amino acid metabolism. For the FB1, except for the FUM15 gene, the other 15 biosynthetic-related genes were highly expressed at 0.98 aw and 15 °C. In addition, the expression pattern analysis of other biosynthetic genes involved in secondary metabolite production and regulation of fumonisins production was conducted to explore how this fungus responds to the stress combinations of temperature and aw. Overall, this study primarily examines the impact of temperature and aw on the growth of F. verticillioides and its production of FB1 using transcriptome data. The findings presented here have the potential to contribute to the development of novel strategies for managing fungal diseases and offer valuable insights for preventing fumonisin contamination in food and feed storage.

MBD4 loss results in global reactivation of promoters and retroelements with low methylated CpG density

BackgroundInherited defects in the base-excision repair gene MBD4 predispose individuals to adenomatous polyposis and colorectal cancer, which is characterized by an accumulation of C > T transitions resulting from spontaneous deamination of 5’-methylcytosine.MethodsHere, we have investigated the potential role of MBD4 in regulating DNA methylation levels using genome-wide transcriptome and methylome analyses. Additionally, we have elucidated its function through a series of in vitro experiments.ResultsHere we show that the protein MBD4 is required for DNA methylation maintenance and G/T mismatch repair. Transcriptome and methylome analyses reveal a genome-wide hypomethylation of promoters, gene bodies and repetitive elements in the absence of MBD4 in vivo. Methylation mark loss is accompanied by a broad transcriptional derepression phenotype affecting promoters and retroelements with low methylated CpG density. MBD4 in vivo forms a complex with the mismatch repair proteins (MMR), which exhibits high bi-functional glycosylase/AP-lyase endonuclease specific activity towards methylated DNA substrates containing a G/T mismatch. Experiments using recombinant proteins reveal that the association of MBD4 with the MMR protein MLH1 is required for this activity.ConclusionsOur data identify MBD4 as an enzyme specifically designed to repair deaminated 5-methylcytosines and underscores its critical role in safeguarding against methylation damage. Furthermore, it illustrates how MBD4 functions in normal and pathological conditions.

Genome-wide prediction and transcriptome analysis of sugar transporters in four ascomycete fungi

The import of plant-derived small sugars by sugar transporters (STs) has received increasing interest due to its important biological role and great industrial potential. STs are important targets of genetic engineering to improve fungal plant biomass conversion. Comparatively analysis of the genome-wide prevalence and transcriptomics of STs was performed in four filamentous fungi: Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens and Trichoderma reesei. Using phylogenetic analysis and literature mining, their predicted STs were divided into ten subfamilies with putative sugar specificities assigned. In addition, transcriptome analysis revealed complex expression profiles among different STs subfamilies and fungal species, indicating a sophisticated transcriptome regulation and functional diversity of fungal STs. Several STs showed strong co-expression with other genes involved in sugar utilization, encoding CAZymes and sugar catabolic enzymes. This study provides new insights into the diversity of STs at the genomic/transcriptomic level, facilitating their biochemical characterization and metabolic engineering.

TMIC-75. SPATIALLY-RESOLVED TRANSCRIPTOME ANALYSIS OF BRAIN METASTATIC BREAST CANCER REVEAL KEY MEDIATORS OF BRAIN-TROPIC METASTATIC POTENTIAL

Abstract BACKGROUND In women with metastatic breast cancer, 30% (HER2+) to 50% (triple negative) develop brain metastases. Breast-to-brain metastasis carries a poor median survival of only 10 months and is accompanied by severe neurologic decline. Halting the progression of breast cancer brain metastases requires modulation of the tumor microenvironment (TME), yet little is known about how the primary breast TME affects brain tropism, or how, once there, metastatic cancer cells interact with normal brain cells (e.g., neurons, astrocytes, microglia). METHODS We constructed a large (n = 253 cores), diverse, clinically-annotated tissue microarray (TMA) of breast cancer brain metastases with surrounding brain (n = 127), associated primary breast tumors (n = 67, with 30 matched), normal brain (n = 23), and staining control (n = 18) tissue samples, from patients (n = 140) treated at Stanford Hospital between 2008 – 2018. We then leveraged a combination of genome-wide transcriptome analysis and high resolution spatial imaging (using GeoMx Digital Spatial Profiling) to interrogate the distinct, spatially-driven gene signatures of tumor, immune, and normal brain cells within the breast cancer brain metastasis TME. RESULTS We revealed novel gene regulatory programs that are associated with metastatic potential from the primary breast to the brain. We also identified not only diverging features of the breast and brain TMEs, but equivalent features which may contribute to preferential honing and outgrowth of metastatic breast cells in the brain microniche. Additionally, we uncovered significant cellular crosstalk between tumor, immune, and brain cell populations, including putative cell-cell interactions and intercellular signaling pathways. CONCLUSION These results served as a starting point for identifying key tumor-immune-brain interactions responsible for priming immune and brain tolerance of malignant cells, allowing outgrowth and progression of breast cancer brain metastases. Further investigation focused on tumor-brain crosstalk, specifically tumor-microglia interactions, will provide promising targets for therapeutic advancements.

