Activator Of Gene Transcription Research Articles

Genome-Wide Identification of CAMTA Gene Family in Oat (Avena sativa) and Expression Analysis under Various Abiotic Stresses

Oat (Avena sativa) is one of the most important cereal crops and cool-season forage grasses in the world. The calmodulin-binding transcription activator (CAMTA) gene family is one of the largest families in plants, and it plays vital roles in multiple biological processes. However, the CAMTA genes in oats, especially those involved in abiotic stress, have not yet been elucidated. Herein, our findings reveal the presence of 20 distinct AsCAMTA genes, which were clustered into three subfamilies based on their gene structure and conserved motifs, indicating functional similarities within each subgroup. Chromosomal mapping indicated an uneven distribution across 10 chromosomes, suggesting a complex evolutionary history marked by potential gene duplication events. The results showed that most AsCAMTA genes contained stress-related cis-elements. The study further investigated the expression patterns of these genes under abiotic stress conditions utilizing RT-qPCR analysis. The results identified three AsCAMTA genes (AsCAMTA5, AsCAMTA7, and AsCAMTA19) that exhibited significant up-regulation under salt stress, with AsCAMTA7 also showing a marked increase in expression under drought stress. These findings suggest a pivotal role of AsCAMTA5, AsCAMTA7, and AsCAMTA19 genes in mediating the responses to various abiotic stresses by integrating multiple stress signals in oats. This investigation provides valuable insights into the potential functions of AsCAMTA genes in the stress response mechanisms of oats, laying a foundation for further functional studies aimed at enhancing abiotic stress tolerance in crops.

Genome-wide analysis of the calmodulin-binding transcription activator (CAMTA) gene family in Sesamum indicum L., and its role in abiotic stress tolerance traits

Calmodulin-binding transcription activator (CAMTA) is one of the key transcription factor families possessing calcium receptors (calmodulins, CaM). It modulates the expression levels of genes associated with ontogeny and various biotic and abiotic stress factors. The CAMTA family genes were known to be involved in different abiotic stress in several crop species. However, their functional relevance in sesame remains unexplored. To understand the role of CAMTA in stress tolerance in sesame, we performed a genome-wide analysis to identify the members of the SiCAMTA gene family. We have identified and reported here the five SiCAMTA genes localized on four chromosomes within the sesame genome. In silico analysis of the putative 2-kilobase (kb) promoter regions for these five SiCAMTA genes showed that phytohormone and stress response-related cis-elements were predominated in SiCAMTA2 and SiCAMTA5. Also, we studied its modulated expression levels, with special reference to drought and waterlogging stress. It revealed that the SiCAMTA5 and SiCAMTA2 genes were the most responsive to the studied stress factors. The target prediction and network analysis suggested that SiCAMTAs could bind the CGCG cis element in the target gene promoters and predicted 1202 SiCAMTA target genes in the sesame genome, including abiotic stress-responsive genes viz. LEA, PIP1–2, PPO1, SAP, ARF17, and GA3OX1. These findings were validated using qPCR analysis for five CAMTA and 10 CAMTA target genes and establish a foundation for future functional research of SiCAMTA genes towards sesame stress tolerance.

NucMACC: An MNase-seq pipeline to identify structurally altered nucleosomes in the genome.

Micrococcal nuclease sequencing is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Data analysis is complex due to the AT-dependent sequence bias of the endonuclease and the requirement for high sequencing depth. Here, we present the nucleosome-based MNase accessibility (nucMACC) pipeline unveiling the regulatory chromatin landscape by measuring nucleosome accessibility and stability. The nucMACC pipeline represents a systematic and genome-wide approach for detecting unstable ("fragile") nucleosomes. We have characterized the regulatory nucleosome landscape in Drosophila melanogaster, Saccharomyces cerevisiae, and mammals. Two functionally distinct sets of promoters were characterized, one associated with an unstable nucleosome and the other being nucleosome depleted. We show that unstable nucleosomes present intermediate states of nucleosome remodeling, preparing inducible genes for transcriptional activation in response to stimuli or stress. The presence of unstable nucleosomes correlates with RNA polymerase II proximal pausing. The nucMACC pipeline offers unparalleled precision and depth in nucleosome research and is a valuable tool for future nucleosome studies.

