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Aims/hypothesis Beta cells control glucose homeostasis via regulated production and secretion of insulin. This function arises from a highly specialised gene expression programme that is established during development and then sustained, with limited flexibility, in terminally differentiated cells. Dysregulation of this programme is seen in type 2 diabetes but mechanisms that preserve gene expression or underlie its dysregulation in mature cells are not well resolved. This study investigated whether methylation of histone H3 lysine 4 (H3K4), a marker of gene promoters with unresolved functional importance, is necessary for the maintenance of mature beta cell function. Methods Beta cell function, gene expression and chromatin modifications were analysed in conditional Dpy30 knockout mice, in which H3K4 methyltransferase activity is impaired, and in a mouse model of diabetes. Results H3K4 methylation maintains expression of genes that are important for insulin biosynthesis and glucose responsiveness. Deficient methylation of H3K4 leads to a less active and more repressed epigenome profile that locally correlates with gene expression deficits but does not globally reduce gene expression. Instead, developmentally regulated genes and genes in weakly active or suppressed states particularly rely on H3K4 methylation. We further show that H3K4 trimethylation (H3K4me3) is reorganised in islets from the Leprdb/db mouse model of diabetes in favour of weakly active and disallowed genes at the expense of terminal beta cell markers with broad H3K4me3 peaks. Conclusions/interpretation Sustained methylation of H3K4 is critical for the maintenance of beta cell function. Redistribution of H3K4me3 is linked to gene expression changes that are implicated in diabetes pathology. Graphical abstract

In this work, we show using density functional theory calculations that controlling the spin state of the surface of magnetic metals has a substantial effect on their chemical properties. For a range of adsorbates, the adsorption energy is shown to be stronger on non-spin polarized surfaces than on spin polarized ground state surfaces. This is true for Fe, Co, and Ni surfaces, and the result is the same for three commonly used exchange-correlation functionals. We further discuss the origin of the effect in terms of the surface electronic structure and show that a simple model based on the d-band model of adsorption can explain the effect. Finally, we discuss how spin effects may be used to control surface reactivity and provide guidance on how to alter the surface spin state, e.g., adding a metal promotor.

Gallic acid is a prevalent secondary plant metabolite distinguished as one of the most effective free-radical scavengers among phenolic acids. This compound is also known for its cytotoxic, anti-inflammatory, and antimicrobial activities. Bulk quantities of gallic acid are conventionally produced by acid hydrolysis of tannins, a costly and environmentally hazardous process. With the aim to develop more sustainable approaches, microbial bioproduction strategies have been attempted recently. To advance synthetic biology and metabolic engineering of microorganisms for gallic acid production, we characterize here a transcription factor-based inducible system PpGalR/PPP_RS13150 that responds to the extracellular gallic acid in a dose-dependent manner in Pseudomonas putida KT2440. Surprisingly, this compound does not mediate induction when PpGalR/PPP_RS13150 is used in non-native host background. We show that the activation of the inducible system requires gallate dioxygenase activity encoded by galA gene. The 4-oxalomesaconic acid, an intermediate of gallic acid-metabolism, is identified as the effector molecule that interacts with the transcription factor GalR mediating activation of gene expression. Introduction of galA gene along galR enables development of biosensors suitable for detection and monitoring of gallic acid extracellularly using non-native hosts such as E. coli and C. necator. Moreover, the P. putida-based biosensor's applicability is demonstrated by detecting and measuring gallic acid in extracts of Camellia sinensis leaves. This study reports the strategy, which can be applied for developing gallic acid biosensors using bacterial species outside Pseudomonas genus.

We explored the most expedient pathway for phenolic compound preparation using a combination of arylboronic acids, a green oxidant (H2O2) and a catalytic amount of readily available medicinal materials (TYLENOL® and ASPIRIN®). The arylboronic acids were successfully transformed into the corresponding phenols in high yields under metal- and base-free aqueous aerobic conditions. We demonstrated that enhanced availability and sustainability are some advantages associated with the use of medicinal supports.

DNA supercoiling, the level of under- or overwinding of the DNA polymer around itself, is widely recognized as an ancestral regulation mechanism of gene expression in bacteria. Higher levels of negative supercoiling facilitate the opening of the DNA double helix at gene promoters and thereby increase gene transcription rates. Different levels of supercoiling have been measured in bacteria exposed to different environments, leading to the hypothesis that variations in supercoiling could be a response to changes in the environment. Moreover, DNA transcription has been shown to generate local variations in the supercoiling level and, therefore, to impact the transcription rate of neighboring genes. In this work, we study the coupled dynamics of DNA supercoiling and transcription at the genome scale. We implement a genome-wide model of gene expression based on the transcription-supercoiling coupling. We show that, in this model, a simple change in global DNA supercoiling is sufficient to trigger differentiated responses in gene expression levels via the transcription-supercoiling coupling. Then, studying our model in the light of evolution, we demonstrate that this non-linear response to different environments, mediated by the transcription-supercoiling coupling, can serve as the basis for the evolution of specialized phenotypes.

Spermatogenesis is a complex process in which spermatogonial stem cells differentiate and develop into mature spermatozoa. The transcriptional regulatory network involved in fish spermatogenesis remains poorly understood. Here, we demonstrate in Nile tilapia that the Sox transcription factor family member Sox30 is specifically expressed in the testes and mainly localizes to spermatocytes and spermatids. CRISPR/Cas9-mediated sox30 mutation results in abnormal spermiogenesis, reduction of sperm motility, and male subfertility. Comparative transcriptome analysis shows that sox30 mutation alters the expression of genes involved in spermatogenesis. Further chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), ChIP-PCR, and luciferase reporter assays revealed that Sox30 positively regulates the transcription of ift140 and ptprb, two genes involved in spermiogenesis, by directly binding to their promoters. Our data, taken together, indicate that Sox30 plays an essential role in Nile tilapia spermatogenesis by directly regulating the transcription of the spermiogenesis-related genes ift140 and ptprb.

Background: Glioblastoma (GBM) is the most common but lethal brain cancer with poor prognosis. The developing brain homeobox 2 (DBX2) has been reported to play important roles in tumor growth. However, the mechanisms of DBX2 in GBM are still unknown. Objective: This study aims to investigate the function and mechanisms of DBX2 in GBM. Methods: The expressions of DBX2 and REST in GBM were measured by analyzing data from databases, and the results were checked by qPCR and/or western blot of GBM cell lines. Cell proliferation was determined by CCK8 assay, immunohistochemistry and colony formation assay. ChIP-qPCR was used to determine the binding sites of DBX2 on REST. Results: In this study, we found that the expression of DBX2 was upregulated in the GBM cell lines. The cell proliferation was damaged after blocking DBX2 expression in U87 and U251 GBM cell lines. The expression level of DBX2 had a positive relationship with that of REST. Our ChIP-qPCR results showed that DBX2 is directly bound to the promoter region of REST. Additionally, the increased GBM cell proliferation caused by DBX2 overexpression can be rescued by REST loss of function. Conclusion: DBX2 could promote cell proliferation of GBM by binding to the promoter region of REST gene and increasing REST expression.

