Protein Transcription Factors Research Articles

A computational analysis of the molecular mechanisms underlying the effects of ibuprofen and dibutyl phthalate on gene expression in fish

The impact of emerging pollutants such as ibuprofen and dibutyl phthalate on aquatic species is a growing concern and the need for proper assessment and evaluation of these toxicants is imperative. The objective of this study was to examine the toxicogenomic impacts of ibuprofen and dibutyl phthalate on Clarias gariepinus, a widely distributed African catfish species. Results showed that exposure to the test compounds caused significant changes in gene expression, including upregulation of growth hormone, interleukin, melatonin receptors, 17β-Hydroxysteroid Dehydrogenase, heat shock protein, doublesex, and mab-3 related transcription factor. On the other hand, expression of forkhead Box Protein L2 and cytochrome P450 was downregulated, revealing a potential to induce female to male sex reversal. The binding affinities and hydrophobic interactions of the test compounds with the reference genes were also studied, showing that ibuprofen had the lowest binding energy and the highest affinity for the docked genes. Both compounds revealed a mutual molecular interaction with amino acids residues within the catalytic cavity of the docked genes. These results provide new insights into the toxic effects of ibuprofen and dibutyl phthalate on Clarias gariepinus, contributing to a better understanding of the environmental impact of these pollutants.

Apple Glycosyltransferase MdUGT73AR4 Glycosylates ABA to Regulate Stomatal Movement Involved in Drought Stress.

Abscisic acid (ABA) is a drought-stress-responsive hormone that plays an important role in the stomatal activity of plant leaves. Currently, ABA glycosides have been identified in apples, but their glycosyltransferases for glycosylation modification of ABA are still unidentified. In this study, the mRNA expression of glycosyltransferase gene MdUGT73AR4 was significantly up-regulated in mature apple leaves which were treated in drought stress by Real-Time PCR. It was hypothesised that MdUGT73AR4 might play an important role in drought stress. In order to further characterise the glycosylation modification substrate of glycosyltransferase MdUGT73AR4, we demonstrated through in vitro and in vivo functional validation that MdUGT73AR4 can glycosylate ABA. Moreover, the overexpression lines of MdUGT73AR4 significantly enhance its drought stress resistance function. We also found that the adversity stress transcription factor AREB1B might be an upstream transcription factor of MdUGT73AR4 by bioinformatics, EMSA, and ChIP experiments. In conclusion, this study found that the adversity stress transcription factor AREB1B was significantly up-regulated at the onset of drought stress, which in turn positively regulated the downstream glycosyltransferase MdUGT73AR4, causing it to modify ABA by mass glycosylation and promoting the ABA synthesis pathway, resulting in the accumulation of ABA content, and displaying a stress-resistant phenotype.

Transcription Factor ETV4 Activates AURKA to Promote PD-L1 Expression and Mediate Immune Escape in Lung Adenocarcinoma

Introduction: The occurrence and progression of lung adenocarcinoma (LUAD) impair T-cell immune responses, causing immune escape and subsequently affecting the efficacy of immunotherapy in patients. Aurora kinase A (AURKA) is upregulated in varying cancers, but its role in LUAD immune escape is elusive. This work attempted to explore molecular mechanisms of AURKA regulation in LUAD immune escape. Methods: Through bioinformatics analysis, AURKA level in LUAD was evaluated, and potential upstream transcription factors of AURKA were predicted using hTFtarget. ETS variant transcription factor 4 (ETV4) expression in LUAD was analyzed through The Cancer Genome Atlas. Pearson’s correlation analysis was then utilized to test the correlation between AURKA and ETV4. Interaction and binding between AURKA and ETV4 were validated through dual-luciferase assay and chromatin immunoprecipitation. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) tested relative mRNA expression of AURKA and ETV4 in LUAD cells, cell counting kit-8 assayed cell viability, and Western blot analysis was conducted to determine the protein level of programmed death-ligand 1 (PD-L1). Coculture of LUAD cells with activated CD8+ T cells was carried out, and an LDH assay was used to assess the cytotoxicity of CD8+ T cells against LUAD cells. Interferon-γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) levels in the coculture system were assessed by enzyme-linked immunosorbent assay (ELISA). Western blot assessed protein levels of JAK2, p-JAK2, STAT3, and p-STAT3. Results: Compared to normal tissues, AURKA and ETV4 were upregulated in tumor tissues, and AURKA presented a negative association with CD8+ T-cell immune infiltration but a positive association with PD-L1. qRT-PCR unveiled significantly upregulated mRNA of AURKA and ETV4 in LUAD cells compared to normal lung epithelial cells. Knockdown of AURKA significantly decreased cell viability and PD-L1 protein level in LUAD cells, enhanced cytotoxicity of CD8+ T cells against LUAD cells and IFN-γ, IL-2, and TNF-α expression, while overexpression of AURKA yielded opposite results. Furthermore, the knockdown of ETV4 could reverse the oncogenic characteristics of cells caused by AURKA overexpression. Conclusion: Our study illustrated that ETV4/AURKA axis promoted PD-L1 expression, suppressed CD8+ T-cell activity, and mediated immune escape in LUAD by regulating the JAK2/STAT3 signaling pathway.