Chromatin modifier Hmga2 promotes adult hematopoietic stem cell function and blood regeneration in stress conditions

The molecular mechanisms governing the response of hematopoietic stem cells (HSCs) to stress insults remain poorly defined. Here, we investigated effects of conditional knock-out or overexpression of Hmga2 (High mobility group AT-hook 2), a transcriptional activator of stem cell genes in fetal HSCs. While Hmga2 overexpression did not affect adult hematopoiesis under homeostasis, it accelerated HSC expansion in response to injection with 5-fluorouracil (5-FU) or in vitro treatment with TNF-α. In contrast, HSC and megakaryocyte progenitor cell numbers were decreased in Hmga2 KO animals. Transcription of inflammatory genes was repressed in Hmga2-overexpressing mice injected with 5-FU, and Hmga2 bound to distinct regions and chromatin accessibility was decreased in HSCs upon stress. Mechanistically, we found that casein kinase 2 (CK2) phosphorylates the Hmga2 acidic domain, promoting its access and binding to chromatin, transcription of anti-inflammatory target genes, and the expansion of HSCs under stress conditions. Notably, the identified stress-regulated Hmga2 gene signature is activated in hematopoietic stem progenitor cells of human myelodysplastic syndrome patients. In sum, these results reveal a TNF-α/CK2/phospho-Hmga2 axis controlling adult stress hematopoiesis.

Brief research report: ETS-1 blockade increases ICAM-1 expression in activated human retinal endothelial cells.

Intercellular adhesion molecule 1 (ICAM-1) is a central cell adhesion molecule for retinal transendothelial migration of the leukocytes in non-infectious posterior uveitis. Inhibiting ICAM1 gene transcription reduces induction of ICAM-1 in inflamed retinal endothelium. Based on published literature implicating transcription factor ETS-1 as an activator of ICAM1 gene transcription, we investigated the effect of ETS-1 blockade on ICAM-1 levels in cytokine-stimulated human retinal endothelial cells. We first examined ICAM1 and ETS1 transcript expression in human retinal endothelial cells exposed to tumor necrosis factor-alpha (TNF-α) or interleukin-1beta (IL-1β). ICAM1 and ETS1 transcripts were increased in parallel in primary human retinal endothelial cell isolates (n = 5) after a 4-hour stimulation with TNF-α or IL-1β (p ≤ 0.012 and ≤ 0.032, respectively). We then assessed the effect of ETS-1 blockade by small interfering (si)RNA on cellular ICAM1 transcript and membrane-bound ICAM-1 protein. ETS1 transcript was reduced by greater than 90% in cytokine-stimulated and non-stimulated human retinal endothelial cell monolayers following a 48-hour treatment with two ETS-1-targeted siRNA, in comparison to negative control non-targeted siRNA (p ≤ 0.0002). The ETS-1 blockade did not reduce ICAM1 transcript expression nor levels of membrane-bound ICAM-1 protein, rather it increased both for a majority of siRNA-treatment and cytokine-stimulation conditions (p ≤ 0.018 and ≤ 0.004, respectively). These unexpected findings indicate that ETS-1 blockade increases ICAM-1 transcript and protein levels in human retinal endothelial cells. Thus ETS-1-targeting would be expected to promote rather than inhibit retinal transendothelial migration of leukocytes in non-infectious posterior uveitis.

Dof1.7 and NIGT1 transcription factors mediate multilayered transcriptional regulation for different expression patterns of NITRATE TRANSPORTER2 genes under nitrogen deficiency stress.