In recent years, a plethora of new synthetic biology tools for use in cyanobacteria have been published; however, their reported characterizations often cannot be reproduced, greatly limiting the comparability of results and hindering their applicability. In this interlaboratory study, the reproducibility of a standard microbiological experiment for the cyanobacterial model organism Synechocystis sp. PCC 6803 was assessed. Participants from eight different laboratories quantified the fluorescence intensity of mVENUS as a proxy for the transcription activity of the three promoters PJ23100, PrhaBAD, and PpetE over time. In addition, growth rates were measured to compare growth conditions between laboratories. By establishing strict and standardized laboratory protocols, reflecting frequently reported methods, we aimed to identify issues with state-of-the-art procedures and assess their effect on reproducibility. Significant differences in spectrophotometer measurements across laboratories from identical samples were found, suggesting that commonly used reporting practices of optical density values need to be supplemented by cell count or biomass measurements. Further, despite standardized light intensity in the incubators, significantly different growth rates between incubators used in this study were observed, highlighting the need for additional reporting requirements of growth conditions for phototrophic organisms beyond the light intensity and CO2 supply. Despite the use of a regulatory system orthogonal to Synechocystis sp. PCC 6803, PrhaBAD, and a high level of protocol standardization, ∼32% variation in promoter activity under induced conditions was found across laboratories, suggesting that the reproducibility of other data in the field of cyanobacteria might be affected similarly German Research Foundation 390713860, 239748522 European Union 760994 Ministerio de Ciencia e Innovación PID2020-112645GB-I00 UK Biotechnology and Biological Sciences Research Council (BBSRC) BB/S020128/1

Background and aims Gastric cancer (GC) is a malignant tumor that seriously affects human health and Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a molecular subtype of GC. This study aims to determine the relationship between the methylation status of the TMEM130 gene and GC, and to explore the influence of EBV infection. Methods qRT-PCR was conducted to investigate the transcriptional expression of TMEM130 in GC. BSP and MSP assays were used to detect the methylation level of the TMEM130 promoter. The cell migration ability was detected by Transwell and western blot after transfection of TMEM130 plasmids in GC cells. Results The transcriptional expression of TMEM130 decreased in GC with hypermethylation of the promoter region. The DNA methyltransferase inhibitor could increase the mRNA expression of TMEM130. Moreover, hypermethylation of the TMEM130 promoter in GC tissues was associated with EBV infection. Overexpression of TMEM130 in GC cell lines suppresses cell migration ability. Conclusion This study was the first to research the expression and function of TMEM130 and found that TMEM130 gene hypermethylation might contribute to GC migration and EBV infection as a cause of hypermethylation of the TMEM130 gene. TMEM130 is a promising biomarker for the diagnosis of GC.

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

Promoters and enhancers are sites of transcription initiation (TSSs) and carry active histone modifications, including H3K4me1, H3K4me3, and H3K27ac. Yet, the principles governing the boundaries of such regulatory elements are still poorly characterized. Alu elements are good candidates for a boundary function, being highly abundant in gene-rich regions, while essentially excluded from regulatory elements. Here, we show that the interval from the TSS to the first upstream Alu accommodates essentially all H3K4me3 marks, while excluding DNA methylation. In contrast, enhancer-enriched H3K4me1 and H3K27ac marks eventually cross the first-Alu limit, consistent with enhancer-annotation occasionally overlapping with Alu elements. Remarkably, the average length of TSS-to-first Alu intervals greatly varies in-between tissues, being longer in stem- and shorter in immune-cells. Shortest TSS-to-Alu intervals were observed at promoters active in T cells, particularly at immune genes, correlating with serendipitous RNA polymerase II transcription and accumulation of H3K4me1 signal at the first-Alu. At several T-cell first-Alus, the DNA methylation was further found to evolved with age, regressing from young to middle-aged, then recovering later in life. Thus, the first-Alu upstream of TSSs functions as a dynamic boundary for regulatory elements, initiating the upstream DNA-methylation landscape, while also participating in the recording of immune gene transcriptional events.

Objectives: This study aims to investigate the relationship between the interferon-gamma receptor 1 (IFNGR1) polymorphism and susceptibility to lung sarcoidosis. Patients and methods: The study included a total of 55 patients (13 males, 42 females; mean age: 46.5±9.1 years; range, 22 to 66 years) with lung sarcoidosis and 28 healthy controls (6 males, 22 females; mean age: 43.9±5.9 years; range 22 to 60 years) selected from the Turkish population. The polymerase chain reaction was used for genotyping of participants to determine single-nucleotide polymorphisms. Hardy-Weinberg equilibrium, which is considered an important tool for detecting genotyping errors, was tested. Allele and genotype frequencies of patients and controls were compared using logistic regression analysis. Results: The analyses showed no correlation between the tested IFNGR1 single-nucleotide polymorphism (rs2234711) and lung sarcoidosis (p>0.05). The categorization analysis according to the clinical features, laboratory, and radiographic characteristics showed no correlation between the tested polymorphism of IFNGR1 (rs2234711) and these characteristics (p>0.05). Conclusion: The results of the study showed that the tested gene polymorphism (rs2234711) of IFNGR1 was not associated with lung sarcoidosis. More comprehensive studies are needed to verify our results.

Increasing evidence shows that promoters and enhancers could be related to 3D chromatin structure, thus affecting cellular functions. Except for their roles in forming canonical chromatin loops, promoters and enhancers have not been well studied regarding the maintenance of broad chromatin organization. Here, we focused on the active promoters/enhancers predicted to form many 3D contacts with other active promoters/enhancers (referred to as hotspots) and identified dozens of loci essential for cell growth and survival through CRISPR screening. We found that the deletion of an essential hotspot could lead to changes in broad chromatin organization and the expression of distal genes. We showed that the essentiality of hotspots does not result from their association with individual genes that are essential for cell viability but rather from their association with multiple dysregulated non-essential genes to synergistically impact cell fitness.

Many items affect the yields of every recombinant protein production. Promoters are one of the key regulatory elements which control the level of recombinant protein expression in the host. Although in some studies (Hepatitis B Surface Antigen) HBsAg was cloned in E. coli, in many of them common promoters were used. In this study two co-vectors (PHK and PHGK) were designed and used. Both of them were shuttle types based on phycocyanin-specific promoter and each was a combination of two types of vectors: a plasmid and a transposon. ELISA results, in line with Western blot results, showed the ability of phycocyanin promoter as well as revealed the expression level of HBsAg in transgenes from the Top10 PHK vector is higher than those of Top10 PHGK. The expression of HBsAg under the phycocyanin promoter in the present study is 7.5 µg/lit. In the current study as a pilot step, our attitude and objective of bacteria expression are to evaluate the function of the phycocyanin promoter based on the HBsAg gene. Due to the nature of the shuttle vector, in continuation of the present study to reduce the non-responsive populations, high antigen expression, meet the need for renewal and, other benefits, expression of HBsAg in other than bacterial host is under investigation.

Background Optimal non-invasive biomarkers for diagnosis and treatment of nonalcoholic fatty liver disease (NAFLD) remain to be identified. Aims To identify potential DNA methylation biomarkers for NAFLD. Methods Genome-wide DNA methylation profiling was performed to identify differentially methylated CpG sites in peripheral blood leukocytes. Differentially methylated regions were validated using the MassCLEAVE assay. The expression levels of candidate genes were explored by Gene Expression Omnibus database. Results The hypomethylation of PRKCE CpG 4.5 and CpG 18.19 was associated with nonalcoholic fatty liver (NAFL), the odds ratio (OR) and 95% confidence interval (CI) were 0.129 (0.026–0.639) and 0.231 (0.069–0.768). The methylation level of CpG 1.2 and average methylation level of SEC14L3 were correlated with NAFL, with OR (95% CI) being 0.283 (0.093–0.865) and 0.264 (0.087–0.799). PRKCE CpG 4.5 and cg17802464 of SEC14L3 were correlated with body mass index, waist circumference, total triglyceride, high-density lipoprotein cholesterol, alanine aminotransferase and aspartate aminotransferase. All selected datasets showed high expression levels of PRKCE and SEC14L3 in patients with NAFLD. Conclusions Our findings suggest that the hypomethylation of PRKCE and SEC14L3 promoters represent attractive biomarkers for NAFLD. Further studies are warranted to validate these biomarkers as molecular tools for diagnosis of NAFLD and therapeutic targets.

