General Transcription Factor Research Articles

Comprehensive Analysis of Prognostic and Genetic Signatures for General Transcription Factor III (GTF3) in Clinical Colorectal Cancer Patients Using Bioinformatics Approaches.

Colorectal cancer (CRC) has the fourth-highest incidence of all cancer types, and its incidence has steadily increased in the last decade. The general transcription factor III (GTF3) family, comprising GTF3A, GTF3B, GTF3C1, and GTFC2, were stated to be linked with the expansion of different types of cancers; however, their messenger (m)RNA expressions and prognostic values in colorectal cancer need to be further investigated. To study the transcriptomic expression levels of GTF3 gene members in colorectal cancer in both cancerous tissues and cell lines, we first performed high-throughput screening using the Oncomine, GEPIA, and CCLE databases. We then applied the Prognoscan database to query correlations of their mRNA expressions with the disease-specific survival (DSS), overall survival (OS), and disease-free survival (DFS) status of the colorectal cancer patient. Furthermore, proteomics expressions of GTF3 family members in clinical colorectal cancer specimens were also examined using the Human Protein Atlas. Finally, genomic alterations of GTF3 family gene expressions in colorectal cancer and their signal transduction pathways were studied using cBioPortal, ClueGO, CluePedia, and MetaCore platform. Our findings revealed that GTF3 family members’ expressions were significantly correlated with the cell cycle, oxidative stress, WNT/β-catenin signaling, Rho GTPases, and G-protein-coupled receptors (GPCRs). Clinically, high GTF3A and GTF3B expressions were significantly correlated with poor prognoses in colorectal cancer patients. Collectively, our study declares that GTF3A was overexpressed in cancer tissues and cell lines, particularly colorectal cancer, and it could possibly step in as a potential prognostic biomarker.

Structures of mammalian RNA polymerase II pre-initiation complexes.

The initiation of transcription is a focal point for the regulation of gene activity during mammalian cell differentiation and development. To initiate transcription, RNA polymerase II (PolII) assembles with general transcription factors into a pre-initiation complex (PIC) that opens promoter DNA. Previous work provided the molecular architecture of the yeast1-9 and human10,11 PIC and a topological model for DNA opening by the general transcription factor TFIIH12-14. Here we report the high-resolution cryo-electron microscopy structure of PIC comprising human general factors and Sus scrofa domesticus PolII, which is 99.9% identical to human PolII. We determine the structures of PIC with closed and opened promoter DNA at 2.5-2.8Å resolution, and resolve the structure of TFIIH at 2.9-4.0Å resolution. We capture the TFIIH translocase XPB in the pre- and post-translocation states, and show that XPB induces and propagates a DNA twist to initiate the opening of DNA approximately 30 base pairs downstream of the TATA box. We also provide evidence that DNA opening occurs in two steps and leads to the detachment of TFIIH from the core PIC, which may stop DNA twisting and enable RNA chain initiation.

Structure of the human Mediator-RNA polymerase II pre-initiation complex.

Mediator is a conserved coactivator complex that enables the regulated initiation of transcription at eukaryotic genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate the phosphorylation of RNA polymeraseII (PolII) and promoter escape1-6. Here we prepare a recombinant version of human Mediator, reconstitute a 50-subunit Mediator-PIC complex and determine the structure of the complex by cryo-electron microscopy. The head module of Mediator contacts the stalk of PolII and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions observed in the corresponding complex in yeast7-9. The metazoan subunits MED27-MED30 associate with exposed regions in MED14 and MED17 to form the proximal part of the Mediator tail module that binds activators. Mediator positions the flexibly linked cyclin-dependent kinase (CDK)-activating kinase of the general transcription factor TFIIH near the linker to the C-terminal repeat domain of PolII. The Mediator shoulder domain holds the CDK-activating kinase subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook domains reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of the CDK7 kinase to allosterically stimulate phosphorylationof the C-terminal domain.