Neuron mapping in the Molly fish optic tectum: An emphasis on the adult neurogenesis process.

Teleost fish exhibit the most pronounced and widespread adult neurogenesis. Recently, functional development and the fate of newborn neurons have been reported in the optic tectum (OT) of fish. To determine the role of neurogenesis in the OT, this study used histological, immunohistochemical, and electron microscopic investigations on 18 adult Molly fish specimens (Poecilia sphenops). The OT of the Molly fish was a bilateral lobed structure located in the dorsal part of the mesencephalon. It exhibited a laminated structure made up of alternating fiber and cellular layers, which were organized into six main layers. The stratum opticum (SO) was supplied by optic nerve fibers, in which the neuropil was the main component. Radial bipolar neurons that possessed bundles of microtubules were observed in the stratum fibrosum et griseum superficiale (SFGS). Furthermore, oligodendrocytes with their processes wrapped around the nerve fibers could be observed. The stratum album centrale (SAC) consisted mainly of the axons of the stratum griseum centrale (SGC) and the large tectal, pyriform, and horizontal neurons. The neuronal cells of the SO and large tectal cells of the SAC expressed autophagy-related protein-5 (APG5). Interleukin-1β (IL-1β) was expressed in both neurons and glia cells of SGC. Additionally, inducible nitric oxide synthase (iNOS) was expressed in the neuropil of the SAC synaptic layer and granule cells of the stratum periventriculare (SPV). Also, transforming growth factor beta (TGF-β), SRY-box transcription factor 9 (SOX9), and myostatin were clearly expressed in the proliferative neurons. In all strata, S100 protein and Oligodendrocyte Lineage Transcription Factor 2 (Olig2) were expressed by microglia, oligodendrocytes, and astrocytes. In conclusion, it was possible to identify different varieties of neurons in the optic tectum, each with a distinct role. The existence of astrocytes, proliferative neurons, and stem cells highlights the regenerative capacity of OT. RESEARCH HIGHLIGHTS: The OT of the Molly fish exhibited a laminated structure made up of alternating fiber and cellular layers, which were organized into six main layers. Radial bipolar neurons that possessed bundles of microtubules were observed in the stratum fibrosum et griseum superficiale (SFGS). The stratum album central (SAC) consisted mainly of the axons of the stratum griseum centrale (SGC) and the large tectal, pyriform, and horizontal neurons. Inducible nitric oxide synthase (iNOS) was expressed in the neuropil of the SAC synaptic layer and granule cells of the stratum periventricular (SPV). Also, transforming growth factor beta (TGF-β), SRY-box transcription factor 9 (SOX9), and myostatin were clearly expressed in the proliferative neurons. The existence of astrocytes, proliferative neurons, and stem cells highlights the regenerative capacity of OT.

