Activator Of Gene Transcription Research Articles

Stochastic scanning events on the GCN4 mRNA 5' untranslated region generate cell-to-cell heterogeneity in the yeast nutritional stress response.

Gene expression stochasticity is inherent in the functional properties and evolution of biological systems, creating non-genetic cellular individuality and influencing multiple processes, including differentiation and stress responses. In a distinct form of non-transcriptional noise, we find that interactions of the yeast translation machinery with the GCN4 mRNA 5'UTR, which underpins starvation-induced regulation of this transcriptional activator gene, manifest stochastic variation across cellular populations. We use flow cytometry, fluorescence-activated cell sorting and microfluidics coupled to fluorescence microscopy to characterize the cell-to-cell heterogeneity of GCN4-5'UTR-mediated translation initiation. GCN4-5'UTR-mediated translation is generally not de-repressed under non-starvation conditions; however, a sub-population of cells consistently manifests a stochastically enhanced GCN4 translation (SETGCN4) state that depends on the integrity of the GCN4 uORFs. This sub-population is eliminated upon deletion of the Gcn2 kinase that phosphorylates eIF2α under nutrient-limitation conditions, or upon mutation to Ala of the Gcn2 kinase target site, eIF2α-Ser51. SETGCN4 cells isolated using cell sorting spontaneously regenerate the full bimodal population distribution upon further growth. Analysis of ADE8::ymRuby3/ GCN4::yEGFP cells reveals enhanced Gcn4-activated biosynthetic pathway activity in SETGCN4 cells under non-starvation conditions. Computational modeling interprets our experimental observations in terms of a novel translational noise mechanism underpinned by natural variations in Gcn2 kinase activity.

Mechanism of Zn2+ regulation of cellulase production in Trichoderma reesei Rut-C30

BackgroundTrichoderma reesei Rut-C30 is a hypercellulolytic mutant strain that degrades abundant sources of lignocellulosic plant biomass, yielding renewable biofuels. Although Zn2+ is an activator of enzymes in almost all organisms, its effects on cellulase activity in T. reesei have yet to be reported.ResultsAlthough high concentrations of Zn2+ severely suppressed the extension of T. reesei mycelia, the application of 1–4 mM Zn2+ enhanced cellulase and xylanase production in the high-yielding cellulase-producing Rut-C30 strain of T. reesei. Expression of the major cellulase, xylanase, and two essential transcription activator genes (xyr1 and ace3) increased in response to Zn2+ stimulation. Transcriptome analysis revealed that the mRNA levels of plc-e encoding phospholipase C, which is involved in the calcium signaling pathway, were enhanced by Zn2+ application. The disruption of plc-e abolished the cellulase-positive influence of Zn2+ in the early phase of induction, indicating that plc-e is involved in Zn2+-induced cellulase production. Furthermore, treatment with LaCl3 (a plasma membrane Ca2+ channel blocker) and deletion of crz1 (calcineurin-responsive zinc finger transcription factor 1) indicated that calcium signaling is partially involved in this process. Moreover, we identified the zinc-responsive transcription factor zafA, the transcriptional levels of which declined in response to Zn2+ stress. Deletion of zafA indicates that this factor plays a prominent role in mediating the Zn2+-induced excessive production of cellulase.ConclusionsFor the first time, we have demonstrated that Zn2+ is toxic to T. reesei, although promotes a marked increase in cellulase production. This positive influence of Zn2+ is facilitated by the plc-e gene and zafA transcription factor. These findings provide insights into the role of Zn2+ in T. reesei and the mechanisms underlying signal transduction in cellulase synthesis.

NLRC5-CIITA Fusion Protein as an Effective Inducer of MHC-I Expression and Antitumor Immunity.