Introduction Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. Objectives We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. Methods G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. Results PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. Conclusion G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.

Transposable elements are an abundant source of transcription factor binding sites, and favorable genomic integration may lead to their recruitment by the host genome for gene regulatory functions. However, it is unclear how frequent co-option of transposable elements as regulatory elements is, to which regulatory programs they contribute and how they compare to regulatory elements devoid of transposable elements. Here, we report a transcription initiation-centric, in-depth characterization of the transposon-derived regulatory landscape of mouse embryonic stem cells. We demonstrate that a substantial number of transposable element insertions, in particular endogenous retroviral elements, are associated with open chromatin regions that are divergently transcribed into unstable RNAs in a cell-type specific manner, and that these elements contribute to a sizable proportion of active enhancers and gene promoters. We further show that transposon subfamilies contribute differently and distinctly to the pluripotency regulatory program through their repertoires of transcription factor binding site sequences, shedding light on the formation of regulatory programs and the origins of regulatory elements.

Background LINC00152 (long intergenic non-protein coding RNA 152) was identified as an oncogenic lncRNA in multiple cancers. In the current study, we aimed to explore the transcriptional profile of LINC00152 in oral squamous cell carcinoma (OSCC) and its regulations at the transcriptional level. Methods Bioinformatic analysis was performed by extracting the OSCC subset from The Cancer Genome Atlas (TCGA)-Head and Neck Squamous Cell Carcinoma (HNSC). LINC00152 subcellular localization and its interacting transcriptional factors (TFs) were explored. Dual-luciferase assay and ChIP-qPCR were applied to study transcriptional regulation. In vitro and in-vivo tumor cell growth models were used for functional assays. Results NR_024206.2 was the dominant isoform that accounts for 80% of all transcripts of LINC00152. LINC00152 upregulation was associated with unfavorable survival of patients with OSCC. LINC00152 knockdown significantly impaired OSCC cell growth in vitro and in vivo. RNA FISH assay confirmed nuclear and cytoplasmic distribution of LINC00152. It physically interacted with Upstream Transcription Factor 1 (USF1), a common transcription factor in mammalian cells. USF1 could bind to the promoter region of MRPL52 (Mitochondrial Ribosomal Protein L52) and activate its transcription. LINC00152 could enhance the binding, thereby indirectly elevating MRPL52 expression. USF1 or MRPL52 knockdown slowed the proliferation of OSCC cells and partly canceled LINC00152 mediated growth-promoting effects. Conclusion This study revealed a novel LINC00152-USF1/MRPL52 axis promoting OSCC tumor growth.

Photoperiod responsiveness is a key factor limiting the geographic distribution of cultivated soybean and its wild ancestor. In particular, the genetic basis of the adaptation in wild soybean remains poorly understood. In this study, by combining whole-genome resequencing and genome-wide association studies we identified a novel locus, Time of Flowering 5 (Tof5), which promotes flowering and enhances adaptation to high latitudes in both wild and cultivated soybean. By genomic, genetic and transgenic analyses we showed that Tof5 encodes a homolog of Arabidopsis thaliana FRUITFULL (FUL). Importantly, further analyses suggested that different alleles of Tof5 have undergone parallel selection. The Tof5H1 allele was strongly selected by humans after the early domestication of cultivated soybean, while Tof5H2 allele was naturally selected in wild soybean, and in each case facilitating adaptation to high latitudes. Moreover, we found that the key flowering repressor E1 suppresses the transcription of Tof5 by binding to its promoter. In turn, Tof5 physically associates with the promoters of two important FLOWERING LOCUS T (FT), FT2a and FT5a, to upregulate their transcription and promote flowering under long photoperiods. Collectively, our findings provide insights into how wild soybean adapted to high latitudes through natural selection and indicate that cultivated soybean underwent changes in the same gene but evolved a distinct allele that was artificially selected after domestication.

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most devastating citrus diseases worldwide. Generating disease-resistant citrus varieties is considered one of the most efficient and environmentally friendly measures for controlling canker. X. citri subsp. citri causes canker symptoms by inducing the expression of canker susceptibility gene LOB1 via PthA4, a transcription activator-like (TAL) effector, by binding to the effector binding element (EBE) in the promoter region. In previous studies, canker-resistant plants were generated by mutating the coding region or the EBE of LOB1. However, homozygous or biallelic canker-resistant plants have not been generated for commercial citrus varieties, such as grapefruit (Citrus paradisi), which usually contain two alleles of LOB1 and thus, have two types of LOB1 promoter sequences: TI LOBP and TII LOBP. Two different sgRNAs were used to target both EBE types. Both 35S promoter and Yao promoter were used to drive the expression of SpCas9p to modify EBEPthA4-LOBP in grapefruit. Using ‘Duncan’ grapefruit epicotyls as explants, 19 genome-edited grapefruit plants were generated with one biallelic mutant line (#DunYao7). X. citri subsp. citri caused canker symptoms on wild-type and nonbiallelic mutant plants but not on #DunYao7. XccPthA4 mutant containing the designer TAL effector dLOB1.5, which recognizes a conserved sequence in both wild-type and #DunYao7, caused canker symptoms on both wild-type and #DunYao7. No off-target mutations were detected in #DunYao7. This study represents the first time that CRISPR-mediated genome editing has been successfully used to generate disease-resistant plants for ‘Duncan’ grapefruit, paving the way for using disease-resistant varieties to control canker.

Background Palustrin-2CE2 and brevinin-2CE3 are antimicrobial peptides from Rana chensinensis. In R. chensinensis tadpoles, the expression of prepropalustrin-2CE2 and preprobrevinin-2CE3 increased with the developmental stage. In addition, the expression of the two genes was dramatically upregulated with stimulation by Escherichia coli, Staphylococcus aureus, and the chemical lipopolysaccharide (LPS). The genomic organization of the two antimicrobial peptide genes was confirmed. Both prepropalustrin-2CE2 and preprobrevinin-2CE3 contain three exons separated by two large introns. Additionally, several presumed transcription factor binding sites were identified in the promoter sequence. Functional analysis of the promoter was performed using a luciferase reporter system, and further confirmed by yeast one-hybrid experiment and EMSA assay. The results indicated that the transcription factors NF-κB and RelA are involved in regulating the expression of prepropalustrin-2CE2 and preprobrevinin-2CE3. As amphibian populations decline globally, this study provides new data demonstrating how frogs defend against pathogens from the environment by regulating AMP expression. For amphibians, antimicrobial peptides are innate immune molecules that resist adverse external environmental stimuli. However, the regulation mechanism of antimicrobial peptide gene expression in frogs is still unclear. Objective The two antimicrobial peptides, palustrin-2CE2 and brevinin-2CE3, are produced under external stimulation in Rana chensinensis. Using this model, we analyzed the gene structure and regulatory elements of the two antimicrobial peptide genes and explored the regulatory effects of related transcription factors on the two genes. Method Different stimuli such as E. coli, S. aureus, and chemical substance lipopolysaccharide (LPS) were applied to Rana chensinensis tadpoles at different developmental stages, and antimicrobial peptide expression levels were detected by RT-PCR. Bioinformatics analysis and 5'-RACE and genome walking technologies were employed to analyze the genome structure and promoter region of the antimicrobial peptide genes. With dual-luciferase reporter gene assays, yeast one-hybrid experiment and EMSA assays, we assessed the regulatory effect of the endogenous regulators of the cell on the antimicrobial peptide promoter. Results The transcription levels of prepropalustrin-2CE2 and preprobrevinin-2CE3 were significantly upregulated after different stimulations. Genomic structure analysis showed that both genes contained three exons and two introns. Promoter analysis indicated that there are binding sites for regulatory factors of the NF-κB family in the promoter region, and experiments showed that endogenous NF-κB family regulatory factors in frog cells activate the promoters of the antimicrobial peptide genes. Yeast one-hybrid experiment and EMSA assay demonstrated that RelA and NF-κB1 might interact with specific motifs in the prepropalustrin-2CE2 promoter. Conclusion In this paper, we found that the gene expression levels of the antimicrobial peptides, palustrin-2CE2 and brevinin-2CE3, in R. chensinensis will increase under environmental stimuli, and we verified that the changes in gene expression levels are affected by the transcription factors RelA and NF-κB1. The yeast one-hybrid experiment and EMSA assay confirmed that RelA and NF-κB1 could directly interact with the frog antimicrobial peptide gene promoter, providing new data for the regulatory mechanism of antimicrobial peptides in response to environmental stimuli.