RUNX1-induced upregulation of PTGS2 enhances cell growth, migration and invasion in colorectal cancer cells

Colorectal cancer (CRC) arises via the progressive accumulation of dysregulation in key genes including oncogenes and tumor-suppressor genes. Prostaglandin-endoperoxide synthase 2 (PTGS2, also called COX2) acts as an oncogenic driver in CRC. Here, we explored the upstream transcription factors (TFs) responsible for elevating PTGS2 expression in CRC cells. The results showed that PTGS2 silencing repressed cell growth, migration and invasion in HCT116 and SW480 CRC cells. The two fragments (499–981 bp) and (1053–1434 bp) were confirmed as the core TF binding profiles of the PTGS2 promoter. PTGS2 expression positively correlated with RUNX1 level in colon adenocarcinoma (COAD) samples using the TCGA-COAD dataset. Furthermore, RUNX1 acted as a positive regulator of PTGS2 expression by promoting transcriptional activation of the PTGS2 promoter via the 1086–1096 bp binding motif. In conclusion, our study demonstrates that PTGS2 upregulation induced by the TF RUNX1 promotes CRC cell growth, migration and invasion, providing an increased rationale for the use of PTGS2 inhibitors in CRC prevention and treatment.

Differential expressions of endoplasmic reticulum stress-associated genes in aortic dissection and their correlation with immune cell infiltration

To explore differentially expressed endoplasmic reticulum stress-associated genes (ERSAGs) in aortic dissection (AD) and their correlations with immune cell infiltration to identify new therapeutic targets for AD. Two AD mRNA expression datasets (GSE190635 and GSE98770) were downloaded from GEO database for analysis of differentially expressed genes between the aorta of AD patients and normal aorta using R software. ERSAGs dataset was downloaded from GeneCards website, and GeneMANIA database was used to analyze the protein-protein interaction network of the differentially expressed ERSAGs and the proteins interacting with these genes. Based on GSE98770 dataset we analyzed the distributions of 22 immune cells within the aortic wall of AD patients using CIBERSORT package of R software. Surgical aortic wall specimens were obtained from 10 AD patients and 10 non-AD patients for detecting AGER mRNA expression using qRTPCR, and the upstream transcriptional factors, miRNAs, and chemicals targeting AGER were analyzed using the TRRUST database and NetworkAnalyst database. Bioinformatic analysis suggested significant differential expression of AGER in AD, which interacted with 20 proteins involved in pattern recognition receptor signaling pathway, positive regulation of DNA-binding transcription factor activity, myeloid leukocyte migration, leukocyte migration, and regulation of the I-κB kinase/NF-κB signaling. In AD, AGER expression level was positively correlated with Treg cell abundance (r=0.59, P < 0.05). The results of qRT-PCR demonstrated significantly lower expression of AGER mRNA in AD than in non-AD patients (1.00±0.30 vs 1.76±0.68, P < 0.05). ROC curve analysis showed that at the cut-off value of 1.335, AGER had an AUC of 0.86 (95% CI: 0.67-1.00, P= 0.0073) for predicting AD. Three transcriptional factors, 3 miRNAs, and 27 chemicals were predicted in the AGER regulatory network. AGER is lowly expressed in the aorta of AD patients and may influence the occurrence of AD through Treg cells.

Implications of GCLC in prognosis and immunity of lung adenocarcinoma and multi-omics regulation mechanisms