Aggressive tumors evade cytotoxic T lymphocytes by suppressing MHC class-I (MHC-I) expression that also compromises tumor responsiveness to immunotherapy. MHC-I defects strongly correlate to defective expression of NLRC5, the transcriptional activator of MHC-I and antigen processing genes. In poorly immunogenic B16 melanoma cells, restoring NLRC5 expression induces MHC-I and elicits antitumor immunity, raising the possibility of using NLRC5 for tumor immunotherapy. As the clinical application of NLRC5 is constrained by its large size, we examined whether a smaller NLRC5-CIITA fusion protein, dubbed NLRC5-superactivator (NLRC5-SA) as it retains the ability to induce MHC-I, could be used for tumor growth control. We show that stable NLRC5-SA expression in mouse and human cancer cells upregulates MHC-I expression. B16 melanoma and EL4 lymphoma tumors expressing NLRC5-SA are controlled as efficiently as those expressing full-length NLRC5 (NLRC5-FL). Comparison of MHC-I-associated peptides (MAPs) eluted from EL4 cells expressing NLRC5-FL or NLRC5-SA and analyzed by mass spectrometry revealed that both NLRC5 constructs expanded the MAP repertoire, which showed considerable overlap but also included a substantial proportion of distinct peptides. Thus, we propose that NLRC5-SA, with its ability to increase tumor immunogenicity and promote tumor growth control, could overcome the limitations of NLRC5-FL for translational immunotherapy applications.

HSP27 modulates tumoural immune evasion by enhancing STAT3-mediated upregulation of PD-L1 and NLRC5 in ovarian cancer.

Multiple preclinical studies have demonstrated that the addition of hyperthermia (HT) to immunotherapy could enhance tumour immunogenicity and stimulate an antitumour immune response, primarily via heat shock proteins (HSPs). However, antitumour immune responses are often impeded by immune evasion mechanisms, such as the overexpression of programmed death-ligand1 (PD-L1) and the loss of major histocompatibility complex class 1 (MHC-1) expression. In this context, we sought to investigate the effect of HT on PD-L1 and NOD-like receptor family CARD domain containing 5 (NLRC5) identified as the key transcriptional activator of MHC-1 genes, and their interaction in ovarian cancer. A coculture of ovarian cancer cell lines (IGROV1 and SKOV3) with peripheral blood mononuclear cells was set up. Then, culture media conditioned with IGROV1 or SKOV3 subjected to HT was tested on untreated cell cultures. Knocking down heat shock protein B1 (HSPB1 or HSP27), heat shock protein A1 (HSPA1 or HSP70), and pharmacological inhibition of STAT3 phosphorylation were performed. Subsequently, we measured expression levels of PD-L1, NLRC5, and proinflammatory cytokines. The correlation between PD-L1 and NLRC5 expression in ovarian cancer was evaluated using the Cancer Genome Atlas database. We found that HT produces a concomitant decrease in PD-L1 and NLRC5 expression in coculture. Notably, however, the conditioned media by heat-shocked cells increases their expression. HSP27 knockdown can reverse this increase. Adding STAT3 phosphorylation inhibitor significantly enhanced the expression inhibition of PD-L1 and NLRC5 induced by HSP27 silencing. Correlation analysis showed a positive correlation in ovarian cancer between NLRC5 and PD-L1. These findings demonstrate that HSP27 modulates PD-L1 and NLRC5 expression through the activation of a common regulator 'STAT3'. Moreover, the positive correlation between PD-L1 and NLRC5 led us to conclude that the upregulation of PD-L1 and the downregulation of MHC class I are two mutually exclusive mechanisms of immune evasion in ovarian cancer.

The pleiotropic AMPK-CncC signaling pathway regulates the trade-off between detoxification and reproduction.

The association of decreased fecundity with insecticide resistance and the negative sublethal effects of insecticides on insect reproduction indicates the typical trade-off between two highly energy-demanding processes, detoxification and reproduction. However, the underlying mechanisms are poorly understood. The energy sensor adenosine monophosphate-activated protein kinase (AMPK) and the transcription factor Cap "n" collar isoform C (CncC) are important regulators of energy metabolism and xenobiotic response, respectively. In this study, using the beetle Tribolium castaneum as a model organism, we found that deltamethrin-induced oxidative stress activated AMPK, which promoted the nuclear translocation of CncC through its phosphorylation. The CncC not only acts as a transcription activator of cytochrome P450 genes but also regulates the expression of genes coding for ecdysteroid biosynthesis and juvenile hormone (JH) degradation enzymes, resulting in increased ecdysteroid levels as well as decreased JH titer and vitellogenin (Vg) gene expression. These data show that in response to xenobiotic stress, the pleiotropic AMPK-CncC signaling pathway mediates the trade-off between detoxification and reproduction by up-regulating detoxification genes and disturbing hormonal homeostasis.