Identification of NTRK3 as a potential prognostic biomarker associated with tumor mutation burden and immune infiltration in bladder cancer

BackgroundBladder cancer (BLCA) is a common malignant tumor of urinary system with high morbidity and mortality. In recent years, immunotherapy has played a significant role in the treatment of BLCA. Tumor mutation burden (TMB) has been reported to be a powerful biomarker for predicting tumor prognosis and efficacy of immunotherapy. Our study aimed to explore the relationship between TMB, prognosis and immune infiltration to identify the key genes in BLCA.MethodsClinical information, somatic mutation and gene expression data of BLCA patients were downloaded from The Cancer Genome Atlas (TCGA) database. Patients were divided into high and low TMB groups according to their calculated TMB scores. Gene Set Enrichment Analysis (GSEA) was performed to screen for significantly enriched pathways. Differentially expressed genes (DEGs) between the two groups were identified. Univariate Cox analysis and Kaplan-Meier survival analysis were applied for screening key genes. Immune infiltration was performed for TMB groups and NTRK3.ResultsHigher TMB scores were related with poor survival in BLCA. After filtering, 36 DEGs were identified. NTRK3 had the highest hazard ratio and significant prognostic value. Co-expressed genes of NTRK3 were mainly involved in several pathways, including DNA replication, basal transcription factors, complement and coagulation cascades, and ribosome biogenesis in eukaryotes. There was a significant correlation among TMB scores, NTRK3 expression and immune infiltration.ConclusionsOur results suggest that NTRK3 is a TMB-related prognostic biomarker, which lays the foundation for further research on the immunomodulatory effect of NTRK3 in BLCA.

A review on herbal Nrf2 activators with preclinical evidence in cardiovascular diseases.

Cardiovascular diseases (CVDs) are an ever-growing problem and are the most common cause of death worldwide. The uncontrolled production of reactive oxygen species (ROS) and the activation of ROS associated with various cell signaling pathways with oxidative cellular damage are the most common pathological conditions connected with CVDs including endothelial dysfunction, hypercontractility of vascular smooth muscle, cardiac hypertrophy and heart failure. The nuclear factor E2-related factor 2 (Nrf2) is a basic leucine zipper redox transcription factor, together with its negative regulator, kelch-like ECH-associated protein 1 (Keap1), which serves as a key regulator of cellular defense mechanisms to combat oxidative stress and associated diseases. Multiple lines of evidence described here support the cardiac protective property of Nrf2 in various experimental models of cardiac related disease conditions. In this review, we emphasized the molecular mechanisms of Nrf2 and described the detailed outline of current findings on the therapeutic possibilities of the Nrf2 activators specifically from herbal origin in various CVDs. Based on evidence from various preclinical experimental models, we have highlighted the activation of Nrf2 pathway as a budding therapeutic option for the prevention and treatment of CVDs, which needs further investigation and validation in the clinical settings.

A homozygous variant in TBPL2 was identified in women with oocyte maturation defects and infertility.

What are the genetic causes of oocyte maturation defects? A homozygous splicing variant (c.788 + 3A>G) in TATA-box binding protein like 2 (TBPL2) was identified as a contributory genetic factor in oocyte maturation defects. TBPL2, a vertebrate oocyte-specific general transcription factor, is essential for oocyte development. TBPL2 variants have not been studied in human oocyte maturation defects. Two infertile families characterized by oocyte maturation defects were recruited for whole-exome sequencing (WES). Genomic DNA was extracted from peripheral blood for WES analysis. Sanger sequencing was performed for data validation. Pathogenicity of variants was predicted by in silico analysis. Minigene assay and single-oocyte RNA sequencing were performed to investigate the effects of the variant on mRNA integrity and oocyte transcriptome, respectively. A homozygous splicing variant (c.788 + 3A>G) in TBPL2 was identified in two unrelated families characterized by oocyte maturation defects. Haplotype analysis indicated that the disease allele of Families 1 and 2 was independent. The variant disrupted the integrity of TBPL2 mRNA. Transcriptome sequencing of affected oocytes showed that vital genes for oocyte maturation and fertilization were widely and markedly downregulated, suggesting that a mutation in the transcriptional factor, TBPL2, led to global gene alterations in oocytes. Limitations include the lack of direct functional evidence. Owing to the scarcity of human oocyte samples, only two immature MI oocytes were obtained from the patients, and we could only investigate the effect of the mutation at the transcriptional level by high-throughput sequencing technology. No extra oocytes were obtained to assess the transcriptional activity of the mutant oocytes by immunofluorescence, or investigate the effects on the binding of TBPL2 caused by the mutation. Our findings highlight a critical role of TBPL2 in female reproduction and identify a homozygous splicing mutation in TBPL2 that might be related to defects in human oocyte maturation. This information will facilitate the genetic diagnosis of infertile individuals with repeated failures of IVF, providing a basis for genetic counseling. This study was supported by the National Key Research and Development Program of China (2018YFC1004000, 2017YFC1001504 and 2017YFC1001600), the National Natural Science Foundation of China (81871168, 31900409 and 31871509), the Foundation for Distinguished Young Scholars of Shandong Province (JQ201816), the Innovative Research Team of High-Level Local Universities in Shanghai (SSMU-ZLCX20180401) and the Fundamental Research Funds of Shandong University. The authors have no competing interests to declare. N/A.