BackgroundFerroptosis is an iron-dependent type of regulated cell death, and has been implicated in lung adenocarcinoma (LUAD). Evidence has proved the key role of glutamate-cysteine ligase catalytic subunit (GCLC) in ferroptosis, but its role in LUAD remains unclear. Herein, we explored the implications of GCLC and relevant genes in LUAD prognosis and immunity as well as underlying molecular mechanisms.MethodsThis work gathered mRNA, miRNA, DNA methylation, somatic mutation and copy-number variation data from TCGA-LUAD. WGCNA was utilized for selecting GCLC-relevant genes, and a GCLC-relevant prognostic signature was built by uni- and multivariate-cox regression analyses. Immune compositions were estimated via CIBERSORT, and two immunotherapy cohorts of solid tumors were analyzed. Multi-omics regulatory mechanisms were finally assessed.ResultsOur results showed that GCLC was overexpressed in LUAD, and potentially resulted in undesirable survival. A prognostic model was generated, which owned accurate and independent performance in prognostication. GCLC, and relevant genes were notably connected with immune compositions and immune checkpoints. High GCLC expression was linked with better responses to anti-PD-L1 and anti-CTLA-4 treatment. Their possible DNA methylation sites were inferred, e.g., hypomethylation in cg19740353 might contribute to GCLC up-regulation. Frequent genetic mutations also affected their expression. Upstream transcription factors (E2F1/3/4, etc.), post-transcriptional regulation of miRNAs (hsa-mir-30c-1, etc.), lncRNAs (C8orf34-AS1, etc.), and IGF2BP1-mediated m6A modification were identified. It was also found NOP58-mediated SUMOylation post-translational modification.ConclusionsTogether, we show that GCLC and relevant genes exert crucial roles in LUAD prognosis and immunity, and their expression can be controlled by complex multi-omics mechanisms.

Construction of ferroptosis-related prediction model for pathogenesis, diagnosis and treatment of ruptured abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a dangerous cardiovascular disease, which often brings great psychological burden and economic pressure to patients. If AAA rupture occurs, it is a serious threat to patients' lives. Therefore, it is of clinical value to actively explore the pathogenesis of ruptured AAA and prevent its occurrence. Ferroptosis is a new type of cell death dependent on lipid peroxidation, which plays an important role in many cardiovascular diseases. In this study, we used online data and analysis of ferroptosis-related genes to uncover the formation of ruptured AAA and potential therapeutic targets. We obtained ferroptosis-related differentially expressed genes (Fe-DEGs) from GSE98278 dataset and 259 known ferroptosis-related genes from FerrDb website. Enrichment analysis of differentially expressed genes (DEGs) was performed by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG). Receiver Operating characteristic (ROC) curve was employed to evaluate the diagnostic abilities of Fe-DEGs. Transcription factors and miRNAs of Fe-DEGs were identified through PASTAA and miRDB, miRWalk, TargetScan respectively. Single-sample gene set enrichment analysis (ssGSEA) was used to observe immune infiltration between the stable group and the rupture group. DGIdb database was performed to find potential targeted drugs of DEGs. GO and KEGG enrichment analysis found that DEGs mainly enriched in "cellular divalent inorganic cation homeostasis," "cellular zinc ion homeostasis," "divalent inorganic cation homeostasis," "Mineral absorption," "Cytokine - cytokine receptor interaction," "Coronavirus disease - COVID-19." Two up-regulated Fe-DEGs MT1G and DDIT4 were found to further analysis. Both single and combined applications of MT1G and DDIT4 showed good diagnostic efficacy (AUC = 0.8254, 0.8548, 0.8577, respectively). Transcription factors STAT1 and PU1 of MT1G and ARNT and MAX of DDIT4 were identified. Meanwhile, has_miR-548p-MT1G pairs, has_miR-53-3p/has_miR-181b-5p/ has_miR-664a-3p-DDIT4 pairs were found. B cells, NK cells, Th2 cells were high expression in the rupture group compared with the stable group, while DCs, Th1 cells were low expression in the rupture group. Targeted drugs against immunity, GEMCITABINE and INDOMETHACIN were discovered. We preliminarily explored the clinical significance of Fe-DEGs MT1G and DDIT4 in the diagnosis of ruptured AAA, and proposed possible upstream regulatory transcription factors and miRNAs. In addition, we also analyzed the immune infiltration of stable and rupture groups, and found possible targeted drugs for immunotherapy.

Minimization and complete loss of general transcription factor proteins in the intracellular parasite Encephalitozoon cuniculi

Minimization and complete loss of general transcription factor proteins in the intracellular parasite <i>Encephalitozoon cuniculi</i>

Myosin VI in the nucleolus of neurosecretory PC12 cells: its involvement in the maintenance of nucleolar structure and ribosome organization.