Functional characterization of transcriptional activator gene SIARRI in tomato reveals its role in fruit growth and ripening.

Auxins regulate several characteristics of plant development and growth. Here, we characterized a new transcriptional activator SIARRI which binds specific DNA sequences and was revealed in Arabidopsis (ARR1). SIARRI acts as a two-component response regulator and its Arabidopsis homologous gene is AT3G16857. It belongs to the subfamily of type-B response regulators in the cytokinin signaling pathway. The study aimed to characterize the transgenic Micro-Tom plants by the overexpression of Solanum lycopersicum two-component response regulator ARR1. Overexpression of SIARRI results in a pleiotropic phenotype during fruit development and ripening. This study indicates that SIARRI is a primary regulator of leaf morphology and fruit development. Moreover, overexpressed plants showed variations in growth related to auxin as well as shorter hypocotyl elongation, enlarged leaf vascularization, and decreased apical dominance. The qRT-PCR investigation revealed that expression was downregulated at the breaker stage and high at Br+6 at various stages of fruit growth and ripening. In contrast to the fruit color, lycopene and β-carotene concentrations in red-yellow overexpression line fruits were reduced significantly, and also slightly reduced in some red fruits. The quantity of β-carotene in the transgenic fruits was lower than that of lycopene. This study showed that this gene might be a new transcriptional activator in fruit development and ripening. Furthermore, this study will provide new insights into tomato fruit ripening.

Regulation of pseurotin A biosynthesis by GliZ and zinc in Aspergillus fumigatus

Recently, we reported that zinc regulates gliotoxin biosynthesis via ZafA, which is a zinc-responsive transcriptional activator. From an HPLC analysis of culture media of Aspergillus fumigatus, we found a trend of decreasing gliotoxin production but increasing pseurotin A and fumagillin production in proportion to the zinc concentration. The expression of the genes involved in pseurotin A biosynthesis was upregulated under high zinc concentrations. Furthermore, upregulated expression of pseurotin A biosynthetic genes and higher production of pseurotin A were observed in the zafA deletion strain. Interestingly, the deletion of gliZ, a transcriptional activator of gliotoxin biosynthesis genes, resulted in upregulated expression of pseurotin A biosynthetic genes and increased production of pseurotin A. We detected upregulation of fumR expression in the gliZ and zafA deletion mutants. The overexpression of gliZ observed in the zafA deletion mutant resulted in the failure of the mutant to increase pseurotin A production, which is a phenotype of the zafA deletion mutant. These results suggest that ZafA sequentially regulates pseurotin A biosynthesis through GliZ. Finally, we found through a murine virulence test that the gliZ and fumR double-deletion mutants showed a delayed death rate compared with the single-deletion mutants of either gliZ or fumR. Taken together, these results suggested that the biosynthesis of gliotoxin and pseurotin A are regulated in opposite ways by zinc utilization and that each secondary metabolite is synthesized when the synthesis of another secondary metabolite fails to protect it against the defense system of the host.

