Wild-type Promoter Research Articles

Testosterone Inhibits Lipid Accumulation in Porcine Preadipocytes by Regulating ELOVL3.

Castration is commonly used to reduce stink during boar production. In porcine adipose tissue, castration reduces androgen levels resulting in metabolic disorders and excessive fat deposition. However, the underlying detailed mechanism remains unclear. In this study, we constructed porcine preadipocyte models with and without androgen by adding testosterone exogenously. The fluorescence intensity of lipid droplet (LD) staining and the fatty acid synthetase (FASN) mRNA levels were lower in the testosterone-treated cells than in the untreated control cells. In contrast, the mRNA levels of adipose triglycerides lipase (ATGL) and androgen receptor (AR) were higher than in the testosterone-treated cells than in the control cells. Subsequently, transcriptomic sequencing of porcine preadipocytes incubated with and without testosterone showed that the mRNA expression levels of very long-chain fatty acid elongase 3 (ELOVL3), a key enzyme involved in fatty acids synthesis and metabolism, were high in control cells. The siRNA-mediated knockdown of ELOVL3 reduced LD accumulation and the mRNA levels of FASN and increased the mRNA levels of ATGL. Next, we conducted dual-luciferase reporter assays using wild-type and mutant ELOVL3 promoter reporters, which showed that the ELOVL3 promoter contained an androgen response element (ARE); furthermore, its transcription was negatively regulated by AR overexpression. In conclusion, our study reveals that testosterone inhibits fat deposition in porcine preadipocytes by suppressing ELOVL3 expression. Moreover, our study provides a theoretical basis for further studies on the mechanisms of fat deposition caused by castration.

Interaction of NF-κB and FOSL1 drives glioma stemness

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor; GBM’s inevitable recurrence suggests that glioblastoma stem cells (GSC) allow these tumors to persist. Our previous work showed that FOSL1, transactivated by the STAT3 gene, functions as a tumorigenic gene in glioma pathogenesis and acts as a diagnostic marker and potential drug target in glioma patients. Accumulating evidence shows that STAT3 and NF-κB cooperate to promote the development and progression of various cancers. The link between STAT3 and NF-κB suggests that NF-κB can also transcriptionally regulate FOSL1 and contribute to gliomagenesis. To investigate downstream molecules of FOSL1, we analyzed the transcriptome after overexpressing FOSL1 in a PDX-L14 line characterized by deficient FOSL1 expression. We then conducted immunohistochemical staining for FOSL1 and NF-κB p65 using rabbit polyclonal anti-FOSL1 and NF-κB p65 in glioma tissue microarrays (TMA) derived from 141 glioma patients and 15 healthy individuals. Next, mutants of the human FOSL1 promoter, featuring mutations in essential binding sites for NF-κB were generated using a Q5 site-directed mutagenesis kit. Subsequently, we examined luciferase activity in glioma cells and compared it to the wild-type FOSL1 promoter. Then, we explored the mutual regulation between NF-κB signaling and FOSL1 by modulating the expression of NF-κB or FOSL1. Subsequently, we assessed the activity of FOSL1 and NF-κB. To understand the role of FOSL1 in cell growth and stemness, we conducted a CCK-8 assay and cell cycle analysis, assessing apoptosis and GSC markers, ALDH1, and CD133 under varying FOSL1 expression conditions. Transcriptome analyses of downstream molecules of FOSL1 show that NF-κB signaling pathway is regulated by FOSL1. NF-κB p65 protein expression correlates to the expression of FOSL1 in glioma patients, and both are associated with glioma grades. NF-κB is a crucial transcription factor activating the FOSL1 promoter in glioma cells. Mutual regulation between NF-κB and FOSL1 contributes to glioma tumorigenesis and stemness through promoting G1/S transition and inhibiting apoptosis. Therefore, the FOSL1 molecular pathway is functionally connected to NF-κB activation, enhances stemness, and is indicative that FOSL1 may potentially be a novel GBM drug target.

Therapeutic targeting of PLK1 in TERT promoter-mutant hepatocellular carcinoma.

