Box Promoter Research Articles

Transcriptomic and in silico analysis of BLACE (B-cell acute lymphoblastic leukemia expressed), a new non-coding RNA, as a diagnostic biomarker in B-cell ALL

ALL (acute lymphoblastic leukemia) is a type of hematological malignancy that involves developmental and differentiation arrest at the lymphoblast stage. BLACE, a gene specifically expressed in B-cell acute lymphoblastic leukemia shows little or no expression in mature B-lymphocytes. The current pilot study involves transcriptional analysis of BLACE in B-cell ALL patients. Expression of BLACE was high in both pediatric and adult ALL patients. Promoter analysis of the BLACE gene showed the presence of CAAT and TATA box promoters and G-rich sequences with a potential to form G-quadruplexes. Due to identification of TAL1 transcription factor binding sites within the BLACE promoter region, expression of TAL1 gene was measured and found to correlate with the BLACE expression. The presence of an overlapping G-rich sequence and TAL1 binding site at −1291 bps within BLACE promoter indicated a new target site for controlling BLACE expression. The docking studies performed between BLACE-TAL1 protein showed a binding score of −208.68 kcal/mol and identified 21 BLACE nucleotide - TAL1 residues interacting at the docking interface. Together, our findings suggested that BLACE gene specifically expressed in B-cell ALL could serve as a new therapeutic target. Further investigations are required to get a comprehensive understanding of the BLACE gene mechanism.

CCAAT Promoter element regulates transgenerational expression of the MHC class I gene

Transgenerational gene expression depends on both underlying DNA sequences and epigenetic modifications. The latter, which can result in transmission of variegated gene expression patterns across multiple generations without DNA alterations, has been termed epigenetic inheritance and has been documented in plants, worms, flies and mammals. Whereas transcription factors binding to cognate DNA sequence elements regulate gene expression, the molecular basis for epigenetic inheritance has been linked to histone and DNA modifications and non-coding RNA. Here we report that mutation of the CCAAT box promoter element abrogates NF-Y binding and disrupts the stable transgenerational expression of an MHC class I transgene. Transgenic mice with a mutated CCAAT box in the MHC class I transgene display variegated expression of the transgene among littermates and progeny in multiple independently derived transgenic lines. After 4 generations, CCAAT mutant transgenic lines derived from a single founder stably displayed distinct patterns of expression. Histone modifications and RNA polymerase II binding correlate with expression of CCAAT mutant transgenic lines, whereas DNA methylation and nucleosome occupancy do not. Mutation of the CCAAT box also results in changes to CTCF binding and DNA looping patterns across the transgene that correlate with expression status. These studies identify the CCAAT promoter element as a regulator of stable transgenerational gene expression such that mutation of the CCAAT box results in variegated transgenerational inheritance. Considering that the CCAAT box is present in 30% of eukaryotic promoters, this study provides insights into how fidelity of gene expression patterns is maintained through multiple generations.

FOXO6 transcription inhibition of CTRP3 promotes OGD/R-triggered cardiac microvascular endothelial barrier disruption via SIRT1/Nrf2 signalling.

C1q/TNF-related protein 3 (CTRP3) has been clarified to display its protective roles in cardiac function. The current study is concentrated on exploring the impacts of CTRP3 on myocardial ischaemia. Oxygen and glucose hypoxia/reoxygenation (OGD/R) model was constructed in human cardiac microvascular endothelial cells (HCMECs). Reverse transcription-quantitative polymerase chain reaction and western blot analysis of CTRP3 expression were conducted. CCK-8 assay was to estimate cell activity and lactate dehydrogenase (LDH) assay kit was to test LDH release. TUNEL assay and western blot were to judge apoptosis. Endothelial barrier function was detected by in vitro vascular permeability assay kit. Zonula occludens-1 (ZO-1) expression was evaluated by immunofluorescence assay. The interaction between CTRP3 promoter and Forkhead Box O6 (FOXO6) was predicted by JASPAR database and verified by chromatin immunoprecipitation and luciferase reporter assays. After OGD/R-induced HCMECs were co-transfected with CTRP3 overexpression and FOXO6 overexpression plasmids, the above functional experiments above were conducted again. Lastly, the expression of sirtuin 1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signalling-related proteins was examined by western blot. CTRP3 was down-regulated in OGD/R-induced HCMECs. CTRP3 enhanced the viability and barrier integrity while reduced the apoptosis and permeability of OGD/R-insulted HCMECs. This process may be regulated by FOXO6 transcription. Also, FOXO6 inhibition-mediated CTRP3 up-regulation activated the SIRT1/Nrf2 signalling. FOXO6 transcription inhibition of CTRP3 promotes OGD/R-triggered cardiac microvascular endothelial barrier disruption via SIRT1/Nrf2 signalling.