Elucidating the mechanisms regulating nitrogen (N) deficiency responses in plants is of great agricultural importance. Previous studies revealed that decreased expression of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) transcriptional repressor genes upon N deficiency is involved in N deficiency-inducible gene expression in Arabidopsis thaliana. However, our knowledge of the mechanisms controlling N deficiency-induced changes in gene expression is still limited. Through the identification of Dof1.7 as a direct target of NIGT1 repressors and a novel N deficiency response-related transcriptional activator gene, we here show that NIGT1 and Dof1.7 transcription factors (TFs) differentially regulate N deficiency-inducible expression of three high-affinity nitrate transporter genes, NRT2.1, NRT2.4, and NRT2.5, which are responsible for most of the soil nitrate uptake activity of Arabidopsis plants under N-deficient conditions. Unlike NIGT1 repressors, which directly suppress NRT2.1, NRT2.4, and NRT2.5 under N-sufficient conditions, Dof1.7 directly activated only NRT2.5 but indirectly and moderately activated NRT2.1 and NRT2.4 under N-deficient conditions, probably by indirectly decreasing NIGT1 expression. Thus, Dof1.7 converted passive transcriptional activation into active and potent transcriptional activation, further differentially enhancing the expression of NRT2 genes. These findings clarify the mechanism underlying different expression patterns of NRT2 genes upon N deficiency, suggesting that time-dependent multilayered transcriptional regulation generates complicated expression patterns of N deficiency-inducible genes.

The roles and mechanisms of ETS1 in autoimmune diseases and cancers: A comprehensive review

E26 transformation-specific-1 (ETS1) is a founding member of the ETS transcription factor family. This transcription factor is highly conserved in amino acid sequences and highly expressed in many immune tissues, such as the thymus, spleen, and lymph gland. ETS1 plays multiple regulatory roles in immune-related diseases and acts as a transcriptional activator or inhibitor of many genes to regulate immune cell differentiation, development, apoptosis, and tumor occurrence. The expression level of ETS1 is correlated with disease severity. However, the molecular mechanisms behind disease and tumor progression mediated by ETS1 have not been fully elucidated. In addition, the therapeutic potential of ETS1 in clinical treatment remains to be further explored. Here, we review and summarize the molecular structure and functions of ETS1 and then focus on the roles of ETS1 in the occurrence of autoimmune diseases and cancers. This review provides a reference corroborating ETS1 as a potential therapeutic target for immune-related diseases and cancers.

Targeting class A GPCRs for hard tissue regeneration

G protein-coupled receptors (GPCRs) play important roles in various pathogeneses and physiological regulations. Owing to their functional diversity, GPCRs are considered one of the primary pharmaceutical targets. However, drugs targeting GPCRs have not been developed yet to regenerate hard tissues such as teeth and bones. Mesenchymal stromal cells (MSCs) have high proliferation and multi-lineage differentiation potential, which are essential for hard tissue regeneration. Here, we present a strategy for targeting class A GPCRs for hard tissue regeneration by promoting the differentiation of endogenous MSCs into osteogenic and odontogenic progenitor cells. Through in vitro screening targeted at class A GPCRs, we identified six target receptors (LPAR1, F2R, F2RL1, F2RL2, S1PR1, and ADORA2A) and candidate drugs with potent biomineralization effects. Through a combination of profiling whole transcriptome and accessible chromatin regions, we identified that p53 acts as a key transcriptional activator of genes that modulate the biomineralization process. Moreover, the therapeutic potential of class A GPCR-targeting drugs was demonstrated in tooth pulpotomy and calvarial defect models. The selected drugs revealed potent regenerative effects in both tooth and bone defects, represented by newly formed highly mineralized regions. Consequently, this study provides translational evidence for a new regenerative strategy for damaged hard tissue.

Conserved and Unique Regulatory Mechanisms of RUNX1 in Hematopoietic Stem/Progenitor Cell Subpopulations in Both Normal Hematopoiesis and FPD/AML Development