In Silico Identification of Natural Food Compounds as Potential Quorum-Sensing Inhibitors Targeting the LasR Receptor of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major cause of nosocomial infections and is often associated with biofilm-mediated antibiotic resistance. The LasR protein is a key component of the quorum system in P. aeruginosa, allowing it to regulate its biofilm-induced pathogenicity. When the bacterial population reaches a sufficient density, the accumulation of N-(3-oxododecanoyl) acyl homoserine lactone (3O-C12-HSL) leads to the activation of the LasR receptor, which then acts as a transcriptional activator of target genes involved in biofilm formation and virulence, thereby increasing the bacteria's antibiotic resistance and enhancing its virulence. In this study, we performed a structure-based virtual screening of a natural food database of 10 997 compounds against the crystal structure of the ligand-binding domain of the LasR receptor (PDB ID: 3IX4). This allowed us to identify four molecules, namely ZINC000001580795, ZINC000014819517, ZINC000014708292, and ZINC000004098719, that exhibited a favorable binding mode and docking scores greater than -13 kcal/mol. Furthermore, the molecular dynamics simulation showed that these four molecules formed stable complexes with LasR during the 150-ns molecular dynamics (MD) simulation, indicating their potential for use as inhibitors of the LasR receptor in P. aeruginosa. However, further experimental validation is needed to confirm their activity.

Optimization of CRISPR/Cas9 method for transgenesis of model microalgae <i>Chlamydomonas reinhardtii</i>

In this work we knocked out the LTS3 gene of the microalgae Chlamydomonas reinhardtii using the TIM technique optimized for the available equipment. We achieved transformation efficiency of 68.8%, knockout of this gene lead to the death of C. reinhardtii cells after several division cycles.
 The creation and study of genetically modified organisms in fundamental research allows a deeper understanding of the basic processes in the cells with the prospect of further applying this knowledge in practice. Microalgae are an interesting object for genetic engineering because of the great prospects for their application in biotechnology, but in almost every case it is necessary to develop new strategies and transformation methods for the introduction of genetic constructs into the cell. CRISPR/Cas revolutionized the field of genome editing due to its simplicity, efficiency and accuracy compared to previously used methods, which over time simplified the development of protocols [1]. Currently, the most effective method of transformation is TIM (Targeted Insertional Mutagenesis) [2], developed for the microalgae Chlamydomonas reinhardtii P.A. Dang. model object of photosynthesis genetics.
 To test and optimize the TIM technique [2] in our lab, we carried out a knockout of the LTS3 gene, a transcriptional activator of chlorophyll biosynthesis genes in heterotrophic conditions [3].
 We used glass beads agitation and electroporation (Gene Pulser Xcell, Bio-Rad, USA) methods in order to introduce into C. reinhardtii cells of the CC-125 (wt, mt+) strain the ribonucleoprotein complex SpCas9/sgRNA and double-stranded donor DNA with paromomycin resistance gene.
 The effectiveness of transformation varied from 10.6% to 68.8%. Probably, the LTS3 gene product plays a key role in the pathway of chlorophyll biosynthesis, since its knockout led to the death of C. reinhardtii cells after several division cycles.
 The transformation protocol optimized for the equipment available in our lab can be further refined and used to study the functions of other C. reinhardtii genes.

DOT1L regulates chamber-specific transcriptional networks during cardiogenesis and mediates postnatal cell cycle withdrawal

Mechanisms by which specific histone modifications regulate distinct gene networks remain little understood. We investigated how H3K79me2, a modification catalyzed by DOT1L and previously considered a general transcriptional activation mark, regulates gene expression during cardiogenesis. Embryonic cardiomyocyte ablation of Dot1l revealed that H3K79me2 does not act as a general transcriptional activator, but rather regulates highly specific transcriptional networks at two critical cardiogenic junctures: embryonic cardiogenesis, where it was particularly important for left ventricle-specific genes, and postnatal cardiomyocyte cell cycle withdrawal, with Dot1L mutants having more mononuclear cardiomyocytes and prolonged cardiomyocyte cell cycle activity. Mechanistic analyses revealed that H3K79me2 in two distinct domains, gene bodies and regulatory elements, synergized to promote expression of genes activated by DOT1L. Surprisingly, H3K79me2 in specific regulatory elements also contributed to silencing genes usually not expressed in cardiomyocytes. These results reveal mechanisms by which DOT1L successively regulates left ventricle specification and cardiomyocyte cell cycle withdrawal.

Neuronal HLH-30/TFEB modulates peripheral mitochondrial fragmentation to improve thermoresistance in Caenorhabditis elegans.