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare monogenetic disease, which is characterized by susceptibility to some weakly virulent mycobacteria. Here, we explored the pathogenic genes and molecular mechanisms of MSMD patients. We recruited three patients diagnosed with MSMD from two families. Two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene were identified from two families. The translocation of K410E mutant STAT1 protein into nucleus was not affected. The binding ability between gamma-activating sequence (GAS) and K410E mutant STAT1 protein was significantly reduced, which will reduce the interaction between STAT1 protein with the promoters of target genes. The M691V mutant STAT1 protein cannot translocate into the nucleus after IFN-γ stimulation, which will affect the STAT1 protein form gamma-activating factors (GAF) and bind the GAS in the promoter region of downstream target genes. Taken together, our results showed that the mutation of K410E led to impaired binding of STAT1 to target DNA, and the mutation of M691V prevented the transport of STAT1 into the nucleus, which led to MSMD. Together, we identified two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene in MSMD patients, and deciphered the molecular mechanism of MSMD caused by STAT1 mutations.

Monodirectional tissue P systems with promoters are natural inspired parallel computing paradigms, where only symport rules are permitted, and with the restriction of “monodirectionality”, objects for two given regions are transferred in one direction. In this article, a novel kind of P systems, monodirectional evolutional symport tissue P systems with promoters (MESTP P systems) is raised, where objects may be revised during the movement between two regions. The computational theory of MESTP P systems that rules are employed in a flat maximally parallel pattern is investigated. We prove that finite natural number sets are created by MESTP P systems applying one cell, at most 1 promoter and all evolutional symport rules having a maximal length 2 or with arbitrary number of cells, promoters and all evolutional symport rules having a maximal length 2. MESTP P systems are Turing universal when two cells, at most 1 promoter and all evolutional symport rules having a maximal length 2 are employed. In addition, with the help of cell division mechanism, monodirectional evolutional symport tissue P systems with promoters and cell division (MESTPD P systems) are employed to solve NP -complete (the SAT ) problem, where system uses at most 1 promoter and all evolutional symport rules having a maximal length 3. These results show that MESTP(D) P systems are still computationally powerful even if monodirectionality control mechanism is imposed, thereby developing membrane algorithms for MESTP(D) P systems is theoretically possible as well as potentially exploitable.

International audience; PurposeX-linked inhibitor of apoptosis protein (XIAP) deficiency, also known as the X-linked lymphoproliferative syndrome of type 2 (XLP-2), is a rare immunodeficiency characterized by recurrent hemophagocytic lymphohistiocytosis, splenomegaly, and inflammatory bowel disease. Variants in XIAP including missense, non-sense, frameshift, and deletions of coding exons have been reported to cause XIAP deficiency. We studied three young boys with immunodeficiency displaying XLP-2-like clinical features. No genetic variation in the coding exons of XIAP was identified by whole-exome sequencing (WES), although the patients exhibited a complete loss of XIAP expression.MethodsTargeted next-generation sequencing (NGS) of the entire locus of XIAP was performed on DNA samples from the three patients. Molecular investigations were assessed by gene reporter expression assays in HEK cells and CRISPR-Cas9 genome editing in primary T cells.ResultsNGS of XIAP identified three distinct non-coding deletions in the patients that were predicted to be driven by repetitive DNA sequences. These deletions share a common region of 839 bp that encompassed the first non-coding exon of XIAP and contained regulatory elements and marks specific of an active promoter. Moreover, we showed that among the 839 bp, the exon was transcriptionally active. Finally, deletion of the exon by CRISPR-Cas9 in primary cells reduced XIAP protein expression.ConclusionsThese results identify a key promoter sequence contained in the first non-coding exon of XIAP. Importantly, this study highlights that sequencing of the non-coding exons that are not currently captured by WES should be considered in the genetic diagnosis when no variation is found in coding exons.

Coordinated spatiotemporal expression of large sets of genes is required for the development and homeostasis of organisms. To achieve this goal, organisms use myriad strategies where they form operons, utilize bidirectional promoters, cluster genes, share enhancers among genes by DNA looping, and form topologically associated domains and transcriptional condensates. Coexpression achieved by these different strategies is hypothesized to have functional importance in minimizing gene expression variability, establishing dosage balance to ensure stoichiometry of protein complexes, and minimizing accumulation of toxic intermediate metabolites. By combining gene-editing tools with computational modeling, recent studies tested the advantages of adjacent genes located in pairs and clusters. We propose that with the advancement of gene editing, single-cell sequencing, and imaging tools, one could readily test the functional importance of different coexpression strategies in a variety of biological processes.

Muscle fibers are syncytial postmitotic cells that can acquire exogenous nuclei from resident muscle stem cells, called satellite cells. Myonuclei are added to muscle fibers by satellite cells during conditions such as load-induced hypertrophy. It is difficult to dissect the molecular contributions of resident versus satellite cell-derived myonuclei during adaptation due to the complexity of labeling distinct nuclear populations in multinuclear cells without label transference between nuclei. To sidestep this barrier, we used a genetic mouse model where myonuclear DNA can be specifically and stably labeled via nonconstitutive H2B-GFP at any point in the lifespan. Resident myonuclei (Mn) were GFP-tagged in vivo before 8 wk of progressive weighted wheel running (PoWeR) in adult mice (>4-mo-old). Resident + satellite cell-derived myonuclei (Mn+SC Mn) were labeled at the end of PoWeR in a separate cohort. Following myonuclear isolation, promoter DNA methylation profiles acquired with low-input reduced representation bisulfite sequencing (RRBS) were compared to deduce epigenetic contributions of satellite cell-derived myonuclei during adaptation. Resident myonuclear DNA has hypomethylated promoters in genes related to protein turnover, whereas the addition of satellite cell-derived myonuclei shifts myonuclear methylation profiles to favor transcription factor regulation and cell-cell signaling. By comparing myonucleus-specific methylation profiling to previously published single-nucleus transcriptional analysis in the absence (Mn) versus the presence of satellite cells (Mn+SC Mn) with PoWeR, we provide evidence that satellite cell-derived myonuclei may preferentially supply specific ribosomal proteins to growing myofibers and retain an epigenetic “memory” of prior stem cell identity. These data offer insights on distinct epigenetic myonuclear characteristics and contributions during adult muscle growth.