Hotspot mutations in the promoter of telomerase reverse transcriptase (TERT) gene are the most common genetic variants in hepatocellular carcinoma (HCC) and associated with poor prognosis of the disease. However, no drug was currently approved for treating TERT promoter mutation positive HCC patients. Here, we aim to explore the potential therapeutic strategy for targeting TERT promoter mutation in HCC. The Liver Cancer Model Repository database was used for screening potential drugs to selectively suppress the growth of TERT promoter mutant HCC cells. RNA-seq, CRISPR-Cas9 technology and siRNA transfection were performed for mechanistic studies. Cell counting kit-8 (CCK8) assay and the xenograft tumour models were used for cell growth detection in vitro and in vivo, respectively. Cell apoptosis and cell cycle arrest were analysed by Annexin V-FITC staining and/or propidium iodide staining. PLK1 inhibitors were remarkably more sensitive to HCC cells harbouring TERT promoter mutation than wild-type cells in vitro and in vivo, which were diminished after TERT promoter mutation was edited to the wild-type nucleotide. Comparing the HCC cells with wild-type promoter of TERT, PLK1 inhibitors specifically downregulated Smad3 to regulate TERT for inducing apoptosis and G2/M arrest in TERT mutant HCC cells. Moreover, knockout of Smad3 counteracted the effects of PLK1 inhibitors in TERT mutant HCC cells. Finally, a cooperative effect of PLK1 and Smad3 inhibition was observed in TERT mutant cells. PLK1 inhibition selectively suppressed the growth of TERT mutant HCC cells through Smad3, thus contributed to discover a novel therapeutic strategy to treat HCC patients harbouring TERT promoter mutations. TERT promoter mutation confers sensitivity to PLK1 inhibitors in HCC. The selective growth inhibition of TERT mutant HCC cells induced by PLK1 inhibitor was mediated by Smad3. Combined inhibition of PLK1 and Smad3 showed a cooperative anti-tumor effect in TERT mutant HCC cells.

Comprehensive evaluation of T7 promoter for enhanced yield and quality in mRNA production

The manufacturing of mRNA vaccines relies on cell-free based systems that are easily scalable and flexible compared with the traditional vaccine manufacturing processes. Typically, standard processes yield 2 to 5 g L−1 of mRNA, with recent process optimisations increasing yields to 12 g L−1. However, increasing yields can lead to an increase in the production of unwanted by-products, namely dsRNA. It is therefore imperative to reduce dsRNA to residual levels in order to avoid intensive purification steps, enabling cost-effective manufacturing processes. In this work, we exploit sequence modifications downstream of the T7 RNA polymerase promoter to increase mRNA yields whilst simultaneously minimising dsRNA. In particular, transcription performance was optimised by modifying the sequence downstream of the T7 promoter with additional AT-rich sequences. We have identified variants that were able to produce higher amounts of mRNA (up to 14 g L−1) in 45 min of reaction. These variants exhibited up to a 30% reduction in dsRNA byproduct levels compared to a wildtype T7 promoter, and have similar EGFP protein expression. The results show that optimising the non-coding regions can have an impact on mRNA production yields and quality, reducing overall manufacturing costs.

Abstract 5388: FOSL1 promotes glioma tumorigenesis and stemness through NF-κB and STAT3 pathways