Transcription factor EB (TFEB) is a conserved master transcriptional activator of autophagy and lysosomal genes that modulates organismal lifespan regulation and stress resistance. As neurons can coordinate organism-wide processes, we investigated the role of neuronal TFEB in stress resistance and longevity. To this end, the Caenorhabditis elegans TFEB ortholog, hlh-30, was rescued panneuronally in hlh-30 loss of function mutants. While important in the long lifespan of daf-2 animals, neuronal HLH-30/TFEB was not sufficient to restore normal lifespan in short-lived hlh-30 mutants. However, neuronal HLH-30/TFEB rescue mediated robust improvements in the heat stress resistance of wildtype but not daf-2 animals. Notably, these mechanisms can be uncoupled, as neuronal HLH-30/TFEB requires DAF-16/FOXO to regulate longevity but not thermoresistance. Through further transcriptomics profiling and functional analysis, we discovered that neuronal HLH-30/TFEB modulates neurotransmission through the hitherto uncharacterized protein W06A11.1 by inducing peripheral mitochondrial fragmentation and organismal heat stress resistance in a non-cell autonomous manner. Taken together, this study uncovers a novel mechanism of heat stress protection mediated by neuronal HLH-30/TFEB.

Synthesis and evaluation of naphthalene derivatives as potent STAT3 inhibitors and agents against triple-negative breast cancer growth and metastasis.

Triple-negative breast cancer (TNBC) represents the worst prognostic subtype of breast cancer and lacks targeted therapeutic drugs. Signal transducer and activator of transcription 3 (STAT3) is overexpressed and constitutively activated in TNBCs and associated with poor patient outcomes. However, no agents targeting STAT3 have been successfully developed and marketed. Selective Estrogen Receptor Modulators (SERMs) have been reported as potential inhibitors of the IL-6/STAT3 signaling pathway. Naphthalene compounds have good pharmacological activity and significant anti-cancer activity. In this study, we synthesized a new series of naphthalene derivatives with the general structure of SERM and evaluated their effects on TNBC and STAT3 signals. A new series of compounds based on the scaffold of SERMs and an amino group were designed and screened based on the structure-activity relationship by MTT assay. The binding activity of SMY002 to STAT3 was predicted and validated by docking and SPR. The STAT3 signaling target and anti-cancer effects of SMY002 were evaluated with three TNBC cell lines and the mice transplanted tumor model. Among the compounds, SMY002 displayed the most potent activity, which could directly interact with STAT3 SH2-domain, and strongly inhibit the phosphorylation, dimerization, nuclear distribution, transcriptional activity, and target genes expression of STAT3. Furthermore, SMY002 markedly suppressed migration, invasion, survival, growth, and metastasis of TNBC cells in vitro and in vivo via down-regulating the expression of Cyclin D1 and MMP9. SMY002 can significantly inhibit the growth and metastasis of TNBC cells by targeting the STAT3 signal.

The ethylene response factor SlERF.B8 triggers jasmonate biosynthesis to promote cold tolerance in tomato

Cold stress inhibits growth, decreases yields and impairs fruit quality in tomato ( Solanum lycopersicum ). Previous studies have shown that cold stress leads to increased JA accumulation, however, how jasmonate biosynthesis is regulated upon cold stress remains largely unknown. Here, we report an ethylene response factor SlERF.B8 that acts as a crucial regulator of JA biosynthesis in tomato under cold stress. Transcription of SlERF.B8 was markedly enhanced by JA and cold stress, and silencing of SlERF.B8 led to decreased JA accumulation and attenuated cold tolerance in tomato. ChIP-qPCR and dual-luciferase assays showed that SlERF.B8 functions as a transcriptional activator of JA biosynthesis genes, including LOXD , AOC and OPR3 . Furthermore, MYC2, a critical mediator of JA signaling, activates the transcription of SlERF.B8 , thus forming a positive feedback loop, which amplifies the output of JA signaling. Taken together, our data favor that SlERF.B8 is a key regulator of JA biosynthesis and cold tolerance in tomato plants. • Transcription of SlERF.B8 was markedly induced by JA and cold stress. • Silencing of SlERF.B8 decreases JA accumulation and attenuates cold tolerance. • SlERF.B8 activates the transcription of JA biosynthesis genes. • JA signaling activates the transcription of SlERF.B8 . • JA and ET synergistically regulate tomato cold tolerance.