Upregulation of glucosamine-phosphate N-acetyltransferase 1 is a promising diagnostic and predictive indicator for poor survival in patients with lung adenocarcinoma.

Lung adenocarcinoma, a type of non-small cell lung cancer, is the leading cause of cancer death worldwide. Great efforts have been made to identify the underlying mechanism of adenocarcinoma, especially in relation to oncogenes. The present study by integrating computational analysis with western blotting, aimed to understand the role of the upregulation of glucosamine-phosphate N-acetyltransferase 1 (GNPNAT1) in carcinogenesis. In the present study, publicly available gene expression profiles and clinical data were downloaded from The Cancer Genome Atlas to determine the role of GNPNAT1 in lung adenocarcinoma (LUAD). In addition, the association between LUAD susceptibility and GNPNAT1 upregulation were analyzed using Wilcoxon signed-rank test and logistic regression analysis. In LUAD, GNPNAT1 upregulation was significantly associated with disease stage [odds ratio (OR)=2.92, stage III vs. stage I], vital status (dead vs. alive, OR=1.89), cancer status (tumor status vs. tumor-free status, OR=1.85) and N classification (yes vs. no, OR=1.75). Cox regression analysis and the Kaplan-Meier method were utilized to evaluate the association between GNPNAT1 expression and overall survival (OS) time in patients with LUAD. The results demonstrated that patients with increased GNPNAT1 expression levels exhibited a reduced survival rate compared with those with decreased expression levels (P=8.9×10−5). In addition, Cox regression analysis revealed that GNPNAT1 upregulation was significantly associated with poor OS time [hazard ratio (HR): 1.07; 95% confidence interval (CI): 1.04–1.10; P<0.001]. The gene set enrichment analysis revealed that ‘cell cycle’, ‘oocyte meiosis’, ‘pyrimidine mediated metabolism’, ‘ubiquitin mediated proteolysis’, ‘one carbon pool by folate’, ‘mismatch repair progesterone-mediated oocyte maturation’ and ‘basal transcription factors purine metabolism’ were differentially enriched in the GNPNAT1 high-expression samples compared with GNPNAT1 low-expression samples. The aforementioned pathways are involved in the pathogenesis of LUAD. The findings of the present study suggested that GNPNAT1 upregulation may be considered as a promising diagnostic and prognostic biomarker in patients with LUAD. In addition, the aforementioned pathways may be pivotal pathways perturbed by the abnormal expression of GNPNAT1 in LUAD. The findings of the present study demonstrated the therapeutic value of the regulation of GNPNAT1 in lung adenocarcinoma.

Planarian stem cells specify fate yet retain potency during the cell cycle.