Abstract Background: Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor, GBM’s inevitable recurrence suggests that glioblastoma stem cells (GSC) render these tumors persistent. Our previous work showed that FOSL1, which is transactivated by the STAT3 gene, functions as a tumorigenic gene in glioma pathogenesis and is a diagnostic marker and potential drug target for glioma patients. Accumulating evidence shows that STAT3 and NF-κB cooperate to promote the development and progression of various cancers. The link between STAT3 and NF-κB suggests that NF-κB can also transcriptionally regulate FOSL1 and contribute to gliomagenesis. Methods: 1). To investigate downstream molecules of FOSL1, we analyzed the transcriptome after overexpression of FOSL1 in a PDX-L14 line, characterized by very low FOSL1 expression 2). We conducted immunohistochemical staining for FOSL1 and p65 using rabbit polyclonal anti-FOSL1 and p65 in glioma tissue microarrays (TMA) derived from 76 glioma patients and 10 normal individuals. 3). Mutants of the human FOSL1 promoter, featuring mutations in essential binding sites for NF-κB and STAT3, were generated using a Q5 site-directed mutagenesis kit. Subsequently, we examined luciferase activity in glioma cells and compared it to the wild-type FOSL1 promoter. 4). We explore the mutual regulation between NF-κB signaling and FOSL1 by modulating the expression of NF-κB or FOSL1. Subsequently, we assessed the activity of FOSL1 and NF-κB. 5) To understand the role of FOSL1 in cell growth and stemness, we conducted a CCK-8 assay, cell cycle analysis, assessing apoptosis and GSC markers, ALDH1 and CD133 under varying FOSL1 expression conditions. Results: 1) Transcriptome analyses of downstream molecules of FOSL1 show that NF-κB signaling pathways are regulated by FOSL1. 2) p65 protein expression correlates to the expression of FOSL1 in glioma patients. 3) NF-κB is a crucial transcription factor activating the FOSL1 promoter in glioma cells. 4) Mutual regulation between NF-κB and FOSL1 contributes to glioma tumorigenesis and stemness through promoting G1/S transition and inhibition of apoptosis. Conclusion: The FOSL1 molecular pathway is functionally connected to NF-κB and STAT3 activation, enhances stemness, and FOSL1 is a novel GBM drug target. Citation Format: Mingli Liu, Vanajothi Ramar, Azam Khedri, BreAnna Hudson, Shanchun Guo. FOSL1 promotes glioma tumorigenesis and stemness through NF-κB and STAT3 pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5388.

A Universal Strategy for the Efficient Expression of Nanobodies in Pichia pastoris

In recent years, nanobodies have played an increasingly crucial role in virus neutralization, ELISA detection, and medical imaging. This study aimed to explore a universal expression strategy in Pichia pastoris using three nanobodies, denoted Va, Vb, and Vc, as model proteins. Initially, plasmids pLD-AOXα and pLD-AOX were engineered to minimize the risk of antibiotic resistance gene drift. Optimization of promoters and signal peptides resulted in a 1.38-fold and 1.89-fold increase in Va production. Further optimization of gene dosage led to an additional 1.39-fold enhancement in Va yield. Subsequently, 25 molecular chaperones were co-expressed with Va under the control of the wild-type AOX1 promoter, with HAC1 further increasing Va yield by 1.5-fold. By fine-tuning the promoter strength for HAC1, Va production was increased by 2.41-fold under the control of the 55p promoter. Finally, through high-density fermentation, the Va yield reached 2.13 g/L, representing a 49.8-fold increase compared to the initial strain 1-AOXα-Va in shake-flask culture. Integration of pLD-55p-HAC1 into the GS115 genome resulted in the H55 host, and the transformation of multicopy plasmids into this host led to a 1.98-fold increase in Vb yield and a 2.34-fold increase in Vc yield, respectively. The engineering of antibiotic-free parental plasmids, modification of expression components, gene dosage optimization, and the H55 host are regarded as a composite strategy which will pave the way for efficient expression of nanobodies in the future.

Human_SNP_TATAdb: a database of SNPs that statistically significantly change the affinity of the TATA-binding protein to human gene promoters: genome-wide analysis and use cases

Human_SNP_TATAdb: a database of SNPs that statistically significantly change the affinity of the TATA-binding protein to human gene promoters: genome-wide analysis and use cases

Enhancing the antibacterial function of probiotic Escherichia coli Nissle: when less is more.