Homeobox A3 and KDM6A cooperate in transcriptional control of aerobic glycolysis and glioblastoma progression.

Alterations in transcriptional regulators of glycolytic metabolism have been implicated in brain tumor growth, but the underlying molecular mechanisms remain poorly understood. Knockdown and overexpression cells were used to explore the functional roles of HOXA3 in cell proliferation, tumor formation, and aerobic glycolysis. Chromatin immunoprecipitation, luciferase assays, and western blotting were performed to verify the regulation of HK2 and PKM2 by HOXA3. PLA, Immunoprecipitation, and GST-pull-down assays were used to examine the interaction of HOXA3 and KDM6A. We report that transcription factor homeobox A3 (HOXA3), which is aberrantly highly expressed in glioblastoma (GBM) patients and predicts poor prognosis, transcriptionally activates aerobic glycolysis, leading to a significant acceleration in cell proliferation and tumor growth. Mechanically, we identified KDM6A, a lysine-specific demethylase, as an important cooperator of HOXA3 in regulating aerobic glycolysis. HOXA3 activates KDM6A transcription and recruits KDM6A to genomic binding sites of glycolytic genes, targeting glycolytic genes for transcriptional activation by removing the suppressive histone modification H3K27 trimethylation. Further evidence demonstrates that HOXA3 requires KDM6A for transcriptional activation of aerobic glycolysis and brain tumor growth. Our findings provide a novel molecular mechanism linking HOXA3-mediated transactivation and KDM6A-coupled H3K27 demethylation in regulating glucose metabolism and GBM progression.

Harmine, an inhibitor of the type III secretion system of Salmonella enterica serovar Typhimurium.

New therapeutic strategies for clinical Salmonella enterica serovar Typhimurium (S. Typhimurium) infection are urgently needed due to the generation of antibiotic-resistant bacteria. Inhibition of bacterial virulence has been increasingly regarded as a potential and innovative strategy for the development of anti-infection drugs. Salmonella pathogenicity island (SPI)-encoded type III secretion system (T3SS) represents a key virulence factor in S. Typhimurium, and active invasion and replication in host cells is facilitated by the secretion of T3SS effector proteins. In this study, we found that harmine could inhibit T3SS secretion; thus, its potential anti-S. Typhimurium infection activity was elucidated. Harmine inhibits the secretion and expression of T3SS effector proteins and consequently attenuates the S. Typhimurium invasion function of HeLa cells. This inhibition may be implemented by reducing the transcription of pathogenesis-related SPI-1 transcriptional activator genes hilD, hilC, and rtsA. Harmine improves the survival rate and bacterial loads of mice infected with S. Typhimurium. In summary, harmine, an effective T3SS inhibitor, could be a leading compound for the development of treatments for Salmonella infection.

LuxR-Type SCO6993 Negatively Regulates Antibiotic Production at the Transcriptional Stage by Binding to Promoters of Pathway-Specific Regulatory Genes in Streptomyces coelicolor.

SCO6993 (606 amino acids) in Streptomyces coelicolor belongs to the large ATP-binding regulators of the LuxR family regulators having one DNA-binding motif. Our previous findings predicted that SCO6993 may suppress the production of pigmented antibiotics, actinorhodin, and undecylprodigiosin, in S. coelicolor, resulting in the characterization of its properties at the molecular level. SCO6993-disruptant, S. coelicolor ΔSCO6993 produced excess pigments in R2YE plates as early as the third day of culture and showed 9.0-fold and 1.8-fold increased production of actinorhodin and undecylprodigiosin in R2YE broth, respectively, compared with that by the wild strain and S. coelicolor ΔSCO6993/SCO6993+. Real-time polymerase chain reaction analysis showed that the transcription of actA and actII-ORF4 in the actinorhodin biosynthetic gene cluster and that of redD and redQ in the undecylprodigiosin biosynthetic gene cluster were significantly increased by SCO6993-disruptant. Electrophoretic mobility shift assay and DNase footprinting analysis confirmed that SCO6993 protein could bind only to the promoters of pathway-specific transcriptional activator genes, actII-ORF4 and redD, and a specific palindromic sequence is essential for SCO6993 binding. Moreover, SCO6993 bound to two palindromic sequences on its promoter region. These results indicate that SCO6993 suppresses the expression of other biosynthetic genes in the cluster by repressing the transcription of actII-ORF4 and redD and consequently negatively regulating antibiotic production.