RUNX1 is a RUNT domain core-binding factor indispensable for the establishment of definitive hematopoiesis in vertebrate. In humans, heterozygous RUNX1 germline mutations are closely associated with Familial Platelet Disorder with propensity to develop AML (FPD/AML), a rare autosomal dominant disorder characterized by thrombocytopenia, platelet dysfunction, and markedly elevated risk for myelodysplastic syndrome and leukemia. RUNX1 regulates hematopoietic genes directly or indirectly, acting as a context-dependent activator or repressor of gene transcription. Here we have examined how RUNX1 regulates various subpopulations of hematopoietic stem/progenitor cells (HSPCs) to provide important insights into its physiologic and pathogenic roles and mechanisms.We used two complementary mouse models: a conditional RUNX1 knockout mouse, i,e, Mx-Cre;Runx1flox/flox, and a tetracycline-inducible FPD/AML patient derived RUNX1 S291fsX300 mutation knock-in crossed to a MLL-PTD knock-in mouse, i.e. MLL-PTD;RUNX1 S291fsX300. We monitored hematopoiesis and/or disease progression in both models by tracking CBCs in peripheral blood and analyzing bone marrow cells by flow cytometry. The bone marrow samples were sorted by flow cytometry to yield HSPC subpopulations: HSC (LSK CD34-Flt3-), GMP (LK FCRII/III+CD34+), and MEP (LK FCRII/III-CD34-). Cells were analyzed for transcriptome by deep RNA-seq, epigenetics by ATAC-seq, and gene targets by anti-RUNX1 CHIP-seq. Focusing on the HSC, GMP, and MEP subpopulations, we found that RUNX1 deletion in the hematopoietic system causes thrombocytopenia and HSC engraftment defects, and dramatic changes in HSPC subpopulations: RUNX1 deficiency resulted in an increase MegaK-primed HSCs via blocking their direct differentiation to megakaryocytes leading to thrombocytopenia, and caused a marked increase of GMPs and a significant decrease of MEPs. Induction of RUNX1 S291fsX300 mutant in MLL-PTD;RUNX1 S291fsX300 mice mimicked that of RUNX1 deletion, resulting similarly in thrombocytopenia, an expansion of Mega-HSCs, increased GMPs, and decreased MEPs. “Correction” of the RUNX1S291fsX300 mutation by doxycycline withdraw reversed the FPD phenotype as well as the cellular distribution changes in HSCs, GMPs and MEPs. RNA-seq analyses of DEGs found that RUNX1 acts both as an activator and a suppressor in gene transcription in HSPCs. In HSCs, among 1879 DEGs, 608 are upregulated and 1271 are downregulated in Runx1 -/- compared with WT cells. In GMPs, 2042 DEGs were seen, among which 908 are upregulated and 1134 are downregulated whereas in MEPs, 316 transcripts were upregulated and 1214 were downregulated amongst 1530 DEGs in Runx1 -/- cells. Combining the DEGs from RNA-seq with annotated ATAC-seq peaks, we discovered through gene ontology analyses that a set of systemic development genes are commonly regulated by RUNX1 via chromosome accessibility in all HSPC subpopulations, with potentially shared targets such as Jam2, Bcar1, Irf4 Insrr , Cxcl12, Tnfsf4, Rap1gap, Aldh1a1. In contrast, in HSCs, a unique set of hemostasis/platelet regulatory genes such as Itga2b, Gp5, Pdgfb and Vwf were seen as potential targets, consistent with our recent findings ( Blood Adv 7:2590). In GMPs, RUNX1 appears to specifically regulate inflammatory and innate immunity genes with potential targets such as Gfi1b, Ets1, Tnfrsf11a while in MEP, RUNX1 specifically regulates cell motility and migration gene sets affecting targets such as Notch1, Ifitm1, Hoxa7, Cd300a Cxcl10 Fut7, suggesting a role of RUNX1 in modulating MEP movement/location in the bone marrow. RUNX1 CHIP-seq analyses are under way to further clarify the potential targets in each HSPC subpopulation. Our studies indicate that RUNX1 regulates common as well as distinct transcriptional events in various HSPC subpopulations by context-dependent activation and suppression mechanisms. RUNX1 plays a universal role in the systemic developmental program of various HSPC subpopulations but appears to engage in unique lineage specific factors to deliver subpopulation-specific functions such as mega-K cell fate-priming of HSCs, inflammatory program in GMPs, and bone marrow location signals in MEPs. Ongoing analyses of the FPD/AML mouse genomic data in this context are expected to reveal contributions of patient RUNX1 mutations to the disease development.