Probiotic bacteria confer multiple health benefits, including preventing the growth, colonization, or carriage of harmful bacteria in the gut. Bacteriocins are antibacterial peptides produced by diverse bacteria, and their production is tightly regulated and coordinated at the transcriptional level. A popular strategy for enhancing the antibacterial properties of probiotic bacteria is to retrofit them with the ability to overproduce heterologous bacteriocins. This is often achieved from non-native constitutive promoters or in response to host or pathogen signal from synthetic promoters. How the dysregulated overproduction of heterologous bacteriocins affects the fitness and antibacterial efficacy of the retrofitted probiotic bacteria is often overlooked. We have conferred the prototypical probiotic Escherichia coli strain Nissle (EcN) the ability to produce microcin C (McC) from the wild-type promoter and two mutant promoters that allow, relative to the wild-type promoter, high and low amounts of McC production. This was done by introducing specific changes to the sequence of the wild-type promoter driving transcription of the McC operon while ensuring that the modified promoters respond to native regulation. By studying the transcriptomic responses and antibacterial efficacy of the retrofitted EcN bacteria in a Galleria mellonella infection model of enterohemorrhagic E. coli, we show that EcN bacteria that produce the lowest amount of McC display the highest antibacterial efficacy with little-to-none undesired collateral impact on their fitness. The results highlight considerations researchers may take into account when retrofitting probiotic bacteria with heterogenous gene products for therapeutic, prophylactic, or diagnostic applications. Bacteria that resist killing by antibiotics are a major risk to modern medicine. The use of beneficial "probiotic" bacteria to make antibiotic-like compounds at the site of infection in the body is emerging as a popular alternative to the use of conventional antibiotics. A potential drawback of engineering probiotic bacteria in this way is that producing antibiotic-like compounds could impart undesired side effects on the performance of such bacteria, thereby compromising their intended use. This study highlights considerations researchers may take into account when engineering probiotic bacteria for therapeutic, prophylactic, or diagnostic applications.

Downregulation of a transcription factor associated with resistance to Bt toxin Vip3Aa in the invasive fall armyworm.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized control of some major pests. However, more than 25 cases of field-evolved practical resistance have reduced the efficacy of transgenic crops producing crystalline (Cry) Bt proteins, spurring adoption of alternatives including crops producing the Bt vegetative insecticidal protein Vip3Aa. Although practical resistance to Vip3Aa has not been reported yet, better understanding of the genetic basis of resistance to Vip3Aa is urgently needed to proactively monitor, delay, and counter pest resistance. This is especially important for fall armyworm (Spodoptera frugiperda), which has evolved practical resistance to Cry proteins and is one of the world's most damaging pests. Here, we report the identification of an association between downregulation of the transcription factor gene SfMyb and resistance to Vip3Aa in S. frugiperda. Results from a genome-wide association study, fine-scale mapping, and RNA-Seq identified this gene as a compelling candidate for contributing to the 206-fold resistance to Vip3Aa in a laboratory-selected strain. Experimental reduction of SfMyb expression in a susceptible strain using RNA interference (RNAi) or CRISPR/Cas9 gene editing decreased susceptibility to Vip3Aa, confirming that reduced expression of this gene can cause resistance to Vip3Aa. Relative to the wild-type promoter for SfMyb, the promoter in the resistant strain has deletions and lower activity. Data from yeast one-hybrid assays, genomics, RNA-Seq, RNAi, and proteomics identified genes that are strong candidates for mediating the effects of SfMyb on Vip3Aa resistance. The results reported here may facilitate progress in understanding and managing pest resistance to Vip3Aa.

Glucose Starvation Stimulates the Promoting Strength of a Novel Evolved Suc2 Promoter.

Promoters are essential for designing Saccharomyces cerevisiae cell factories. Identifying novel promoters tuned to produce specific metabolites under increasingly diverse industrial stresses is required to improve the economic feasibility of whole fermentation processes. In this study, a positively evolved Suc2 promoter (SUC 2p) with promoter activity stronger than that of the wild-type Suc2 promoter (SUC 2wtp) was obtained. Quantitative real-time PCR, fluorescence analysis, Western blotting, and a β-galactosidase activity assay revealed that SUC 2p is a medium-strength promoter compared with eight reported promoters at a medium glucose concentration (2% (w/v)). Different glucose concentrations modulated the strength of SUC 2p. Low glucose concentrations (0.05 and 0.5% (w/v)) enhanced the promoter strength of SUC 2p dramatically, with promoter activity higher than that of reported strong promoters. Glucose starvation resulted in the formation of a new Msn2/4 binding site on SUC 2p. Our work should facilitate the development of promoters with novel fine-tuning properties and the construction of S. cerevisiae cell factories suitable for the industrial production of essential chemicals under glucose-deprived conditions.