Abstract 1370: Targeting LSD1 rescues MHC-I antigen presentation in small cell lung cancer

Abstract Purpose: Small cell lung cancer (SCLC) is a highly aggressive cancer with early primary resistance and modest clinical benefit to immune checkpoint blockade (ICB). Mutation or transcriptional repression of the major histocompatibility complex class I (MHC-I) is a key mechanism driving resistance to T cell-based therapies. Lysine Demethylase 1 (LSD1) regulates gene expression through modulating mono- and di- methylated lysine 4 and 9 of histone H3. LSD1 is a therapeutic target of interests in SCLC due to its oncogenic requirement for tumor growth, and there has been growing evidence pointing to LSD1 as a repressor of tumor-intrinsic immunogenicity. Here investigated the role of LSD1 as a transcriptional regulator of antigen presentation in SCLC. Method: To perturb LSD1 function, we employed the pharmacological inhibitor ORY-1001 and shRNA-mediated knockdown. We then assessed changes in MHC-I expression on SCLC cell lines by flow cytometry and western blot. To analyze transcriptional changes following LSD1 inhibition, we performed RNA-seq on SCLC cells pre- and post-treatment with ORY-1001. To observe for antigen-specific T cell killing, we engineered SCLC cells to express endogenous antigens and co-cultured these cells with primary cognate CD8+ T cells. Finally, we treated genetically engineered mouse model (GEMM)-derived tumors on immunocompetent syngeneic mice with ORY-1001 with or without anti-PD-L1 blockade to assess for anti-tumor effects. Results: We discovered a significant and strong negative correlation between expression of LSD1 and genes encoding the MHC-I antigen presentation pathway. Inhibition of LSD1 induces expression of MHC-I genes and restores expression of genes encoding the antigen presentation machinery. Perturbation of LSD1 further activates interferon signaling and interferon-inducible expression of NLRC5, a well-characterized transcriptional activator of MHC-I genes. We further showed that targeting LSD1 sensitizes SCLC cells to MHC-I-restricted T cell-dependent cytolysis and enhances ICB efficacy in refractory SCLC tumors. Conclusion: Our data define a role for LSD1 as a potent negative regulator of MHC-I-mediated antigen presentation and provide rationale for the use of LSD1 inhibitors to augment response to immunotherapy in SCLC. Citation Format: Minh N. Nguyen, Hirokazu Taniguchi, Andrew Chow, Yingqian A. Zhan, Triparna Sen, Charles M. Rudin. Targeting LSD1 rescues MHC-I antigen presentation in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1370.

Genome-Wide Identification and Characterization of the Calmodulin-Binding Transcription Activator (CAMTA) Gene Family in Plants and the Expression Pattern Analysis of CAMTA3/SR1 in Tomato under Abiotic Stress.