We have previously shown that unconventional myosin VI (MVI), a unique actin-based motor protein, shuttles between the cytoplasm and nucleus in neurosecretory PC12 cells in a stimulation-dependent manner and interacts with numerous proteins involved in nuclear processes. Among the identified potential MVI partners was nucleolin, a major nucleolar protein implicated in rRNA processing and ribosome assembly. Several other nucleolar proteins such as fibrillarin, UBF (upstream binding factor), and B23 (also termed nucleophosmin) have been shown to interact with MVI. A bioinformatics tool predicted the presence of the nucleolar localization signal (NoLS) within the MVI globular tail domain, and immunostaining confirmed the presence of MVI within the nucleolus. Depletion of MVI, previously shown to impair PC12 cell proliferation and motility, caused disorganization of the nucleolus and rough endoplasmic reticulum (rER). However, lack of MVI does not affect nucleolar transcription. In light of these data, we propose that MVI is important for nucleolar and ribosome maintenance but not for RNA polymerase 1-related transcription.

Molecular Responses of the Eukaryotic Cell Line INT407 on the Internalized Campylobacter jejuni-The Other Side of the Coin.

Campylobacter jejuni is a zoonotic bacterium with the capacity to invade the epithelial cells during the pathogenic process. Several bacterial factors have been identified to contribute to this process, but our knowledge is still very limited about the response of the host. To reveal the major routes of this response, a whole-transcriptome analysis (WTA) was performed where gene expressions were compared between the 1st and the 3rd hours of internalization in INT407 epithelial cells. From the 41,769 human genes tested, altogether, 19,060 genes were shown through WTA to be influenced to different extents. The genes and regulation factors of transcription (296/1052; 28%), signal transduction (215/1052; 21%), apoptosis (153/1052; 15%), immune responses (97/1052; 9%), transmembrane transport (64/1052; 6%), cell-cell signaling (32/1052; 3%), cell-cell adhesions (29/1052; 3%), and carbohydrate metabolism (28/1052; 3%) were the most affected biological functions. A striking feature of the gene expression of this stage of the internalization process is the activation of both immune functions and apoptosis, which convincingly outlines that the invaded cell faces a choice between death and survival. The seemingly balanced status quo between the invader and the host is the result of a complex process that also affects genes known to be associated with postinfectious pathological conditions. The upregulation of TLR3 (3.79×) and CD36 (2.73×), two general tumor markers, and SERPINEB9 (11.37×), FNDC1 (7.58×), and TACR2 (8.84×), three factors of tumorigenesis, confirms the wider pathological significance of this bacterium.

Transcriptomic responses to drought stress in the Patagonian southern beech Nothofagus alpina

BackgroundDeciphering the genetic architecture of drought tolerance could allow the candidate genes identification responding to water stress. In the Andean Patagonian forest, the genus Nothofagus represents an ecologically relevant species to be included in different genomic studies. These studies are scarce in South American ecosystems however represent an important source of genomic data in order to interpret future climate-change environment scenarios of these emblematic forests. Here, we achieved the assemblage of the transcriptome of N. alpina while searching for key genes of activated or suppressed metabolic pathways in response to drought stress.ResultsDe novo transcriptome assembly resulted in 104,030 transcripts. Following confirmation of drought conditions, based on reduction of leaf water potential and stomatal conductance, a differential gene expression analysis resulted in 2720 significantly expressed genes (1601 up-regulated and 1119 down-regulated). Enrichment analysis (over-representation analysis and gene set enrichment analysis) resulted in more than one hundred stress-responsive term ontologies (i.e. biological processes) and pathways. Terms such as response to abscisic acid and pathways such as plant hormone signal transduction or starch and sucrose metabolism were over-represented. Protein–protein interaction assessment resulted in networks with significantly expressed top common hub gene clusters (e.g. plant-type cell wall biogenesis among down-regulated or ABA-signalling among up-regulated). These networks evidenced important regulators at gene expression such as transcriptional factors.ConclusionsResponses of N. alpina seedlings to drought stress were evidenced by the activation of several genes linked to GO biological processes and KEGG pathways, which were mainly based on over-expression of specific protein kinases, phosphatases, synthases and transcription factors. This suggests an up-regulation of signalling pathways, triggered through plant hormones such as abscisic acid or auxin, which could counteract the osmotic stress created as a probable immediate response to drought. On the other hand, groups of carbon fixation genes related to the galactose metabolism, photosynthesis, secondary wall biogenesis, and fatty acid biosynthesis degradation were down-regulated under drought. Overall, our results provide new genomic data for understanding how non-model long-lived trees of Patagonian forests would acclimate to environmental changes.