Transcriptional activation of membrane-related Toll-like receptor 5 through the NF-κB subunit p65 and Vibrio parahaemolyticus flagellin in orange-spotted grouper (Epinephelus coioides)

Toll-like receptor 5 (TLR5) is an important pattern recognition receptor (PRR) for bacterial flagellin. There are two kinds of subtypes in fish, the membrane-associated Toll-like receptor 5 (TLR5M) and the soluble form of TLR5 (TLR5S). Among them, TLR5M is a homolog of mammalian TLR5, and TLR5S is fish-specific. TLR5M in orange-spotted grouper (Epinephelus coioides) was involved in recognizing flagellin and activating the nuclear factor (NF)-κB signalling pathway while EcTLR5S negatively regulated the NF-κB pathway. In mammals, the NF-κB pathway is critical to the expression of inflammatory cytokines and effectors. To better understand the activity of the TLR5M gene, we characterized the TLR5M 5′-flanking sequence region from E. coioides and assayed the TLR5M promoter luciferase reporter activity in HEK 293 T cells and grouper spleen cells (GS). The 5′-flanking region of TLR5M included three NF-κB binding sites. To elucidate the molecular basis of TLR5M gene expression, the activity of the TLR5M gene promoter was characterized and the deleted or site-directed mutants were generated to explore the functional significance of these kinds of binding sites. The luciferase activity of the TLR5M promoter in HEK 293 T cells was detected after flagellin was treated. The overexpression of NF-κB/p65 greatly increased the wild-type TLR5M promoter luciferase activity higher than the mutants. TLR5M promoter luciferase activity was increased more in the presence of p65 overexpression and Vibrio parahaemolyticus Flagellin C (vpFlaC) stimulation. These results suggest that NF-κB may be the important transcription factor and vpFlaC may act as a ligand, they could maximally increase the TLR5M promoter luciferase activity.

Rapid production of novel beneficial alleles for improving rice appearance quality by targeting a regulatory element of SLG7.

Rapid production of novel beneficial alleles for improving rice appearance quality by targeting a regulatory element of SLG7.

Deep Learning Prediction of TERT Promoter Mutation Status in Thyroid Cancer Using Histologic Images.

Background and objectives: Telomerase reverse transcriptase (TERT) promoter mutation, found in a subset of patients with thyroid cancer, is strongly associated with aggressive biologic behavior. Predicting TERT promoter mutation is thus necessary for the prognostic stratification of thyroid cancer patients. Materials and Methods: In this study, we evaluate TERT promoter mutation status in thyroid cancer through the deep learning approach using histologic images. Our analysis included 13 consecutive surgically resected thyroid cancers with TERT promoter mutations (either C228T or C250T) and 12 randomly selected surgically resected thyroid cancers with a wild-type TERT promoter. Our deep learning model was created using a two-step cascade approach. First, tumor areas were identified using convolutional neural networks (CNNs), and then TERT promoter mutations within tumor areas were predicted using the CNN-recurrent neural network (CRNN) model. Results: Using the hue-saturation-value (HSV)-strong color transformation scheme, the overall experiment results show 99.9% sensitivity and 60% specificity (improvements of approximately 25% and 37%, respectively, compared to image normalization as a baseline model) in predicting TERT mutations. Conclusions: Highly sensitive screening for TERT promoter mutations is possible using histologic image analysis based on deep learning. This approach will help improve the classification of thyroid cancer patients according to the biologic behavior of tumors.

The diagnostic value of ADC histogram and direct ADC measurements for coexisting isocitrate dehydrogenase mutation and O6-methylguanine-DNA methyltransferase promoter methylation in glioma.