Jia Yang,1,&ast; Yingying Wang,2,&ast; Shao Zhang,3 Yu Li,4 Chuanyin Li,1 Weipeng Liu,1 Shuyuan Liu,1 Yan Liang,1 Xinwen Zhang,1 Zhiling Yan,3 Li Shi,1 Yufeng Yao1 1Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming, Yunnan, 650118, People’s Republic of China; 2School of Basic Medical Science, Kunming Medical University, Kunming, 650500, People’s Republic of China; 3Department of Gynaecologic Oncology, The No. 3 Affiliated Hospital of Kunming Medical University, Kunming, 650118, People’s Republic of China; 4Department of Obstetrics, The No. 1 People’s Hospital of Kunming, Kunming, 650011, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yufeng YaoInstitute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming, 650118, Yunnan, People’s Republic of ChinaEmail leoyyf@gmail.com; yufeng_yao@imbcams.com.cnBackground: The tumour necrosis factor-α (TNF-α) gene plays an important role in the host immune response, which will influence the development and clearance of cancer. Polymorphism of the TNF-α promoter region is considered to influence its transcription and be a risk factor for tumorigenesis. In the current study, we evaluated the role of TNF-α promoter region polymorphisms in susceptibility to cervical intraepithelial neoplasia (CIN) and cervical cancer (CC).Methods: A total of 2732 subjects, including 1173 healthy controls, 579 patients with CIN and 980 patients with CC in a Chinese Han population, were selected for the current study. Five SNPs in the TNF-α promoter, rs1799964 (− 1031 C>T), rs1800630 (− 863 A>C), rs1799724 (− 857 C>T), rs1800629 (− 308 A>G) and rs361525 (− 238 A>G), were selected and genotyped using TaqMan Assays. The association of these SNPs with CIN and cervical cancer was evaluated among healthy controls, CIN and CC patients.Results: The frequency distribution of rs1800629 and rs361525 alleles was significantly different between the CC group and the control group (P=0.009 and P=0.002). The rs1800629 A allele was found to be a protective factor for CC (OR=0.72; 95% CI=0.56– 0.92). The rs361525 A allele was found to be a risk factor for CC (OR=1.69; 95% CI=1.21– 2.38). After pathological subtyping of CC, the allele distribution of rs1800629 and rs361525 were both significantly different between the cervical squamous cell carcinoma and control groups (P=0.002 and P< 0.001). The rs1800629 A allele was protective factor for cervical squamous cell carcinoma (OR=0.66; 95% CI=0.50– 0.86). The rs361525 A allele was a risk factor for cervical squamous cell carcinoma (OR=1.87; 95% CI=1.32– 2.65). Moreover, the genotypic frequency of rs361525 was significantly different between cervical cancer stage I and stage II (P=0.003).Conclusion: The rs1800629 and rs361525 in the TNF-α promoter are associated with susceptibility to CC in the Chinese Han population.Keywords: cervical intraepithelial neoplasia, CIN, cervical cancer, CC, promoter, single-nucleotide polymorphism, SNP, tumour necrosis factor-α, TNF-&#x03B1

It is well-known that the major reason for the rapid proliferation of cancer cells are the hypomethylation of the whole cancer genome and the hypermethylation of the promoter of particular tumor suppressor genes. Locating 5-methylcytosine (5mC) sites in promoters is therefore a crucial step in further understanding of the relationship be-tween promoter methylation and the regulation of mRNA gene expression. High throughput identification of DNA 5mC in wet lab is still time-consuming and labor-extensive. Thus, finding the 5mC site of genome-wide DNA pro-moters is still an important task. We compared the effectiveness of the most popular and strong machine learning techniques namely XGBoost, Random Forest, Deep Forest, and Deep Feedforward Neural Network in predicting the 5mC sites of genome-wide DNA promoters. A feature extraction method based on k-mers embeddings learned from a language model were also applied. Overall, the performance of all the surveyed models surpassed deep learning models of the latest studies on the same dataset employing other encoding scheme. Furthermore, the best model achieved AUC scores of 0.962 on both cross-validation and independent test data. We concluded that our approach was efficient for identifying 5mC sites of promoters with high performance.

Abstract: Inherited beta-thalassemia is caused by irregular production of hemoglobin through reducing beta-globin chains. It has been observed that increasing fetal hemoglobin (HbF) production improves symptoms in the patients; thus, it has been an operative approach to treat patients with betathalassemia. This review represents compounds with biological activities and pharmacological properties that can be useful in promoting the HBF level in β-thalassemia patients. Various natural products with different mechanisms of action can be helpful in this medication cure. Clinical trials were efficient in improving the signs of patients. Association of in vivo, and in vitro studies of HbF induction and γ-globin mRNA growth displays that in vitro experiments could be an indicator of the in vivo response. The current study resulted that; (a) HbF inducers can be grouped into several classes based on their chemical structures and mechanism of actions; (b) According to several clinical trials, wellknown drugs such as hydroxyurea and decitabine are useful HbF inducers. (c) The cellular biosensor K562 carrying genes under the control of the human γ-globin and β-globin gene promoters were applied during the researches. (d) New natural products and lead compounds were found based on various studies as HbF inducers.

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), which required developing novel therapies targeting the inhibition of HBV transcription and replication due to current limited treatment options. We explored novel target for the development of novel therapies targeting the inhibition of HBV replication and transcription. The expression of Id1 and E2F4 in HCC cells and tissues was detected by qRT-PCR and western blot. We investigated the Id1 and E2F4-mediated transcription of HBV infection by using HepG2.2.15, HepAD38, HepG2-NTCP cell lines and AAV/HBV-infected mice. Interactions between the two host proteins and viral covalently closed circular DNA (cccDNA) were assessed using subcellular localization, protein-protein interaction, chromatin immunoprecipitation, and luciferase assays. Ectopic Id1 significantly reduced HBV transcription and replication in both HBV-expressing cells and AAV/HBV-infected mice. Id1 and E2F4 could form a heterodimer to prevent E2F4 from promoting HBV transcription and replication. E2F4 could directly bind to cccDNA and activate the HBV core promoter in cell lines. Furthermore, in vitro binding experiments confirmed that the sequence 1758'-TTAAAGGTC-1766', which is highly conserved among HBV genotypes, is the target site of the E2F4 homodimer. The findings suggest that E2F4 function as novel cccDNA-binding protein to directly activate HBV transcription by binding to Cp promoter region. Our results highlight the ability that E2F4 represent a pan-potential therapeutic target against HBV transcription and provide more clues to better understand the life cycle of HBV.

Cotton that produces natural fiber materials for the textile industry is one of the most important crops in the world. Class II KNOX proteins are often considered as transcription factors in regulating plant secondary cell wall (SCW) formation. However, the molecular mechanism of the KNOX transcription factors-regulated SCW synthesis in plants (especially in cotton) remains unclear in details so far. In this study, we show a cotton class II KNOX protein (GhKNL1) as a transcription repressor functioning in fiber development. The GhKNL1-silenced transgenic cotton produced longer fibers with thicker SCWs, whereas GhKNL1 dominant repression transgenic lines displayed the opposite fiber phenotype, compared with controls. Further experiments revealed that GhKNL1 could directly bind to promoters of GhCesA4-2/4-4/8-2 and GhMYB46 for modulating cellulose synthesis during fiber SCW development of cotton. On the other hand, GhKNL1 could also suppress expressions of GhEXPA2D/4A-1/4D-1/13A through binding to their promoters for regulating fiber elongation of cotton. Taken together, these data revealed GhKNL1 functions in fiber elongation and SCW formation by directly repressing expressions of its target genes related to cell elongation and cellulose synthesis. Thus, our data provide an effective clue for potentially improving fiber quality by genetic manipulation of GhKNL1 in cotton breeding. This article is protected by copyright. All rights reserved.