ETHE1 Accelerates Triple-Negative Breast Cancer Metastasis by Activating GCN2/eIF2α/ATF4 Signaling.

Triple-negative breast cancer (TNBC) is the most fatal subtype of breast cancer; however, effective treatment strategies for TNBC are lacking. Therefore, it is important to explore the mechanism of TNBC metastasis and identify its therapeutic targets. Dysregulation of ETHE1 leads to ethylmalonic encephalopathy in humans; however, the role of ETHE1 in TNBC remains elusive. Stable cell lines with ETHE1 overexpression or knockdown were constructed to explore the biological functions of ETHE1 during TNBC progression in vitro and in vivo. Mass spectrometry was used to analyze the molecular mechanism through which ETHE1 functions in TNBC progression. ETHE1 had no impact on TNBC cell proliferation and xenograft tumor growth but promoted TNBC cell migration and invasion in vitro and lung metastasis in vivo. The effect of ETHE1 on TNBC cell migratory potential was independent of its enzymatic activity. Mechanistic investigations revealed that ETHE1 interacted with eIF2α and enhanced its phosphorylation by promoting the interaction between eIF2α and GCN2. Phosphorylated eIF2α in turn upregulated the expression of ATF4, a transcriptional activator of genes involved in cell migration and tumor metastasis. Notably, inhibition of eIF2α phosphorylation through ISRIB or ATF4 knockdown partially abolished the tumor-promoting effect of ETHE1 overexpression. ETHE1 has a functional and mechanistic role in TNBC metastasis and offers a new therapeutic strategy for targeting ETHE1-propelled TNBC using ISRIB.

YY1 is regulated by ALKBH5-mediated m6A modification and promotes autophagy and cancer progression through targeting ATG4B.

YY1 affects tumorigenesis and metastasis in multiple ways. However, the function of YY1 and the potential mechanisms through which it operates in gastric cancer (GC) progression by regulating autophagy remains poorly understood. This study aimed to assess the essential transcription factors (TFs) involved in autophagy regulation in GC. Western blot, RFP-GFP-LC3 double fluorescence and transmission electron microscopy (TEM) assays were used to probe autophagy activity in GC cells. Methylated RNA immunoprecipitation (MeRIP) was utilized to evaluate the ALKBH5-regulated m6A levels of YY1. Gain- and loss-of-function assays were employed in the scrutiny of the biological effects of the ALKBH5/YY1/ATG4B axis on cancer cell proliferation and invasion abilities in vitro. Per the findings, YY1 was identified as a crucial transcriptional activator of cancer autophagy-related genes and promoted the proliferation and aggressiveness of cancer cells associated with enhanced ATG4B-mediated autophagy. However, ectopic ALKBH5 expression abolished the YY1-induced effect via m6A modification. Importantly, YTHDF1 facilitated the mRNA stability of YY1 through m6A recognition. Collectively, this study found that YY1 was regulated by ALKBH5 and YTHDF1-mediated m6A modification and served as an autophagy-dependent tumor driver to accelerate cancer progression through ATG4B transactivation, providing an exploitable therapeutic target for GC.

Hnrnpk is essential for embryonic limb bud development as a transcription activator and a collaborator of insulator protein Ctcf

Proper development of the limb bud relies on the concordance of various signals, but its molecular mechanisms have not yet been fully illustrated. Here we report that heterogeneous nuclear ribonucleoprotein K (hnRNPK) is essential for limb bud development. Its ablation in the limb bud results in limbless forelimbs and severe deformities of the hindlimbs. In terms of mechanism, hnRNPK functions as a transcription activator for the vital genes involved in the three regulatory axes of limb bud development. Simultaneously, for the first time we elucidate that hnRNPK binds to and coordinates with the insulator protein CCCTC binding factor (CTCF) to maintain a three-dimensional chromatin architecture. Ablation of hnRNPK weakens the binding strength of CTCF to topologically associating domain (TAD) boundaries, then leading to the loose TADs, and decreased interactions between promoters and enhancers, and further decreased transcription of developmental genes. Our study establishes a fundamental and novel role of hnRNPK in regulating limb bud development.