Planarian whole-body regeneration is enabled by stem cells called neoblasts. At least some neoblasts are individually pluripotent. Neoblasts are also heterogeneous, with subpopulations of specialized neoblasts having different specified fates. Fate specification in neoblasts is regulated by fate-specific transcription factor (FSTF) expression. Here, we find that FSTF expression is common in neoblast S/G2/M cell-cycle phases but less common in G1. We find that specialized neoblasts can divide to produce progeny with asymmetric cell fates, suggesting that they could retain pluripotency. Furthermore, no known neoblast class was present in all neoblast colonies, suggesting that pluripotency is not the exclusive property of any known class. We tested this possibility with single-cell transplantations, which indicate that at least some specialized neoblasts are likely clonogenic. On the basis of these findings, we propose a model for neoblast pluripotency in which neoblasts can undergo specialization during the cell cycle without loss of potency.

A simple protocol to purify human TFIID free of the MED26 subunit of mediator complex

The general transcription factor TFIID is a multiprotein complex that is essential for specific transcription initiation by RNA polymerase II. It is composed of the TATA box-binding protein (TBP) and ~13 different TBP-associated factors (TAFs). Purification of TFIID free of other general transcription factors and coactivators is essential to analyze the transcription regulatory mechanisms in reconstituted systems in vitro. A breakthrough in TFIID purification was the generation of HeLa cell lines that express a FLAG epitope-tagged TBP subunit and immunopurification protocols with monoclonal anti-FLAG antibodies. Purification of TFIID from HeLa nuclear extracts generally required a two-step purification procedure involving phosphocellulose P11 chromatography followed by anti-flag M2 affinity purification (Chiang et al., 1993; Ge et al., 1996) [1,2]. Here we show first that the MED26 (CRSP70) coactivator subunit of Mediator co-purifies with TFIID in the above two-step protocol and interacts strongly with TFIID under high salt conditions. We further show that a MED26-free TFIID complex can be obtained by including a simple additional DE52 chromatography step following P11 fractionation. Thus, we demonstrate that MED26 strongly interacts with TFIID and recommend the use of a P11-DE52-M2 resin affinity three-step purification procedure to obtain MED26-free TFIID for analyzing Mediator-dependent transcription regulatory mechanisms in purified transcription systems in vitro.

Expression Analysis of MIST1 and EMT Markers in Primary Tumor Samples Points to MIST1 as a Biomarker of Cervical Cancer.

BackgroundMist1 is a basic transcription factor, which plays an important role in the development of multiple organs, and may also regulate tumor progression by mediating epithelial-mesenchymal transformation. However, there is lack of research on its role of squamous cell carcinoma, especially in cervical squamous cell carcinoma.MethodsBioinformatic methods were used to analyze gene expression, correlation, and patient survival according to the TCGA database. Thirty pairs of cancer tissues and distal cancer tissues from cervical cancer patients who received radical surgery were enrolled in the study. The expression of Mist1 was analyzed using Western blot. Furthermore, the potential associations among Mist1 expression, EMT biomarkers and various clinicopathological characteristics were investigated. All statistical tests employed in this study were two-sided, and P values <0.05 were deemed statistically significant.ResultsOverall survival data were obtained from TCGA-CESC dataset, containing 3 control samples and 305 tumor samples. The expression of Mist1 was significantly higher in primary tumor than in normal tissues (P<0.001). The samples were divided into a low Mist1 expression group (n=144) and a high Mist1 expression group (n=146) according to the median expression level. Kaplan–Meier survival analysis revealed that high expression of Mist1 was significantly correlated with poor overall survival (P=0.032). We further explored the relationships between Mist1 and EMT. Among the 30 primary cervical cancer specimens investigated, the difference in Mist1 expressed statuses between cervical cancer tissues and distal noncancerous cervical tissues was significant (P=0.001). And the epithelial cell marker E-cadherin was downregulated in Mist1 overexpressed cervical cancer cells; however, the mesenchymal marker N-Cadherin and Twist was upregulated.ConclusionOur study found that Mist1 seemed to play the role of oncogene in cervical squamous cell carcinoma and could be a potential biomarker.

Functional genomics study of protein inhibitor of activated STAT1 in mouse hippocampal neuronal cells revealed by RNA sequencing.