We previously identified Wiz in a mouse screen for epigenetic modifiers. Due to its known association with G9a/GLP, Wiz is generally considered a transcriptional repressor. Here, we provide evidence that it may also function as a transcriptional activator. Wiz levels are high in the brain, but its function and direct targets are unknown. ChIP-seq was performed in adult cerebellum and Wiz peaks were found at promoters and transcription factor CTCF binding sites. RNA-seq in Wiz mutant mice identified genes differentially regulated in adult cerebellum and embryonic brain. In embryonic brain most decreased in expression and included clustered protocadherin genes. These also decreased in adult cerebellum and showed strong Wiz ChIP-seq enrichment. Because a precise pattern of protocadherin gene expression is required for neuronal development, behavioural tests were carried out on mutant mice, revealing an anxiety-like phenotype. This is the first evidence of a role for Wiz in neural function. DOI: http://dx.doi.org/10.7554/eLife.15082.001

To prepare various root active promoters for expressing transgenes and prevent gene silencing caused by the repeated use of the same promoter, the expression characteristics of various root active promoters were comparatively evaluated using GUS as a reporter gene. The high-affinity potassium transporter (HKT1;1), the Shaker family potassium ion channel (SKOR), the Shaker family inward rectifying potassium channel (AKT1), the major facilitator superfamily protein (MFS1), and the senescence associated gene 14 (SAG14) promoter from Arabidopsis (Arabidopsis thaliana) were used, and for comparison, four additional constitutive or green tissue specific promoters in the expression vectors were also employed. As the Gateway cloning technology provided by Invitrogen can offer high efficiency and cloning reliability, and easy manipulation of fusion constructs in vitro, our expression vectors are based on binary (destination) vectors compatible with this cloning technique. These destination vectors are also advantageous for stable expression of the transgene, as the heat shock protein terminator is utilized. The AtHKT1;1, SKOR, AKT1, MFS1 and SAG14 promoters were all active in roots but showed slightly different tissue specificities: AtHKT1;1, SKOR, and MFS1 were dominantly active in vascular bundle tissue, while AtHKT1;1 and MFS1— but not SKOR, AKT1, and SAG14—were active in root tips. SKOR showed the strongest root-specificity, and SAG14 showed the highest activity among the five root active promoters. The activity of MFS was developmentally regulated. These destination vectors are now available to express multiple transgenes in transgenic plants, especially in roots.

INTRODUCTION Urokinase plasminogen activator (uPA) is a serine protease and it also demonstrates proinflammatory properties. We thus hypothesized that uPA is involved in immunity of Sertoli cells in the rat testis. MATERIALS AND METHODS The uPA gene(PLAU) promoter was cloned by RT-PCR and the transcriptional activation of core domain was screened by using Dual-Luciferase Reporter Assay System. The Sertoli cells were harvested from 20-day-old Sprague-Dawley male rats and total RNA was isolated. The uPA mRNA levels and MyD88 pathway were tested by qPCR. RESULTS We successfully cloned the 1517-bp rat uPA gene and screened the core domain (-455/+40) in five different fragments of uPA promoter. The uPA expression and uPA promoter activity were upregulated in lipopolysaccharide (LPS)-stimulated Sertoli cells. Furthermore, the uPA expression was regulated through the MyD88-independent pathway by interdicting IRF3 and interferon β. CONCLUSION uPA expression is likely induced by LPS through the core promoter domain of uPA. This finding implied that uPA played a role in the immune function of Sertoli cells in rat testis, which might provide the development of new treatments for male infertility.

CaWRKY40 in pepper (Capsicum annuum) promotes immune responses to Ralstonia solanacearum infection (RSI) and to high-temperature, high-humidity (HTHH) stress, but how it interacts with upstream signalling components remains poorly understood. Here, using approaches of reverse genetics, biochemical and molecular biology we functionally characterised the relationships among the WRKYGMK-containing WRKY protein CaWRKY27b, the calcium-dependent protein kinase CaCDPK29, and CaWRKY40 during pepper response to RSI or HTHH. Our data indicate that CaWRKY27b is upregulated and translocated from the cytoplasm to the nucleus upon phosphorylation of Ser137 in the nuclear localisation signal by CaCDPK29. Using electrophoretic mobility shift assays and microscale thermophoresis, we observed that, due to the replacement of Q by M in the conserved WRKYGQK, CaWRKY27b in the nucleus failed to bind to W-boxes in the promoters of immunity- and thermotolerance-related marker genes. Instead, CaWRKY27b interacted with CaWRKY40 and promoted its binding and positive regulation of the tested marker genes including CaNPR1, CaDEF1 and CaHSP24. Notably, mutation of the WRKYGMK motif in CaWRKY27b to WRKYGQK restored the W-box binding ability. Our data therefore suggest that CaWRKY27b is phosphorylated by CaCDPK29 and acts as a transcriptional activator of CaWRKY40 during the pepper response to RSI and HTHH.

Lysine-specific demethylase 1 (LSD1) is an important histone demethylase that mediates epithelial to mesenchymal transition (EMT). The E239K mutation of LSD1 was identified in a luminal breast cancer patient from the COSMIC Breast Cancer dataset. To investigate the functional effects of the E239K mutation of LSD1, a stable LSD1 knockdown MCF7 cell line was generated. Rescue with WT LSD1, but not E239K mutated LSD1, suppressed the invasion and migration of the LSD1 knockdown cells, indicating that the E239K mutation abolished the suppressive effects of LSD1 on the invasion and migration of MCF7 cells. Further analysis showed that the E239K mutation abolished LSD1-mediated invasion and migration of MCF7 cells through downregulation of estrogen receptor α (ERα). Most importantly, the E239K mutation disrupted the interaction between LSD1 and GATA3, which reduced the enrichment of LSD1 at the promoter region of the ERα gene; the reduced enrichment of LSD1 at the promoter region of the ERα gene caused enhanced histone H3K9 methylation, which subsequently suppressed the transcription of the ERα gene. In summary, the E239K mutation abolishes the suppressive function of LSD1 on migration and invasion of breast cancer cells by disrupting the interaction between LSD1 and GATA3.

Objective Genetic variants spanning the ubiquitin-conjugating enzyme E2 L3 (UBE2L3) gene are associated with increased expression of the UBE2L3-encoded E2 ubiquitin-conjugating enzyme, UbcH7, that facilitates activation of proinflammatory NF-κB signaling, and susceptibility to autoimmune diseases. This study aims to delineate how genetic variants carried on the UBE2L3-YDJC autoimmune risk haplotype function to drive hypermorphic UBE2L3 expression. Methods We used bioinformatic analyses, electrophoretic mobility shift assays, and luciferase reporter assays to identify and functionally characterize allele-specific effects of risk variants positioned in chromatin accessible regions of immune cells. Chromatin conformation capture (3C)-qPCR, ChIP-qPCR, and siRNA knockdown assays were performed on patient-derived EBV-transformed B cells homozygous for the UBE2L3-YDJC non-risk or risk haplotype to determine if the risk haplotype increases UBE2L3 expression by altering the regulatory chromatin architecture in the region. Results Five of the seven prioritized variants demonstrated allele-specific increases in nuclear protein binding affinity and regulatory activity. HiChIP and 3C-qPCR uncovered a long-range interaction between the UBE2L3 promoter (rs140490, rs140491, rs11089620) and downstream YDJC promoter (rs3747093) that was strengthened in the presence of the UBE2L3-YDJC risk haplotype, and correlated with the loss of CTCF binding and gain of YY1 binding at the risk alleles. Depleting YY1 by siRNA disrupted the long-range interaction between the two promoters and reduced UBE2L3 expression. Conclusion The UBE2L3-YDJC autoimmune risk haplotype increases UBE2L3 expression through strengthening a YY1-mediated interaction between the UBE2L3 and YDJC promoters.

Transcription activator-like (TAL) effectors are major virulence factors secreted by the type III secretion systems of Xanthomonas oryzae pv. oryzicola (Xoc) and X. oryzae pv. oryzae (Xoo), causing bacterial leaf streak and bacterial blight, respectively, in rice. However, the knowledge of Xoc TAL effector function in promoting bacterial virulence remains limited. Here, we isolated the highly virulent Xoc strain HGA4 from the outbreak region of Huanggang (Hubei, China), which contains four TAL effectors not found in the Chinese model strain RS105. Among these, Tal2b was selected for introduction into RS105, which resulted in a longer lesion length than that in the control. Tal2b directly binds to the promoter region of the gene and activates the expression of OsF3H03g , which encodes 2-oxoglutarate-dependent dioxygenase in rice. OsF3H03g negatively regulates salicylic acid (SA)-related defense by directly reducing SA, and it plays a positive role in susceptibility to both Xoc and Xoo in rice. OsF3H03g interacts with a uridine diphosphate-glycosyltransferase protein (OsUGT74H4), which positively regulates bacterial leaf streak susceptibility and may inactivate SA via glycosylation modification.