Euo is a developmental regulator that represses late genes and activates midcycle genes in Chlamydia trachomatis.

In this study, we developed a correlative approach that combined DNA immunoprecipitation-seq and RNA-seq analyses to define the regulon of the Chlamydia trachomatis transcription factor Euo. We confirmed the proposed role of Euo as a transcriptional repressor of late chlamydial genes but also showed that Euo activates transcription of a subset of midcycle genes and autoregulates its own expression via negative feedback. This study validates and expands the role of Euo as an important developmental regulator in C. trachomatis. In addition, this genome-wide correlative approach can be applied to study transcription factors in other pathogenic bacteria.

Abstract P3033: Thrombospondin-1 Promotes Vascular Smooth Muscle Cell De-differentiation In Diabetes

Diabetes is a major risk factor for atherosclerosis. Diabetic patients are prone to increased vascular smooth muscle cell (VSMC) migration and proliferation, hallmarks of VSMC de-differentiation, critical for the evolution of atherosclerosis. Multiple studies, including our earlier work, support the role of thrombospondin-1 (TSP1), a proatherogenic matricellular protein, as a putative link between diabetes and vascular disease. However, molecular mechanism(s) by which TSP1 promotes diabetic vasculopathy are incompletely understood. The goal of this work was to explore whether smooth muscle TSP1 (smTSP1) regulates VSMC phenotypic switch in diabetes. We generated conditional SMC-specific TSP1 knockout (smTSP1 KO ) mice by crossing female TSP1 fl/fl mice with tamoxifen (tmx)-inducible Myh11-CreER T2 male mice. For Cre recombination, 5-wks-old male TSP1 fl/fl Cre tg and age-matched TSP1 +/+ Cre tg littermate mice were treated with 60 mg/Kg/day tmx i.p. once daily for 5 consecutive days. To induce type 2 diabetes, mice cohorts were placed on western diet at 7-wks-age followed by streptozotocin (50 mg/Kg/day, i.p. x 5 days) at 16-wks-age. Mice were harvested at 20-wks-age for plasma and tissue collection. Immunoblotting of aortic lysates revealed significant decrease in TSP1 expression in tmx-treated TSP1 fl/fl Cre tg , validating smTSP1 KO mice. Lack of smTSP1 increased ACTA2 (SM contractile marker) expression by 2-fold accompanied with 50% reduction in CDK4 (cell cycle regulator) expression in diabetic smTSP1 KO vs. smTSP1 WT aortic vessels. Consistently, in murine aortic SMC (MASMC) primary cultures derived from smTSP1 KO mice, loss of smTSP1 augmented (>65%) LMOD1 (SM contractile marker) and SRF (transcriptional activator of SM contractile genes) expression coupled with diminished (45%) vimentin (SM synthetic marker) expression in 25mM glucose-treated smTSP1 KO vs. smTSP1 WT cells. Further, propidium iodide staining via flow cytometry revealed reduced S-phase cell counts concomitant to increased G 0 -G 1 cell distribution in glucose-treated smTSP1 KO vs. smTSP1 WT MASMC. Together, our data demonstrates a direct role of TSP1 in VSMC phenotypic transition and opens novel avenues for cell-specific TSP1-based therapies for treatment of diabetes.

Molecular dissection studies of TAC1, a transcription activator of Candida drug resistance genes of the human pathogenic fungus Candida albicans.