To non-invasively predict the coexistence of isocitrate dehydrogenase (IDH) mutation and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation in adult-type diffuse gliomas using apparent diffusion coefficient (ADC) histogram and direct ADC measurements and compare the diagnostic performances of the two methods. A total of 118 patients with adult-type diffuse glioma who underwent preoperative brain magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) were included in this retrospective study. The patient group included 40 patients with coexisting IDH mutation and MGMT promoter methylation (IDHmut/MGMTmet) and 78 patients with other molecular status, including 32 patients with IDH wildtype and MGMT promoter methylation (IDHwt/MGMTmet), one patient with IDH mutation and unmethylated MGMT promoter (IDHmut/MGMTunmet), and 45 patients with IDH wildtype and unmethylated MGMT promoter (IDHwt/MGMTunmet). ADC histogram parameters of gliomas were extracted by delineating the region of interest (ROI) in solid components of tumors. The minimum and mean ADC of direct ADC measurements were calculated by placing three rounded or elliptic ROIs in solid components of gliomas. Receiver operating characteristic (ROC) curve analysis and the area under the curve (AUC) were used to evaluate the diagnostic performances of the two methods. The 10th percentile, median, mean, root mean squared, 90th percentile, skewness, kurtosis, and minimum of ADC histogram analysis and minimum and mean ADC of direct measurements were significantly different between IDHmut/MGMTmet and the other glioma group (P < 0.001 to P = 0.003). In terms of single factors, 10th percentile of ADC histogram analysis had the best diagnostic efficiency (AUC = 0.860), followed by mean ADC obtained by direct measurements (AUC = 0.844). The logistic regression model combining ADC histogram parameters and direct measurements had the best diagnostic efficiency (AUC = 0.938), followed by the logistic regression model combining the ADC histogram parameters with statistically significant difference (AUC = 0.916) and the logistic regression model combining minimum ADC and mean ADC (AUC = 0.851). Both ADC histogram analysis and direct measurements have potential value in predicting the coexistence of IDHmut and MGMTmet in adult-type diffuse glioma. The diagnostic performance of ADC histogram analysis was better than that of direct ADC measurements. The combination of the two methods showed the best diagnostic performance.

A Novel in Duck Myoblasts: The Transcription Factor Retinoid X Receptor Alpha (RXRA) Inhibits Lipid Accumulation by Promoting CD36 Expression.

Retinoid X receptor alpha (RXRA) is a well-characterized factor that regulates lipid metabolism; however, the regulatory mechanism in muscle cells of poultry is still unknown. The overexpression and the knockdown of RXRA in myoblasts (CS2 cells), RT-PCR, and western blotting were used to detect the expression levels of genes and proteins related to PPAR-signaling pathways. Intracellular triglycerides (TGs), cholesterol (CHOL), and nonesterified free fatty acids (NEFAs) were detected by the Elisa kit. Fat droplets were stained with Oil Red O. The double-fluorescein reporter gene and chromatin immunoprecipitation (CHIP) were used to verify the relationship between RXRA and candidate target genes. The RXRA gene was highly expressed in duck breast muscle, and its mRNA and its protein were reduced during the differentiation of CS2 cells. The CS2 cells, with the overexpression of RXRA, showed reduced content in TGs, CHOL, NEFAs, and lipid droplets and upregulated the mRNA expression of CD36, ACSL1, and PPARG genes and the protein expression of CD36 and PPARG. The knockdown of RXRA expression in CS2 cells enhanced the content of TGs, CHOL, NEFAs, and lipid droplets and downregulated the mRNA and protein expression of CD36, ACLS1, ELOVL6, and PPARG. The overexpression of the RXRA gene, the activity of the double-luciferase reporter gene of the wild-type CD36 promoter was higher than that of the mutant type. RXRA bound to -860/-852 nt, -688/-680 nt, and -165/-157 nt at the promoter region of CD36. Moreover, the overexpression of CD36 in CS2 cells could suppress the content of TGs, CHOL, NEFAs, and lipid droplets, while the knockdown expression of CD36 increased the content of TGs, CHOL, NEFAs, and lipid droplets. In this study, the transcription factor, RXRA, inhibited the accumulation of TGs, CHOL, NEFAs, and fat droplets in CS2 cells by promoting CD36 expression.