Transcription factor-based whole-cell biosensors have recently become promising alternatives to conventional analytical methods due to their advantage of simplicity, cost-effectiveness, and environmental friendliness. In this study, we used genetic engineering to develop a whole-cell biosensor based on the activation of promoters by CupR via interactions with gold ions, leading to the expression of reporter genes that yield output signals. Altering the promoter sequences was shown to significantly improve the performance of the biosensor strain in terms of gold-specificity. The detection sensitivity of our engineered strains was 42-fold higher than that of wild-type strains. The linear range of the purposed sensor was 125-1000 nM with a limit of detection at 46.5 nM. The effectiveness of the sensor strain was verified in wastewater samples.

KEY MESSAGE Endogenous U6 promoters increase CRISPR/Cas9 editing efficiency in sorghum and may be useful for gene editing applications in other cereals.

Vibrio anguillarum infection can activate NF-κB/TNFα pathway in the immune organs of fish. Fish muscle is also an important immune organ, but the research on its immune function is few. Our aim was to study regulating mechanism of NF-κB and TNFα gene expressions in the muscle of Japanese flounder (Paralichthys olivaceus) which was under Vibrio anguillarum infection (0, 24, 48, 72 and 96 h). The results showed that the expressions of NF-κB and TNFα increased significantly at 48 h, and there was a significant positive correlation between them. In situ hybridization confirmed the co-existence of NF-κB and TNFα genes in Japanese flounder muscle. Interestingly, the expression of the TNFα gene was regulated by the DNA methylation and its methylation level was negatively correlated with the expression. The lowest methylation level of TNFα occurred at 48 h under Vibrio anguillarum infection (P 0.05). And more, when the fragment (-2122 ∼ -730) was deleted on TNFα gene promoter, double luciferase activity was the highest, indicating that fragment (-730-0) was the transcription factor binding region. The site (-78 ~ -69) on the fragment (-730-0) binding NF-κB was mutated, and double luciferase activity decreased significantly. The results confirmed that the site (-78 ~ -69) was indeed an important binding site for NF-κB. In addition, the activity of TNFα in the serum of Japanese flounder changed with the prolongation of vibrio anguillarum infection, and the concentration of other immune factors such as ALP, ALT, AST and LDH also changed in the muscle under vibrio anguillarum infection. They all showed a trend of first increasing and then decreasing. Above studies implied that Japanese flounder responded to Vibrio anguillarum infection at the immune level with the change of its methylation status and the activation of transcription factor. By studying the mechanism of immune pathways, understanding the response to immune stress is great significant to the research of fish breeding for disease resistance.

Transcription factors bind specific sequences upstream of the 5′ end of their target genes to ensure proper spatiotemporal expression of the target gene. This study aims to demonstrate that the transcription factor SP2 regulates expression of the Ski gene, which has specific binding sites for SP2, and thus enables Ski to regulate astrocyte proliferation. The upstream regulation mechanism of astrocyte proliferation was explored to further regulate the formation of glial scar in specific time and space after spinal cord injury. JASPAR and UCSC databases were used to predict transcription factor binding and the threshold was gradually reduced to screen transcription factors upstream of Ski, leading to the identification of SP2. Next, we analyzed the correlation between the expression of SP2 and Ski in normal astrocytes and reactive astrocytes, as well as the changes in astrocyte proliferation. To confirm that SP2 regulates Ski during astrocyte proliferation, astrocytes were transfected siRNA targeting SP2 and then astrocyte proliferation were analyzed. Finally, a dual luciferase reporter assay and Chromatin immunoprecipitation (ChIP) assay confirmed that the promoter region of Ski contained a specific SP2 binding site. This is the first that SP2 has been identified and confirmed to play an important role in astrocyte proliferation by regulating Ski expression. These results may help identify novel targets for the treatment of spinal cord injury.

Lentiviral vectors (LVs) are robust delivery vehicles for gene therapy as they can efficiently integrate transgenes into host cell genomes. However, LVs with lengthy or complex expression cassettes typically are produced at low titers and have reduced gene transfer capacity, creating barriers for clinical and commercial applications. Modifications of the packaging cell line and methods may be able to produce complex vectors at higher titer and infectivity and may improve production of many different LVs. In this study, we identified two host restriction factors in HEK293T packaging cells that impeded LV production, 2′-5′-oligoadenylate synthetase 1 (OAS1) and low-density lipoprotein receptor (LDLR). Knocking out these two genes separately led to ∼2-fold increases in viral titer. We created a monoclonal cell line, CRISPRed HEK293T to Disrupt Antiviral Response (CHEDAR), by successively knocking out OAS1, LDLR, and PKR, a previously identified factor impeding LV titers. Packaging in CHEDAR yielded ∼7-fold increases in physical particles, full-length vector RNA, and vector titers. In addition, overexpressing transcription elongation factors, SPT4 and SPT5, during packaging improved the production of full-length vector RNA, thereby increasing titers by ∼2-fold. Packaging in CHEDAR with over-expression of SPT4 and SPT5 led to ∼11-fold increases of titers. These approaches improved the production of a variety of LVs, especially vectors with low titers or with internal promoters in the reverse orientation, and may be beneficial for multiple gene therapy applications.

The karyopherin α2 subunit gene (KPNA2), an oncogene, is involved in metabolic reprogramming in cancer. This study aimed to explore the function of KPNα2 in the growth and glycolysis in colon cancer (CC) cells. Genes from the Oncomine database that were differentially expressed in multiple CC types were screened. Bioinformatics analysis suggested that KPNA2 was highly expressed in CC, and consequently, high expression of KPNA2 was detected in the CC cell lines. Down-regulation of KPNA2 reduced viability and DNA-replication ability, and increased apoptosis of HCT116 and LoVo cells. It also reduced glucose consumption, extracellular acidification rate, and the ATP production in cells. Centromere protein A (CENPA) was confirmed as an upstream transcription activator of KPNA2. There was significant H3K27ac modification in the promoter region of KPNA2. CENPA primarily recruited histone acetyltransferase general control of amino acid synthesis (GCN)-5 to the promoter region of KPNA2 to induce transcription activation. Overexpression of either CENPA or GCN-5 blocked the role of short hairpin KPNα2 and restored growth and glycolysis in CC cells. To conclude, the findings from this study suggest that CENPA recruits GCN-5 to the promoter region of KPNA2 to induce KPNα2 activation, which strengthens growth and glycolysis in, and augments the development of, CC.

Fatty acid binding protein 4 (FABP4) was crucial to fatty acid uptake and intracellular transport. However, the mechanisms regulating yak (Bos grunniens) FABP4 transcription were not determined. In the current study, predominant expression levels of yak FABP4 were identified in subcutaneous fat and longissimus dorsi muscles by quantitative real-time polymerase chain reactions (qPCR). The CCAAT/enhancer binding protein alpha (CEBPα) and myocyte enhancer factor 2A (MEF2A), as transcriptional activator or repressor in the promoter region of FABP4, were confirmed by both site-directed mutagenesis experiment and chromatin immunoprecipitation assay. Additionally, molecular mechanisms of CEBPɑ regulation were analyzed to explore the transcriptional regulatory property of FABP4, which indicated that transcriptional activity of CEBPɑ depended on CCAAT/ enhancer binding protein beta (CEBPβ) transcription factor. Our results demonstrated that CEBPβ binding directly to the promoter region drove CEBPɑ transcription, improving yak FABP4 transcriptional activity in adipocytes. This mechanism expanded the information on the transcriptional regulatory network of adipogenesis.