Identification of prognostic stemness-related genes in kidney renal papillary cell carcinoma

BackgroundKidney renal papillary cell carcinoma (KIRP) is the second most prevalent malignant cancer originating from the renal epithelium. Nowadays, cancer stem cells and stemness-related genes (SRGs) are revealed to play important roles in the carcinogenesis and metastasis of various tumors. Consequently, we aim to investigate the underlying mechanisms of SRGs in KIRP.MethodsRNA-seq profiles of 141 KIRP samples were downloaded from the TCGA database, based on which we calculated the mRNA expression-based stemness index (mRNAsi). Next, we selected the differentially expressed genes (DEGs) between low- and high-mRNAsi groups. Then, we utilized weighted gene correlation network analysis (WGCNA) and univariate Cox analysis to identify prognostic SRGs. Afterwards, SRGs were included in the multivariate Cox regression analysis to establish a prognostic model. In addition, a regulatory network was constructed by Pearson correlation analysis, incorporating key genes, upstream transcription factors (TFs), and downstream signaling pathways. Finally, we used Connectivity map analysis to identify the potential inhibitors.ResultsIn total, 1124 genes were characterized as DEGs between low- and high-RNAsi groups. Based on six prognostic SRGs (CCKBR, GPR50, GDNF, SPOCK3, KC877982.1, and MYO15A), a prediction model was established with an area under curve of 0.861. Furthermore, among the TFs, genes, and signaling pathways that had significant correlations, the CBX2-ASPH-Notch signaling pathway was the most significantly correlated. Finally, resveratrol might be a potential inhibitor for KIRP.ConclusionsWe suggested that CBX2 could regulate ASPH through activation of the Notch signaling pathway, which might be correlated with the carcinogenesis, development, and unfavorable prognosis of KIRP.

Functional and structural characterization of an IclR family transcription factor for the development of dicarboxylic acid biosensors.

Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work, we functionally and structurally characterize the Pca regulon regulatory protein (PcaR) transcription factor belonging to the IclR transcription factor family. Here, we present in vitro functional analysis of the ligand profile of PcaR and the construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this transcription factor. This work contributes key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors, in addition to providing more insights into the IclR family of regulators.

Prediction of DNA-binding Sites in Transcriptions Factor in Fur-like Proteins Using Machine Learning and Molecular Descriptors

Introduction: Transcription factors are of great interest in biotechnology due to their key role in the regulation of gene expression. One of the most important transcription factors in gramnegative bacteria is Fur, a global regulator studied as a therapeutic target for the design of antibacterial agents. Its DNA-binding domain, which contains a helix-turn-helix motif, is one of its most relevant features. Methods: In this study, we evaluated several machine learning algorithms for the prediction of DNA-binding sites based on proteins from the Fur superfamily and other helix-turn-helix transcription factors, including Support-Vector Machines (SVM), Random Forest (RF), Decision Trees (DT), and Naive Bayes (NB). We also tested the efficacy of using several molecular descriptors derived from the amino acid sequence and the structure of the protein fragments that bind the DNA. A feature selection procedure was employed to select fewer descriptors in each case by maintaining a good classification performance. Results: The best results were obtained with the SVM model using twelve sequence-derived attributes and the DT model using nine structure-derived features, achieving 82% and 76% accuracy, respectively. Conclusion: The performance obtained indicates that the descriptors we used are relevant for predicting DNA-binding sites since they can discriminate between binding and non-binding regions of a protein.