Protein inhibitor of activated STAT1 (PIAS1), a small ubiquitin-like modifier (SUMO) E3 ligase, was considered to be an inhibitor of STAT1 by inhibiting the DNA-binding activity of STAT1 and blocking STAT1-mediated gene transcription in response to cytokine stimulation. PIAS1 has been determined to be involved in modulating several biological processes such as cell proliferation, DNA damage responses, and inflammatory responses, both in vivo and in vitro. However, the role played by PIAS1 in regulating neurodegenerative diseases, including Alzheimer’s disease (AD), has not been determined. In our study, significantly different expression levels of PIAS1 between normal controls and AD patients were detected in four regions of the human brain. Based on a functional analysis of Pias1 in undifferentiated mouse hippocampal neuronal HT-22 cells, we observed that the expression levels of several AD marker genes could be inhibited by Pias1 overexpression. Moreover, the proliferation ability of HT-22 cells could be promoted by the overexpression of Pias1. Furthermore, we performed RNA sequencing (RNA-seq) to evaluate and quantify the gene expression profiles in response to Pias1 overexpression in HT-22 cells. As a result, 285 significantly dysregulated genes, including 79 upregulated genes and 206 downregulated genes, were identified by the comparison of Pias1/+ cells with WT cells. Among these genes, five overlapping genes, including early growth response 1 (Egr1), early growth response 2 (Egr2), early growth response 3 (Egr3), FBJ osteosarcoma oncogene (Fos) and fos-like antigen 1 (Fosl1), were identified by comparison of the transcription factor binding site (TFBS) prediction results for STAT1, whose expression was evaluated by qPCR. Three cell cycle inhibitors, p53, p18 and p21, were significantly downregulated with the overexpression of Pias1. Analysis of functional enrichment and expression levels showed that basic region leucine zipper domain-containing transcription factors including zinc finger C2H2 (zf-C2H2), homeobox and basic/helix-loop-helix (bHLH) in several signaling pathways were significantly involved in PIAS1 regulation in HT-22 cells. A reconstructed regulatory network under PIAS1 overexpression demonstrated that there were 43 related proteins, notably Nr3c2, that directly interacted with PIAS1.

Genetic parameter estimates and identification of SNPs associated with growth traits in Senegalese sole

Breeding programs are essential for aquaculture. The present study was conducted to investigate the genetic parameters of four growth traits in Senegalese sole. Families were produced by mass spawning (n = 2171 offspring) from an industrial broodstock. Weight, total area, standard length and width (the two latter measured directly on the fish and by image analysis) were determined before the on-growing stage (age ~ 400 d) and at harvest (~800 d) in RAS. Phenotypic data are presented and discussed. Females grew faster than males and a high variation of sex ratios by family was observed. Heritabilities were high for all traits ranging from 0.568 to 0.609 at 400 d and from 0.424 to 0.500 at 800d. The genetic correlations between traits were very high (>0.94) at both ages. Genetic estimates for traits measured in vivo and by digital image analysis were similar. To investigate genomic regions associated with these growth traits, a low-density array using a set of 60 single nucleotide polymorphisms (SNPs) distributed in 17 linkage groups was designed. Using samples from wild fish, a total of 49 assays for SNP analysis were validated. The association analysis was carried out using two fast-growth families and two slow-growth families (n = 279). Two significantly-associated SNPs with most of the traits at both ages were detected. They were located in the general transcription factor 3C polypeptide 4 (gtf3c4) and the mitochondrial fission process protein 1 (mtfp1). Results are of high relevance for genetic breeding programs in this species.