Induction of the CtsR regulon improves Xylanase production in Bacillus subtilis

BackgroundThe bacterium Bacillus subtilis is extensively used for the commercial production of enzymes due to its efficient protein secretion capacity. However, the efficiency of secretion varies greatly between enzymes, and despite many years of research, optimization of enzyme production is still largely a matter of trial-and-error. Genome-wide transcriptome analysis seems a useful tool to identify relevant secretion bottlenecks, yet to this day, only a limited number of transcriptome studies have been published that focus on enzyme secretion in B. subtilis. Here, we examined the effect of high-level expression of the commercially important enzyme endo-1,4-β-xylanase XynA on the B. subtilis transcriptome using RNA-seq.ResultsUsing the novel gene-set analysis tool GINtool, we found a reduced activity of the CtsR regulon when XynA was overproduced. This regulon comprises several protein chaperone genes, including clpC, clpE and clpX, and is controlled by transcriptional repression. CtsR levels are directly controlled by regulated proteolysis, involving ClpC and its cognate protease ClpP. When we abolished this negative feedback, by inactivating the repressor CtsR, the XynA production increased by 25%.ConclusionsOverproduction of enzymes can reduce the pool of Clp protein chaperones in B. subtilis, presumably due to negative feedback regulation. Breaking this feedback can improve enzyme production yields. Considering the conserved nature of Clp chaperones and their regulation, this method might benefit high-yield enzyme production in other organisms.

Untying the regulatory roles of miRNAs in CuO-NPs stress response mechanism in maize: A genome-wide sRNA transcriptome analysis

MicroRNAs (miRNAs), a group of tiny non-coding RNAs play pivotal role in plant responses to environmental stress. The present small RNA transcriptome study aims to untie the role of miRNAs in CuO-NPs stress adaptation in maize seedlings. Restricted seedling growth, enhanced ROS generation and higher membrane damage were recorded under CuO-NPs [<50 nm, 8 mM] treatment. Deep sequencing reveals 7 up- and 36 down-regulated known miRNAs from CuO-NPs challenged leaves. Gene ontology study demonstrates involvement of CuO-NPs responsive miRNAs in a variety of biological processes including plant growth (miR159a, miR159b), redox homeostasis (miR156e, miR395a), detoxification of heavy metals (miR156e, miR827), signal transduction (miR156e, miR156d), and cell signalling (miR167b-3p, miR393a). Enhanced transcriptional abundance of ABC transporter G family member 41 isoform X2 and HM-associated isoprenylated plant protein 45 isoform X1 might be involved in sequestration and detoxification of excess Cu, essential for metal homeostasis in maize. The miR528-5p mediated up-regulation of superoxide dismutase does not give much protection against CuO-NPs induced oxidative stress damages as evident after histochemical staining with NBT. Moreover, CuO-NPs stress mediated down regulation of miR396 could be an underlying cause of the restricted seedling growth. Taken together, our findings provide insights into the miRNA-guided stress regulatory networks involved in plant's adaptive responses to CuO-NPs stress.