Helicases DDX5 and DDX17 promote heterogeneity in HBV transcription termination in infected human hepatocytes

Transcription termination fine-tunes gene expression and contributes to the specification of RNA function in eukaryotic cells. Transcription termination of HBV is subject to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. However, the regulation of this cPAS and its impact on viral gene expression and replication is currently unknown. To unravel the regulation of HBV transcript termination, we implemented a 3' RACE (rapid amplification of cDNA ends)-PCR assay coupled to single molecule sequencing both in invitro-infected hepatocytes and in chronically infected patients. The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication invitro and invivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated with HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of the cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. Our findings identify DDX5 and DDX17 as crucial determinants of HBV transcriptional fidelity and as host restriction factors for HBV replication. HBV covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail for the achievement of HBV cure. Transcriptional fidelity is a cornerstone in the regulation of gene expression. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit recognition of the HBV polyadenylation signal and thereby transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in patients chronically infected with HBV suggests a role for these helicases in HBV persistence invivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.

Genome-wide identification and analyses of ZmAPY genes reveal their roles involved in maize development and abiotic stress responses

Apyrase is a class of enzyme that catalyzes the hydrolysis of nucleoside triphosphates/diphosphates (NTP/NDP), which widely involved in regulation of plant growth and stress responses. However, apyrase family genes in maize have not been identified, and their characteristics and functions are largely unknown. In this study, we identified 16 apyrases (named as ZmAPY1-ZmAPY16) in maize genome, and analyzed their phylogenetic relationships, gene structures, chromosomal distribution, upstream regulatory transcription factors and expression patterns. Analysis of the transcriptome database unveiled tissue-specific and abiotic stress-responsive expression of ZmAPY genes in maize. qPCR analysis further confirmed their responsiveness to drought, heat, and cold stresses. Association analyses indicated that variations of ZmAPY5 and ZmAPY16 may regulate maize agronomic traits and drought responses. Our findings shed light on the molecular characteristics and evolutionary history of maize apyrase genes, highlighting their roles in various biological processes and stress responses. This study forms a basis for further exploration of apyrase functions in maize.

Abstract 2151: VascularRegulationbySuper Enhancer-DerivedLINC00607

Vascular endothelial cells (ECs) play a pivotal role in whole-body homeostasis. Recent advances have revealed enhancer-associated long non-coding RNAs (lncRNAs) as essential regulators in EC function. We investigated LINC00607, a super enhancer-derived lncRNA (SE-lncRNA) in human arteries with an emphasis on ECs. Based on public databases and our single-cell RNA-sequencing (scRNA-seq) data from human arteries collected from healthy and diabetic donors, we found that LINC00607 is abundantly expressed in the arteries and its level is increased in diabetic patients. Furthermore, through transcriptomic and promoter analysis, we identified c-Myc as an upstream transcription factor of LINC00607 in diabetic conditions. Interestingly, we found that N6-methyladenosine modification also contributes to the induced abundance in diabetic patients. Using RNA sequencing, we characterized the transcriptomes regulated by LINC00607 in ECs and vascular smooth muscle cells (VSMCs) and in basal and diabetic conditions in ECs. Finally, using scRNA-seq, we demonstrated that the modified antisense oligonucleotide inhibitor of LINC00607 can reverse dysfunctional changes induced by high glucose and TNF alpha in ECs. Collectively, our study demonstrates a multi-pronged approach to characterize LINC00607 in vascular cells and its gene regulatory networks in ECs and VSMCs. Our findings provide new insights into the regulation and function of SE-derived lncRNAs in both vascular homeostasis and dysfunction in a cell-type and context-dependent manner, which could have a significant impact on our understanding of epigenetic regulation implicated in cardiovascular health and diseases like diabetes.

Abstract 3064: Distinct Regulatory Programs Govern Smooth Muscle Cell Fate In Atherosclerosis