The up-regulation of ABC transporters Cdr1p and Cdr2p that efflux antifungal azole drugs are a leading cause of Multi-Drug Resistance (MDR) in the white fungus Candida albicans. C. albicans was reported to infect patients following the recent Covid-19 pandemic after they were given steroids for recovery. Previously, the TAC1 gene was identified as the transcriptional activator of Candida drug resistance genes (CDR1 and CDR2) and has no known human homologs. This makes it a good target for the development of novel antifungals. We, therefore, carried out the molecular dissection study of TAC1 to understand the functional regulation of the ABC transporter genes (CDR1 and CDR2) under its control. The N-terminal DNA Binding Domain (DBD) of Tac1p interacts with the Drug Responsive Element (DRE) present in the upstream promoter region of CDR1 and CDR2 genes of C. albicans. The interaction between DBD and DRE recruits Tac1p to the promoter of CDR genes. The C-terminal Acidic Activation Domain (AAD) of Tac1p interacts with the TATA box Binding Protein (TBP) and thus recruits TBP to the TATA box of CDR1 and CDR2 genes. Taking a cue from a previous study involving a TAC1 deletion strain that suggested that Tac1p acts as a xenobiotic receptor, in this study, we identified that the Middle Homology Region (MHR) of Tac1p acts as a probable xenobiotic binding domain (XBD) which plays an important role in Candida drug resistance. In addition, we studied the role of Tac1p in the regulation of some lipid profiling genes and stress response genes since they also contain the DRE consensus sequence and found that some of them can respond to xenobiotic stimuli.

Effect of the S008-sgaCD operon on IncC plasmid stability in the presence of SGI1-K or absence of an SGI1 variant

An IncC or IncA plasmid is needed to enable transfer of SGI1 type integrative mobilisable elements but an IncC plasmid does not stably co-exist with SGI1. However, the plasmid is stably maintained with SGI1-K, a natural SGI1 deletion variant that lacks the sgaDC genes (S007 and S006) and the upstream open reading frame (S008) found in the SGI1 backbone. Here, the effect of the sgaDC genes and S008 on the stability of an IncC plasmid in an Escherichia coli strain with or without SGI1-K was examined. Co-transcription of the S008 open reading frame with the downstream sgaDC genes was established. When a strain containing SGI1-K complemented with a pK18 plasmid that included S008-sgaDC or sgaDC expressed from the constitutive pUC promoter was grown without antibiotic selection, the resident IncC plasmid was rapidly lost but loss was slower when S008 was present. In contrast, SGI1-K and the S008-sgaDC or sgaDC plasmid were quite stably maintained for >100 generations. However, the high copy number plasmids carrying the SGI1-derived S008-sgaDC or sgaDC genes constitutively expressed could not be introduced into an E. coli strain carrying the IncC plasmid but without SGI1-K. Using equivalent plasmids with S008-sgaDC or sgaDC genes controlled by an arabinose-inducible promoter, under inducing conditions the IncC plasmid was stable but the plasmid containing the SGI1-derived genes was rapidly lost. This unexpected observation indicates that there are multiple interactions between the IncC plasmid and SGI1 in which the transcriptional activator genes sgaDC play a role. These interactions will require further investigation.

Ornithine is the central intermediate in the arginine degradative pathway and its regulation in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis can utilize several proteinogenic and non-proteinogenic amino acids as sources of carbon, nitrogen, and energy. The utilization of the amino acids arginine, citrulline, and ornithine is catalyzed by enzymes encoded in the rocABC and rocDEF operons and by the rocG gene. The expression of these genes is controlled by the alternative sigma factor SigL. RNA polymerase associated with this sigma factor depends on ATP-hydrolyzing transcription activators to initiate transcription. The RocR protein acts as a transcription activator for the roc genes. However, the details of amino acid metabolism via this pathway are unknown. Here, we investigated the contributions of all enzymes of the Roc pathway to the degradation of arginine, citrulline, and ornithine. We identified the previously uncharacterized RocB protein as responsible for the conversion of citrulline to ornithine. Invitro assays with the purified enzyme suggest that RocB acts as a manganese-dependent N-carbamoyl-L-ornithine hydrolase that cleaves citrulline to form ornithine and carbamate. Moreover, the molecular effector that triggers transcription activation by RocR has not been unequivocally identified. Using a combination of transcription reporter assays and biochemical experiments, we demonstrate that ornithine is the molecular inducer of RocR activity. Taken together, our work suggests that binding of ATP to RocR triggers its hexamerization, and binding of ornithine then allows ATP hydrolysis and activation of roc gene transcription. Thus, ornithine is the central molecule of the roc degradative pathway as it is the common intermediate of arginine and citrulline degradation and the molecular effector of RocR.

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.