Genome-wide association study and transcriptome analysis reveal candidate genes for off-season flowering in blueberry

Off-season flowering in daylength-sensitive plants is an intriguing characteristic that represents an opportunity to expand crop's production timing and regions, and potentially speed up breeding. Southern Highbush Blueberry (SHB) is classified as a short-day plant, with a blooming period in the northern hemisphere ranging from late winter through spring. In Florida, some blueberry genotypes have consistently bloomed during the fall season, suggesting that flower bud initiation occurred during the summer. Such a characteristic indicates a facultative response to daylength or day-neutrality capacity, leading to the off-season flowering observed. To understand the genetic basis of off-season flowering in SHB, we performed a genome-wide association study (GWAS), followed by transcriptomic analysis of contrasting genotypes. Among the 536 genotypes evaluated, 14 % bloomed during the fall, of which seven presented consistently high scores for off-season flowering capacity. The GWAS revealed 12 single nucleotide polymorphisms (SNPs) significantly associated with the off-season flowering trait, which together accounted for 17 % of the phenotypic variance. A gene homologous to an RNA helicase and reported to modulate florescence formation and flowering time in other plant species, was identified within a genomic region associated with the off-season flowering. This gene was up-regulated in a fall-flowering blueberry genotype. Additionally, we detected down-regulation of flowering repressors and up-regulation of MADS-box transcription factors known to be involved in the fate of floral meristems. Our findings provide insights into the genetic and molecular basis of this complex trait in blueberry. These results can be useful for marker-assisted selection and further functional gene validation to accelerate the development of blueberry varieties suitable for off-season production.

Genome-wide transcriptomic and functional analyses provide new insights into the response of spring barley to drought stress.

Drought is a major abiotic stress that impairs the physiology and development of plants, ultimately leading to crop yield losses. Drought tolerance is a complex quantitative trait influenced by multiple genes and metabolic pathways. However, molecular intricacies and subsequent morphological and physiological changes in response to drought stress remain elusive. Herein, we combined morpho-physiological and comparative RNA-sequencing analyses to identify core drought-induced marker genes and regulatory networks in the barley cultivar 'Giza134'. Based on field trials, drought-induced declines occurred in crop growth rate, relative water content, leaf area duration, flag leaf area, concentration of chlorophyll (Chl) a, b and a + b, net photosynthesis, and yield components. In contrast, the Chl a/b ratio, stoma resistance, and proline concentration increased significantly. RNA-sequence analysis identified a total of 2462 differentially expressed genes (DEGs), of which 1555 were up-regulated and 907 were down-regulated in response to water-deficit stress (WD). Comparative transcriptomics analysis highlighted three unique metabolic pathways (carbohydrate metabolism, iron ion binding, and oxidoreductase activity) as containing genes differentially expressed that could mitigate water stress. Our results identified several drought-induced marker genes belonging to diverse physiochemical functions like chlorophyll concentration, photosynthesis, light harvesting, gibberellin biosynthetic, iron homeostasis as well as Cis-regulatory elements. These candidate genes can be utilized to identify gene-associated markers to develop drought-resilient barley cultivars over a short period of time. Our results provide new insights into the understanding of water stress response mechanisms in barley.

Impact of PFAS exposure on immune-mediated diseases and immune transcriptome in Czech adults

Abstract Background Per- and polyfluoroalkyl substances (PFASs) are emerging environmental contaminants with multiple hazardous properties including immunomodulation potency. We aimed to investigate 1) the association between PFAS exposure and immune-mediated diseases (IMDs) and 2) the expression of genes associated with PFAS exposure in a population of Czech adults. Methods This study involved 309 adults from the Czech CELSPAC:YA cohort. The associations of serum concentrations of 9 PFAS with 9 IMDs were assessed by logistic regression. Furthermore, Bayesian kernel machine regression (BKMR) was used to estimate the effect of the PFAS mixture on IMDs. In cases of a statistically significant interaction of PFASs and sex, sex-stratified analyses were performed. To identify transcriptomic profiles, isolated RNA from peripheral mononuclear blood cells was sequenced (QuantSeq) and analysed. Significantly expressed genes associated with multiple PFASs (at least with 4 PFASs) were employed for enrichment analysis by Pathway Studio. All analyses were adjusted for relevant confounders (i.e., sex, age, BMI, smoking, education, and family history of IMDs). Results PFOA, PFOS, and PFUnDA were negatively associated with allergies. Similarly, BKMR modeling showed a negative tendency in the overall effect of PFAS mixture on IMDs. Results of the stratified analysis suggest sex to be an effect modifier in the association of PFOS and atopic eczema. Regarding transcriptomic analysis, 166 statistically significant genes were associated with multiple PFAS exposure. Enrichment analysis of these genes showed involvement of B cell signaling, specifically mechanisms involved in the production of antibody-secreting plasma cells. Conclusions Our results contribute to the body of literature that observes the immunomodulatory effect of PFAS exposure on the level of disease prevalence and further brought new information about underlying deregulatory mechanisms. Key messages • Our study showed an immunosuppressive effect of PFAS exposure on the level of prevalence of immune-mediated diseases, both for PFASs individually and as a mixture. • Genome-wide transcriptomic analysis revealed the involvement of plasma cells as a potential key event of PFAS-induced immunomodulation.