High-throughput transcriptome sequencing and comparative analysis of Escherichia coli and Schizosaccharomyces pombe in respiratory and fermentative growth

In spite of increased complexity in eukaryotes compared to prokaryotes, several basic metabolic and regulatory processes are conserved. Here we explored analogies in the eubacteria Escherichia coli and the unicellular fission yeast Schizosaccharomyces pombe transcriptomes under two carbon sources: 2% glucose; or a mix of 2% glycerol and 0.2% sodium acetate using the same growth media and growth phase. Overall, twelve RNA-seq libraries were constructed. A total of 593 and 860 genes were detected as differentially expressed for E. coli and S. pombe, respectively, with a log2 of the Fold Change ≥ 1 and False Discovery Rate ≤ 0.05. In aerobic glycolysis, most of the expressed genes were associated with cell proliferation in both organisms, including amino acid metabolism and glycolysis. In contrast in glycerol/acetate condition, genes related to flagellar assembly and membrane proteins were differentially expressed such as the general transcription factors fliA, flhD, flhC, and flagellum assembly genes were detected in E. coli, whereas in S. pombe genes for hexose transporters, integral membrane proteins, galactose metabolism, and ncRNAs related to cellular stress were overexpressed. In general, our study shows that a conserved "foraging behavior" response is observed in these eukaryotic and eubacterial organisms in gluconeogenic carbon sources.

Harnessing the IL-21-BATF Pathway in the CD8+ T Cell Anti-Tumor Response.

Simple SummaryIn cancer, CD8+ T cells enter a state of dysfunction within the tumor that prevents them from targeting and killing tumor cells. Our study aims to uncover how CD8+ T cells can be helped by CD4+ T cells or modified in order to improve their effector function against cancer. Thus, allowing them to better fight and control tumors. Our work shows that the protein Basic Leucine Zipper ATF-Like Transcription Factor (BATF) may be a key regulator of CD8+ T cells and their anti-tumor function. These findings can provide further insight for the development of novel therapeutic treatments for cancer patients.In cancer, CD8+ T cells enter a dysfunctional state which prevents them from effectively targeting and killing tumor cells. Tumor-infiltrating CD8+ T cells consist of a heterogeneous population of memory-like progenitor, effector, and terminally exhausted cells that exhibit differing functional and self-renewal capacities. Our recently published work has shown that interleukin (IL)-21-producing CD4+ T cells help to generate effector CD8+ T cells within the tumor, which results in enhanced tumor control. However, the molecular mechanisms by which CD4+ helper T cells regulate the differentiation of effector CD8+ T cells are not well understood. In this study, we found that Basic Leucine Zipper ATF-Like Transcription Factor (BATF), a transcription factor downstream of IL-21 signaling, is critical to maintain CD8+ T cell effector function within the tumor. Using mixed bone marrow chimeras, we demonstrated that CD8+ T cell-specific deletion of BATF resulted in impaired tumor control. In contrast, overexpressing BATF in CD8+ T cells enhanced effector function and resulted in improved tumor control, bypassing the need for CD4+ helper T cells. Transcriptomic analyses revealed that BATF-overexpressing CD8+ T cells had increased expression of costimulatory receptors, effector molecules, and transcriptional regulators, which may contribute to their enhanced activation and effector function. Taken together, our study unravels a previously unappreciated CD4+ T cell-derived IL-21–BATF axis that could provide therapeutic insights to enhance effector CD8+ T cell function to fight cancer.

Circ_0005927 Inhibits the Progression of Colorectal Cancer by Regulating miR-942-5p/BATF2 Axis.

BackgroundColorectal cancer (CRC) is one of the most common aggressive neoplasms worldwide. Circular RNAs (circRNAs) have been involved in the biological process of CRC. This study aimed to explore the effects of circ_0005927 on CRC progression and underneath mechanism.Materials and MethodsThe expression of circ_0005927, microRNA-942-5p (miR-942-5p) and basic leucine zipper ATF-like transcription factor 2 (BATF2) was detected by quantitative real time polymerase chain reaction (qRT-PCR). The protein expression of BATF2 was determined by Western blot. The effects among circ_0005927, miR-942-5p and BATF2 on cell colony-forming ability, apoptosis and migratory and invasive abilities were revealed by cell colony formation, flow apoptosis and transwell assays, respectively. The associated relationship between miR-942-5p and circ_0005927 or BATF2 was predicted by Circinteractome or TargetScan online database, and identified by dual-luciferase reporter or RNA immunoprecipitation (RIP) assay. The impacts of circ_0005927 overexpression on tumor growth in vivo were investigated by in vivo tumor formation assay.ResultsCirc_0005927 expression and the mRNA and protein expression of BATF2 were dramatically downregulated, while miR-942-5p expression was obviously upregulated in CRC tissues or cells compared with control groups. Circ_0005927 overexpression repressed cell colony-forming ability, migration and invasion, whereas induced cell apoptosis in CRC; however, these impacts were hindered by miR-942-5p mimic or BATF2 knockdown. Furthermore, circ_0005927 was a sponge of miR-942-5p, and miR-942-5p bound to BATF2. In addition, circ_0005927 overexpression repressed tumor growth in vivo.ConclusionCirc_0005927 suppressed cell colony-forming ability, migration and invasion, and promoted cell apoptosis by sponging miR-942-5p to induce BATF2 in CRC. The possible mechanism provides a theoretical basis for the study of circRNA-directed therapy for CRC.