Calmodulin-binding transcription activator (CAMTA) plays an important regulatory role in plant growth, development, and stress response. This study identified the phylogenetic relationships of the CAMTA family in 42 plant species using a genome-wide search approach. Subsequently, the evolutionary relationships, gene structures, and conservative structural domain of CAMTA3/SR1 in different plants were analyzed. Meanwhile, in the promoter region, the cis-acting elements, protein clustering interaction, and tissue-specific expression of CAMTA3/SR1 in tomato were identified. The results show that SlCAMTA3/SR1 genes possess numerous cis-acting elements related to hormones, light response, and stress in the promoter regions. SlCAMTA3 might act together with other Ca2+ signaling components to regulate Ca2+-related biological processes. Then, the expression pattern of SlCAMTA3/SR1 was also investigated by quantitative real-time PCR (qRT-PCR) analysis. The results show that SlCAMTA3/SR1 might respond positively to various abiotic stresses, especially Cd stress. The expression of SlCAMTA3/SR1 was scarcely detected in tomato leaf at the seedling and flowering stages, whereas SlCAMTA3/SR1 was highly expressed in the root at the seedling stage. In addition, SlCAMTA3/SR1 had the highest expression levels in flowers at the reproductive stage. Here, we provide a basic reference for further studies about the functions of CAMTA3/SR1 proteins in plants.

Comprehensive analysis of lower mitochondrial complex I expression is associated with cell metastasis of clear cell renal cell carcinoma.

BackgroundClear cell renal cell carcinoma (ccRCC) is characterized by high metastasis potential. It is of great importance to explore the mechanisms underlying ccRCC metastasis and to enable development of potent therapeutics. The mitochondrial complex I (CI) had been considered to play an important role in the development of cancers, but less known in ccRCC.MethodsWe utilized available public databases of ccRCC, including single-cell RNA sequencing (scRNA-seq) data GSE73121 and The Cancer Genome Atlas-kidney renal clear cell carcinoma (TCGA-KIRC). Principal component analysis (PCA) and t-Distributed Stochastic Neighbor Embedding (tSNE) analysis were evaluated the heterogeneity of metastatic renal cell carcinoma (mRCC) and primary renal cell carcinoma (pRCC). Protein-protein interaction (PPI) network identified critical gene. Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) performed to explore the potential biologic pathways.ResultsOur study revealed a significant gene expression heterogeneity between pRCC and mRCC. A PPI network based on differentially expressed genes (DEGs) identified electron transport chain (ETC), especially mitochondrial CI, as the key network hub. Further analysis revealed that the role of mitochondrial CI is associated with tumor metastasis and immune responds of ccRCC. Although CI had low frequency mutations in ccRCC, CI expression is associated with the high frequency mutated genes. A prognosis model included 7 CI genes, and these had a significant effect on overall survival (OS). The area under the curve at 1, 3, and 5 years was 0.717, 0.685, and 0.728, respectively. Transcription factor analysis predicted that PPARG possibly is a potential transcription activator of CI genes in ccRCC.ConclusionsOverall, we found that CI expression is associated with ccRCC progress. CI and PPARG may be potential biomarkers for metastatic ccRCC.

The miR319/TaGAMYB3 module regulates plant architecture and improves grain yield in common wheat (Triticum aestivum).

Plant architecture is a key determinant of crop productivity and adaptation. The highly conserved microRNA319 (miR319) family functions in various biological processes, but little is known about how miR319 regulates plant architecture in wheat (Triticum aestivum). Here, we determined that the miR319/TaGAMYB3 module controls plant architecture and grain yield in common wheat. Repressing tae-miR319 using short tandem target mimics resulted in favorable plant architecture traits, including increased plant height, reduced tiller number, enlarged spikes and flag leaves, and thicker culms, as well as enhanced grain yield in field plot tests. Overexpressing tae-miR319 had the opposite effects on plant architecture and grain yield. Although both TaPCF8 and TaGAMYB3 were identified as miR319 target genes, genetic complementation assays demonstrated that only miR319-resistant TaGAMYB3 (rTaGAMYB3) abolished tae-miR319-mediated growth inhibition of flag leaves and spikes. TaGAMYB3 functions as a transcriptional activator of downstream genes, including TaPSKR1, TaXTH23, TaMADS5 and TaMADS51, by binding to their promoters. Furthermore, TaGAMYB3 physically interacts with TaBA1, an important regulator of spike development, to additively activate the transcription of downstream genes such as TaMADS5. Our findings provide insight into how the miR319/TaGAMYB3 module regulates plant architecture and improves grain yield in common wheat.