RBFOX2 regulated EYA3 isoforms partner with SIX4 or ZBTB1 to control transcription during myogenesis

Alternative splicing is a prevalent gene-regulatory mechanism, with over 95% of multi-exon human genes estimated to be alternatively spliced. Here, we describe a tissue-specific, developmentally regulated, highly conserved, and disease-associated alternative splicing event in exon 7 of the eyes absent homolog 3 (Eya3) gene. We discovered that EYA3 expression is vital to the proliferation and differentiation of myoblasts. Genome-wide transcriptomic analysis and mass spectrometry-based proteomic studies identified SIX homeobox 4 (SIX4) and zinc finger and BTB-domain containing 1 (ZBTB1), as major transcription factors that interact with EYA3 to dictate gene expression. EYA3 isoforms differentially regulate transcription, indicating that splicing aids in temporal control of gene expression during muscle cell differentiation. Finally, we identified RNA-binding fox-1 homolog 2 (RBFOX2) as the main regulator of EYA3 splicing. Together, our findings illustrate the interplay between alternative splicing and transcription during myogenesis.

FRI139 Identification Of Robust Aldosterone-Producing Adenomas-related Genes Using Adrenal Genome-wide Transcriptome Analysis

Abstract Disclosure: S. Lee: None. T. Lee: None. G. Yoo: None. J. Lim: None. Primary aldosteronism (PA), the most common cause of secondary hypertension, is caused by aldosterone overproduction in one or two adrenal glands. It carries a higher risk of cardiovascular disease and mortality compared to essential hypertension. In an effort to identify the underlying molecular mechanisms regarding aldosterone secretion and cell proliferation in aldosterone-producing adenomas (APA), genome-wide transcriptome analyses have been conducted; however, there are still some limitations that need to be addressed since a relatively small number of samples were analyzed at a single center in most studies. In this study, we performed integrative and systematic analysis using adrenal transcriptome datasets to verify candidate genes robustly associated with APA and obtain more generalized signatures involved in the pathophysiology of PA. Three mRNA datasets for adrenal tissues (GSE8514, GSE60042, and GSE64957) were obtained from the Gene Expression Omnibus Database. For the first two datasets, normal adrenal samples from patients with renal cell carcinoma and adrenal tissue samples adjacent to APA were included as controls, respectively. In GSE64957, zona glomerulosa (ZG) and zona fasciculata (ZF) samples were analyzed separately in the current study (GSE64957ZG and GSE64957ZF). Differentially expressed gene (DEG) investigation and comparative analysis were conducted using the limma method. The selection criteria for APA-related genes were as follows: those that are significantly dysregulated in more than three of the four datasets and show a consistent trend of up- or down-regulation in four datasets. 5,375 genes were present in all transcriptome datasets; for the GSE64957ZG, 57 DEGs were identified in the APA group, among which 14, 7, and 37 genes were also dysregulated in the APA group of GSE64957ZF, GSE8514, and GSE60042, respectively. All comparisons among four lists of DEGs exhibited significant enrichment results. The highest degree of overlap was shown in the DEG pair of GSE64957ZG-GSE60042. As a result, approximately 20 genes (12 upregulated and 12 downregulated DEGs), including CYP11B2, PCP4, and ATP2A3, were selected as the final APA-related genes. This study demonstrated several candidate genes that are strongly relevant to APA by examining whole genome disease-related signatures. Besides CYP11B2, it seems that alteration in PCP4 and ATP2A3 genes would be much more involved in the pathogenesis of PA, which warrants further investigation along with validation for the role of the remaining genes. Presentation: Friday, June 16, 2023