Improving isobutanol tolerance and titers through EMS mutagenesis in Saccharomyces cerevisiae.

Improving yeast tolerance toward isobutanol is a critical issue enabling high-titer industrial production. Here, we used EMS mutagenesis to screen Saccharomyces cerevisiae with greater tolerance toward isobutanol. By this method, we obtained EMS39 with high-viability in medium containing 16g/L isobutanol. Then, we metabolically engineered isobutanol synthesis in EMS39. About 2μ plasmids carrying PGK1p-ILV2, PGK1p-ILV3 and TDH3p-cox4-ARO10 were used to over-express ILV2, ILV3 and ARO10 genes, respectively, in EMS39 and wild type W303-1A. And the resulting strains were designated as EMS39-20 and W303-1A-20. Our results showed that EMS39-20 increased isobutanol titers by 49.9% compared to W303-1A-20. Whole genome resequencing analysis of EMS39 showed that more than 59 genes had mutations in their open reading frames or regulatory regions. These 59 genes are enriched mainly into cell growth, basal transcription factors, cell integrity signaling, translation initiation and elongation, ribosome assembly and function, oxidative stress response, etc. Additionally, transcriptomic analysis of EMS39-20 was carried out. Finally, reverse engineering tests showed that overexpression of CWP2 and SRP4039 could improve tolerance of S.cerevisiae toward isobutanol. In conclusion, EMS mutagenesis could be used to increase yeast tolerance toward isobutanol. Our study supplied new insights into mechanisms of tolerance toward isobutanol and enhancing isobutanol production in S. cerevisiae.

Small molecule induces mitochondrial fusion for neuroprotection via targeting CK2 without affecting its conventional kinase activity

Mitochondrial fusion/fission dynamics plays a fundamental role in neuroprotection; however, there is still a severe lack of therapeutic targets for this biological process. Here, we found that the naturally derived small molecule echinacoside (ECH) significantly promotes mitochondrial fusion progression. ECH selectively binds to the previously uncharacterized casein kinase 2 (CK2) α′ subunit (CK2α′) as a direct cellular target, and genetic knockdown of CK2α′ abolishes ECH-mediated mitochondrial fusion. Mechanistically, ECH allosterically regulates CK2α′ conformation to recruit basic transcription factor 3 (BTF3) to form a binary protein complex. Then, the CK2α′/BTF3 complex facilitates β-catenin nuclear translocation to activate TCF/LEF transcription factors and stimulate transcription of the mitochondrial fusion gene Mfn2. Strikingly, in a mouse middle cerebral artery occlusion (MCAO) model, ECH administration was found to significantly improve cerebral injuries and behavioral deficits by enhancing Mfn2 expression in wild-type but not CK2α′+/− mice. Taken together, our findings reveal, for the first time, that CK2 is essential for promoting mitochondrial fusion in a Wnt/β-catenin-dependent manner and suggest that pharmacologically targeting CK2 is a promising therapeutic strategy for ischemic stroke.

Participation of TFIIIB Subunit Brf1 in Transcription Regulation in the Human Pathogen Leishmania major.