The initiation signals are raised around the promoter by one of the general transcription factors, triggering a sequence of events that lead to mRNA transcript formation from target genes. Both specific noncoding DNA regions and transacting, macromolecular assemblies are intricately involved and indispensable. The transition between the subsequent transcriptional stages is accompanied by stage-specific signals and structural changes in the macromolecular assemblies and facilitated by the recruitment/removal of other chromatin and transcription-associated elements. Here, we discuss the choreography of transacting factors around promoter in the establishment and effectuation of the initial phases of transcription such as NDR formation, +1 nucleosome positioning, promoter DNA opening, and RNAPII promoter escape from a structural viewpoint.

The versatile photosynthetic α-proteobacterium Rhodobacter sphaeroides, has recently been extensively engineered as a novel microbial cell factory (MCF) to produce pharmaceuticals, nutraceuticals, commodity chemicals and even hydrogen. However, there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R. sphaeroides. In this study, several native promoters from R. sphaeroides JDW-710 (JDW-710), an industrial strain producing high levels of co-enzyme Q10 (Q10) were selected on the basis of transcriptomic analysis. These candidate promoters were then characterized by using gusA as a reporter gene. Two native promoters, Prsp_7571 and Prsp_6124, showed 620% and 800% higher activity, respectively, than the tac promoter, which has previously been used for gene overexpression in R. sphaeroides. In addition, a Prsp_7571-derived synthetic promoter library with strengths ranging from 54% to 3200% of that of the tac promoter, was created on the basis of visualization of red fluorescent protein (RFP) expression in R. sphaeroides. Finally, as a demonstration, the synthetic pathway of Q10 was modulated by the selected promoter T334* in JDW-710; the Q10 yield in shake-flasks increased 28% and the production reached 226 mg/L. These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R. sphaeroides-derived MCFs.

Deficiency in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression or function is implicated in numerous neurological and psychiatric disorders. PGC-1α is required for the expression of genes involved in synchronous neurotransmitter release, axonal integrity, and metabolism, especially in parvalbumin-positive interneurons. As a transcriptional coactivator, PGC-1α requires transcription factors to specify cell-type-specific gene programs; while much is known about these factors in peripheral tissues, it is unclear if PGC-1α utilizes these same factors in neurons. Here, we identified putative transcription factors controlling PGC-1α-dependent gene expression in the brain using bioinformatics, and then validated the role of the top candidate in a knockout mouse model. We transcriptionally profiled cells overexpressing PGC-1α and searched for over-represented binding motifs in the promoters of upregulated genes. Binding sites of the estrogen-related receptor (ERR) family of transcription factors were enriched and blockade of ERRα attenuated PGC-1α-mediated induction of mitochondrial and synaptic genes in cell culture. Localization in the mouse brain revealed enrichment of ERRα expression in parvalbumin-expressing neurons with tight correlation of expression with PGC-1α across brain regions. In ERRα null mice, PGC-1α-dependent genes were reduced in multiple regions, including neocortex, hippocampus, and cerebellum, though not to the extent observed in PGC-1α null mice. Behavioral assessment revealed ambulatory hyperactivity in response to amphetamine and impairments in sensorimotor gating without the overt motor impairment characteristic of PGC-1α null mice. These data suggest that ERRα is required for normal levels of expression of PGC-1α-dependent genes in neurons, but that additional factors may be involved in their regulation. Significance statement The transcription factors with which PGC-1α interacts determine specificity of the transcriptional program it drives across cell populations, but those mediating its functions in parvalbumin-expressing neurons are unknown. Relative to other PGC-1α-interacting transcription factors, ERRα is enriched in parvalbumin-expressing neurons and shows robust spatial and temporal correlation with PGC-1α expression throughout the brain. ERRα is also necessary for PGC-1α-dependent transcription both in vitro and in vivo for metabolic and neuronal transcripts. These data suggest that ERRα is an important player in cell-specific PGC-1α-dependent transcription in the CNS and may play a role in regulating parvalbumin-expressing neuron maturation and function.

Introduction Osteoporosis, a common skeletal disorder mainly affecting postmenopausal women, is characterized by the imbalance between osteogenesis and osteoclastogenesis. Circ_0134944 has been recently found to be upregulated in postmenopausal osteoporosis (PMOP) patients. However, its role in osteogenesis remains unknown. Here we aimed to explore the role of circ_0134944 in osteogenesis and reveal the underlying mechanism. Methods qRT-PCR was used to determine the expression of circ_0134944, miR-127-5p, PDX1 and SPHK1 in the blood mononuclear cells (BMCs) of PMOP patients. Bone marrow mesenchymal stem cells (BMSCs) were used as the cellular model. Western blotting and qRT-PCR were used to determine the expression of osteogenesis-related genes (Runx2, OPN, OCN). ALP and Alizarin Red S staining were performed to evaluate osteogenic differentiation. The interactions between circ_0134944 and miR-127-5p, miR-127-5p and PDX1, PDX1 and SPHK1 were determined by dual-luciferase reporter and ChIP assay. Results Circ_0134944, PDX1 and SPHK1 were upregulated while miR-127-5p was downregulated in PMOP patients. Enhanced expression of circ_0134944 suppressed osteogenesis, which was then reversed by miR-127-5p overexpression. The binding between circ_0134944 and miR-127-5p, PDX1 and miR-127-5p were confirmed by dual-luciferase reporter assay. Moreover, PDX1 was enriched in the promoter region of SPHK1, and SPHK1 overexpression prevented the promotion of osteogenesis induced by miR-127-5p overexpression. Conclusions Taken together, these results demonstrate that circ_0134944 inhibit osteogenesis via miR-127-5p/PDX1/SPHK1 axis. Thus, the present study offered evidence that circ_0134944/miR-127-5p/PDX1/SPHK1 axis could be a potential therapeutic target for PMOP.

Loss of fatty acid β-oxidation (FAO) in the proximal tubule is a critical mediator of acute kidney injury and eventual fibrosis. However, transcriptional mediators of FAO in proximal tubule injury remain understudied. Krüppel-like factor 15 (KLF15), a highly enriched zinc-finger transcription factor in the proximal tubule, was significantly reduced in proximal tubule cells after aristolochic acid I (AAI) treatment, a proximal tubule-specific injury model. Proximal tubule specific knockout of Klf15 exacerbated proximal tubule injury and kidney function decline compared to control mice during the active phase of AAI treatment, and after ischemia-reperfusion injury. Furthermore, along with worsening proximal tubule injury and kidney function decline, knockout mice exhibited increased kidney fibrosis as compared to control mice during the remodeling phase after AAI treatment. RNA-sequencing of kidney cortex demonstrated increased transcripts involved in immune system and integrin signaling pathways and decreased transcripts encompassing metabolic pathways, specifically FAO, and PPARα signaling, in knockout versus control mice after AAI treatment. In silico and experimental chromatin immunoprecipitation studies collectively demonstrated that KLF15 occupied the promoter region of key FAO genes, CPT1A and ACAA2, in close proximity to transcription factor PPARα binding sites. While the loss of Klf15 reduced the expression of Cpt1a and Acaa2 and led to compromised FAO, induction of KLF15 partially rescued loss of FAO in AAI-treated cells. Klf15, Ppara, Cpt1a, and Acaa2 expression was also decreased in other mouse kidney injury models. Tubulointerstitial KLF15 independently correlated with eGFR, PPARA and CPT1A appearance in expression arrays from human kidney biopsies. Thus, proximal tubule-specific loss of Klf15 exacerbates acute kidney injury and fibrosis, likely due to loss of interaction with PPARα leading to loss of FAO gene transcription.