NF-κB regulates the expression of STING via alternative promoter usage

AimsStimulator of interferon genes (STING) is a transmembrane protein in endoplasmic reticulum and plays crucial roles in autophagy, antiviral and anti-tumor responses. However, there are few studies on the transcriptional regulation mechanism of STING. Main methodsThe 5′ RACE experiment was used to determine the location of STING promoters. Luciferase reporting assay confirmed the activity and core region of STING internal promoter. Site-directed mutagenesis confirmed that NF-κB regulates the activity of STING promoters. The regulation of NF-κB on STING was investigated by real-time quantitative PCR, western blot, chromatin immunoprecipitation assay and lipopolysaccharide (LPS) inflammatory cell model. Key findingsThere was also a transcription start site at the 17 bp sequence upstream of STING second exon. STING-285 was the core region of the internal promoter. After NF-κB binding site mutation, the activity of STING internal promoter decreased significantly. In addition, we found that NF-κB can bind to the promoter region of wild-type STING. Overexpression of NF-κB significantly increased the activity of STING internal promoter and wild-type promoter, while knockdown of endogenous NF-κB significantly inhibited the activity of STING promoters. The binding of NF-κB to STING promoters in vivo were confirmed by chromatin immunoprecipitation assay. Meanwhile, we stimulated HeLa cells with LPS to activate the NF-κB pathway and found that STING expression was up-regulated. SignificanceThese results suggest that transcription factor NF-κB positively regulates the expression of STING via alternative promoter usage. This provides a new basis and potential drug targets for the clinical treatment of STING related diseases.

128. Association of PBP4 Variants and β-lactam Susceptibility in Enterococcus faecalis

Abstract Background Penicillin-binding protein 4 (PBP4) is a low affinity PBP that has been associated with decreased susceptibility to penicillins in Enterococcus faecalis (Efs). In vitro data have shown that changes in the promoter region leading to increased pbp4 gene expression and amino acid changes resulting in active site remodeling contribute to this phenotype. There is limited data on the prevalence of these strains in the United States. We investigated β-lactam susceptibility trends in association with variations in PBP4 (allotypes) and the upstream promoter region. Methods Efs bloodstream isolates (n=184) were selected from the multicenter VENOUS cohort from 2016 to 2021. Whole genome sequencing (WGS) was performed on all isolates, and changes in the pbp4 gene and promoter region 200 bp upstream of the start codon were identified using Efs JH2-2 as reference. Broth microdilution (BMD) testing for ampicillin (AMP), penicillin (PCN), piperacillin (PIP), and imipenem (IMI) was performed for 81 isolates. Analysis of MICs vs. WGS results was performed. Results A total of 31 PBP4 allotypes and 10 promoter variations were identified. ST6 isolates most frequently carried the promoter mutation ΔA117 (P6), previously shown to increase expression of the pbp4 gene, with allotype 1 PBP4 (Fig 1). ST179 isolates most frequently carried the JH2-2 wild type promoter (P1) with the allotype 30 PBP4. All isolates were susceptible to AMP (MIC50 ≤1 µg/mL, MIC90 2 µg/mL) and PCN (MIC50 ≤2 µg/mL, MIC90 4 µg/mL; Table 1). PIP was the least potent β-lactam, with an MIC50 4 µg/mL and an MIC90 of 8 µg/mL. Isolates with the P6 promoter had significantly higher piperacillin MICs (p&amp;lt; 0.0001) as compared to P1. Figure 1.Phylogenetic Tree of E. faecalis Strains Conclusion Changes in the pbp4 gene promoter correlated with an increase in PIP MICs. Caution should be used when choosing β-lactams other than AMP for definitive treatment deep-seated Efs infections. Disclosures Cesar A. Arias, MD, PhD, Entasis Phramceuticals: Grant/Research Support|MeMed Diagnostics: Grant/Research Support|Merck: Grant/Research Support William R. Miller, MD, Entasis Therapeutics: Grant/Research Support|UpToDate: Royalties - Topic Contributor.

Engineering of Bacillus Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit

Bacillus genetics need more versatile promoters for gene circuit engineering. UP elements are widely distributed in noncoding regions and interact with the α-subunit of RNA polymerase (RNAP). They can be applied as a standard element for synthetic biology. Characterization of the binding motif between UP elements and RNAP may assist with rational and effective engineering. In this study, 11 Bacillus constitutive promoters were screened for strength in Bacillus licheniformis. The motif in UP elements from a strong native promoter, PLan, was characterized. The influence of specific sequences on RNAP binding and expression strength was investigated both in vitro and in vivo. It was found that sequences up to 50 base pairs upstream of the consensus motif significantly contributed to α-CTD (the alpha subunit carboxy-terminal domain) association. Meanwhile, two repeats of a proximal subsite were able to more strongly activate the expression (by 8.2-fold) through strengthening interactions between UP elements and RNAP. Based the above molecular basis, a synthetic UP element, UP5-2P, was constructed and applied to nine wild-type promoters. Fluorescence polarization results demonstrated that it had an apparent effect on promoter-α-CTD interactions, and elevated expression strength was observed for all the engineered promoters. The highest improved core promoter, Pacpp, was more strongly activated by 7.4-fold. This work thus develops a novel strategy for Bacillus promoter engineering.