In yeast and higher eukaryotes, transcription factor TFIIIB is required for accurate initiation of transcription by RNA Polymerase III (Pol III), which synthesizes transfer RNAs (tRNAs), 5S ribosomal RNA (rRNA), and other essential RNA molecules. TFIIIB is composed of three subunits: B double prime 1 (Bdp1), TATA-binding protein (TBP), and TFIIB-related factor 1 (Brf1). Here, we report the molecular characterization of Brf1 in Leishmania major (LmBrf1), a parasitic protozoan that shows distinctive transcription characteristics, including the apparent absence of Pol III general transcription factors TFIIIA and TFIIIC. Although single-knockout parasites of LmBrf1 were obtained, attempts to generate LmBrf1-null mutants were unsuccessful, which suggests that LmBrf1 is essential in promastigotes of L. major. Notably, Northern blot analyses showed that the half-lives of the messenger RNAs (mRNAs) from LmBrf1 and other components of the Pol III transcription machinery (Bdp1 and Pol III subunit RPC1) are very similar (~40 min). Stabilization of these transcripts was observed in stationary-phase parasites. Chromatin immunoprecipitation (ChIP) experiments showed that LmBrf1 binds to tRNA, small nuclear RNA (snRNA), and 5S rRNA genes. Unexpectedly, the results also indicated that LmBrf1 associates to the promoter region of the 18S rRNA genes and to three Pol II-dependent regions here analyzed. Tandem affinity purification and mass spectrometry analyses allowed the identification of a putative TFIIIC subunit. Moreover, several proteins involved in transcription by all three RNA polymerases co-purified with the tagged version of LmBrf1.

Molecular Characterization of the Oncogene BTF3 and Its Targets in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most commonly diagnosed and leading causes of cancer mortality worldwide, and the prognosis of patients with CRC remains unsatisfactory. Basic transcription factor 3 (BTF3) is an oncogene and hazardous prognosticator in CRC. Although two distinct functional mechanisms of BTF3 in different cancer types have been reported, its role in CRC is still unclear. In this study, we aimed to molecularly characterize the oncogene BTF3 and its targets in CRC. Here, we first identified the transcriptional targets of BTF3 by applying combined RNA-Seq and ChIP-Seq analysis, identifying CHD1L as a transcriptional target of BTF3. Thereafter, we conducted immunoprecipitation (IP)-MS and E3 ubiquitin ligase analysis to identify potential interacting targets of BTF3 as a subunit of the nascent-polypeptide-associated complex (NAC). The analysis revealed that BTF3 might also inhibit E3 ubiquitin ligase HERC2-mediated p53 degradation. Finally, miRNAs targeting BTF3 were predicted and validated. Decreased miR-497-5p expression is responsible for higher levels of BTF3 post-transcriptionally. Collectively, we concluded that BTF3 is an oncogene, and there may exist a transcription factor and NAC-related proteolysis mechanism in CRC. This study provides a comprehensive basis for understanding the oncogenic mechanisms of BTF3 in CRC.

M6A RNA Methylation Regulators Act as Potential Prognostic Biomarkers in Lung Adenocarcinoma

N6-methyladenosine [m(6)A/m6A] methylation is one of the most common RNA modifications in eukaryotic cell mRNA and plays an important regulatory role in mRNA metabolism, splicing, translocation, stability, and translation. Previous studies have demonstrated that the m6A modification is highly associated with tumor cell proliferation, migration, and invasion. In the present study, five m6A regulatory factors have been revealed, namely heterogeneous nuclear ribonucleoprotein A2/B1(HNRNPA2B1), heterogeneous nuclear ribonucleoprotein C (HNRNPC), Vir like m6A methyltransferase associated protein (KIAA1429/VIRMA), RNA binding motif protein 15 (RBM15) and methyltransferase like 3 (METTL3), which are closely related to the overall survival (OS) of patients with lung adenocarcinoma (LUAD). These five m6A regulatory factors exhibited potential prognostic value for the 1, 3, and 5-years survival outcomes of LUAD patients. Our findings revealed that several signaling pathways, such as cell cycle, DNA replication, RNA degradation, RNA polymerase, nucleotide excision repair and basal transcription factors, are activated in the high-risk group of LUAD patients.