Introduction: During atherosclerosis, some vascular smooth muscle cells (SMCs) undergo phenotypic modulation, de-differentiating and adopting a fibroblast-like (FMC) or a chondrocyte/osteoblast-like phenotype (CMC). Although FMCs and CMCs appear to occur in succession in single-cell RNAseq data, they occupy distinct locations within the lesion and are associated with protective and deleterious plaque features, respectively. Hypothesis: Smooth muscle cell transitions to FMC and CMC occur via discrete transcriptional pathways during atherosclerosis. Methods: We performed single-cell RNA-sequencing (scRNAseq) and assay for transposase-accessible chromatin with sequencing (ATAC-seq) on lineage-traced vascular SMCs isolated from the aortic root of ApoE mice (n=3). Our analysis included pseudotime and RNA velocity analyses of the scRNAseq data, which independently identified a subgroup of Vcam+Cd74+ modulated SMCs as a potential cell state from which FMCs and CMCs appear to originate, each following distinct trajectories. We explored lineage-specific gene expression using the TradeSeq R package and conducted Ingenuity Pathway Analysis on differentially expressed genes to identify relevant upstream transcription factors, which were assayed for lineage-specific motif accessibility. Results: Our analysis showed profound changes in gene expression between the two trajectories as well as differential activation and chromatin accessibility of specific transcription factors at the point where FMCs diverge from CMCs. Within the CMC lineage, we noted heightened activation and chromatin accessibility of transcription factors associated with cellular stress and inflammatory responses, including Tp53, Foxo4, and Irf1. In stark contrast, these factors exhibited diminished activity within the FMC lineage during the same pseudotime interval. Furthermore, coronary artery disease GWAS transcription factors Twist1 and Tcf21 displayed differential motif accessibility over the FMC and CMC trajectories. Conclusions: Our study provides valuable insights into the regulatory mechanisms governing SMC phenotypic transitions in atherosclerosis, and further underscores the importance of GWAS genes in influencing these transitions.

The upregulation and transcriptional regulatory mechanisms of Extra spindle pole bodies like 1 in bladder cancer: An immunohistochemistry and high-throughput screening Evaluation

BackgroundThis study aimed to explore the expression level and transcriptional regulation mechanism of Extra Spindle Pole Bodies Like 1 (ESPL1) in bladder cancer (BC). MethodsA multicentre database of samples (n = 1391) was assayed for ESPL1 mRNA expression in BC and validated at the protein level by immunohistochemical (IHC) staining of in-house samples (n = 202). Single-cell sequencing (scRNA-seq) analysis and enrichment analysis explored ESPL1 distribution and their accompanying molecular mechanisms. ATAC-seq, ChIP-seq and Hi-C data from multiple platforms were used to investigate ESPL1 upstream transcription factors (TFs) and potential epigenetic regulatory mechanisms. Immune-related analysis, drug sensitivity and molecular docking of ESPL1 were also calculated. Furthermore, upstream microRNAs and the binding sites of ESPL1 were predicted. The expression level and early screening efficacy of miR-299-5p in blood (n = 6625) and tissues (n = 537) were examined. ResultsESPL1 was significantly overexpressed at the mRNA level (p < 0.05, SMD = 0.75; 95 % CI = 0.09, 1.40), and IHC staining of in-house samples verified this finding (p < 0.0001). ESPL1 was predominantly distributed in BC epithelial cells. Coexpressed genes of ESPL1 were enriched in cell cycle–related signalling pathways, and ESPL1 might be involved in the communication between epithelial and residual cells in the Hippo, ErbB, PI3K-Akt and Ras signalling pathways. Three TFs (H2AZ, IRF5 and HIF1A) were detected upstream of ESPL1 and presence of promoter-super enhancer and promoter-typical enhancer loops. ESPL1 expression was correlated with various immune cell infiltration levels. ESPL1 expression might promote BC growth and affect the sensitivity and therapeutic efficacy of paclitaxel and gemcitabine in BC patients. As an upstream regulator of ESPL1, miR-299-5p expression was downregulated in both the blood and tissues, possessing great potential for early screening. ConclusionsESPL1 expression was upregulated in BC and was mainly distributed in epithelial cells. Elevated ESPL1 expression was associated with TFs at the upstream transcription start site (TSS) and distant chromatin loops of regulatory elements. ESPL1 might be an immune-related predictive and diagnostic marker for BC, and the overexpression of ESPL1 played a cancer-promoting role and affected BC patients’ sensitivity to drug therapy. miR-299-5p was downregulated in BC blood and tissues and was also expected to be a novel marker for early screening.