Integrated Analysis of Altered lncRNA, circRNA, microRNA, and mRNA Expression in Hepatocellular Carcinoma Carrying TERT Promoter Mutations.

Telomerase reverse transcriptase (TERT) promoter mutations are one of the most common mutations responsible for the development of hepatocellular carcinoma (HCC). Noncoding RNAs (ncRNAs) play a regulatory role in different cancers through the long noncoding RNA (lncRNA)/circular RNA (circRNA)-microRNA (miRNA)-mRNA axis. The aim of the study was to explore the influence of TERT promoter mutations on the ncRNA regulatory network in HCC. Four tumor samples with a wildtype TERT promoter and four tumor samples with TERT promoter mutations (sequencing cohort) were collected from HCC patients for high-throughput next-generation sequencing. Selected ncRNAs and mRNAs were validated by qPCR in 15 HCC tumors with a wildtype TERT promoter and seven HCC tumors with TERT promoter mutations (validation cohort, including the sequencing cohort). In the mutant TERT promoter group, 536 lncRNAs, 21 circRNAs, 41 miRNAs, and 266 mRNAs were significantly up-regulated, while 1745 lncRNAs, 23 circRNAs, 32 miRNAs, and 1117 mRNAs were significantly down-regulated (P < 0.05) compared with the findings in wildtype group. AL360169.3-201, LINC02672-203, hsa_circ_0021412, hsa-miR-29b-1-5p, hsa-miR-4699-5p, hsa-miR-199a-5p, REG3A, SFRP5, and GSTM1 were verified at the RNA expression level to validate the sequencing results. A differentially expressed lncRNA/circRNA-miRNA-mRNA network was constructed to explore the effects of TERT promoter mutations on ncRNA regulation. Two ncRNA regulatory axes associated with TERT promoter mutations (hsa_circ_0003154/hsa_circ_0008952/IGLL5-AS1/LINC576/LINC575-hsa-miR-1260b -CLPTM1L/GSTM1 and hsa_circ_0031584/LINC2101-hsa-miR-214-3p-CD151) had carcinogenic potential. This study provides novel insights into the role of TERT promoter mutations on ncRNAs regulatory network in HCC progression.

Identification and functional analysis of genetic variants of ISL1 gene promoter in human atrial septal defects.

Atrial septal defect (ASD) is a common type of congenital heart disease. A gene promoter plays pivotal role in the disease development. This study was designed to investigate the pathological role of variants of the ISL1 gene promoter region in ASD patients. Total DNA extracted from 625 subjects, including 332 ASD patients and 293 healthy controls, was sequenced to identify variants in the promoter region of ISL1 gene. Further functional analyses of the variants were performed with dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA). All possible binding sites of transcription factor affected by the identified variants were predicted using the JASPAR database. Four variants in the ISL1 gene promoter were found only in patients with ASD by sequencing. Three of the four variants [g.4923 G > C (rs541081886), g.5079 A > G (rs1371835943) and g.5309 G > A (rs116222082)] significantly decreased the transcriptional activities compared with the wild-type ISL1 gene promoter (p < 0.05). The EMSA revealed that these variants [g.4923 G > C (rs541081886), g.5079 A > G (rs1371835943) and g.5309 G > A (rs116222082)] in the ISL1 gene promoter affected the number and affinity of binding sites of transcription factors. Further analysis with the online JASPAR database demonstrated that a cluster of putative binding sites for transcription factors may be altered by these variants. These sequence variants identified from the promoter region of ISL1 gene in ASD patients are probably involved in the development of ASD by affecting the transcriptional activity and altering ISL1 levels. Therefore, these findings may provide new insights into the molecular etiology and potential therapeutic strategy of ASD.