RsaL is a self-regulatory switch that controls alternative biosynthesis of two AHL-type quorum sensing signals in Pseudomonas aeruginosa PA1201.

Pseudomonas aeruginosa is a ubiquitous and metabolically versatile microorganism naturally found in soil and water. It is also an opportunistic pathogen in plants, insects, animals, and humans. In response to increasing cell density, P. aeruginosa uses two acyl-homoserine lactone (AHL) quorum-sensing (QS) signals (i.e.,N-3-oxo-dodecanoyl homoserine lactone [3-oxo-C12-HSL] and N-butanoyl-homoserine lactone [C4-HSL]), which regulate the expression of hundreds of genes. However, how the biosynthesis of these two QS signals is coordinated remains unknown. We studied the regulation of these two QS signals in the rhizosphere strain PA1201. PA1201 sequentially produced 3-oxo-C12-HSL and C4-HSL at the early and late growth stages, respectively. The highest 3-oxo-C12-HSL-dependent elastase activity was observed at the early stage, while the highest C4-HSL-dependent rhamnolipid production was observed at the late stage. The atypical regulator RsaL played a pivotal role in coordinating 3-oxo-C12-HSL and C4-HSL biosynthesis and QS-associated virulence. RsaL repressed lasI transcription by binding the -10 and -35 boxes of the lasI promoter. In contrast, RsaL activated rhlI transcription by binding the region encoding the 5'-untranslated region of the rhlI mRNA. Further, RsaL repressed its own expression by binding a nucleotide motif located in the -35 box of the rsaL promoter. Thus, RsaL acts as a molecular switch that coordinates the sequential biosynthesis of AHL QS signals and differential virulence in PA1201. Finally, C4-HSL activation by RsaL was independent of the Las and Pseudomonas quinolone signal (PQS) QS signaling systems. Therefore, we propose a new model of the QS regulatory network in PA1201, in which RsaL represents a superior player acting at the top of the hierarchy.

Berberine protects hepatocyte from hypoxia/reoxygenation-induced injury through inhibiting circDNTTIP2.

During hepatic ischemia-reperfusion injury, the excessive release of inflammatory cytokines can activate the intracellular signal transduction cascade to induce hepatocyte injury. Apoptosis is an important way of cell death after I/R injury. Berberine, a common quaternary ammonium alkaloid, has anti-inflammatory, anti-oxidative stress, and anti-apoptotic effects. An increasing number of studies have revealed the importance of non-coding RNAs, including microRNA, long non-coding RNAs and circular RNAs (circRNAs), as regulators of the effects of berberine. In this study, we investigated the mechanism of berberine against liver ischemia-reperfusion injury in vitro. In this study, hypoxia-reoxygenation (H/R)-treated L02 cells were pretreated with berberine to study the role and mechanism of berberine in resisting hepatic ischemia-reperfusion injury. The results show that berberine pre-treatment increased the cell viability of H/R-challenged cells, reduced H/R-induced apoptosis and ROS production, reversed H/R-increased on IL-6, IL-1β, TNF-α, and H/R-decreased IL-10 expression. Mechanically, berberine protect hepatocyte from H/R injury, at least partially, through circDNTTIP2. In addition, circDNTTIP2 can bind to the TATA box of caspase3 promoter, thereby promoting caspase 3-related cell apoptosis and the release of inflammatory cytokines. This study found that berberine has a protective effect on H/R-induced hepatocyte damage by inhibiting a novel circRNA, circDNTTIP2. This study provides potential treatment strategies and treatment targets for liver ischemia-reperfusion injury.

Demethylation in promoter region of severely damaged hepatocytes enhances chemokine receptor CXCR4 gene expression.

The liver is known to possess remarkable regenerative potential, but persistent inflammation or severe acute injury can lead to liver fibrosis and incomplete regeneration, ultimately resulting in liver failure. Recent studies have shown that the axis of two types of CXCL12 receptors, CXCR4 and CXCR7, plays a crucial role in liver fibrosis and regeneration. The present study aimed to investigate the regulatory factors involved in CXCR4 expression in injured liver. Immunohistochemical screening of liver tissue samples collected during liver transplantation revealed a reciprocal expression pattern between CXCR4 and MeCP2. An in vitro system involving cultured cell lines and H2O2 treatment was established to study the impact of oxidative stress on signaling pathways and epigenetic alterations that affect CXCR4 mRNA expression. Operating through distinct signaling pathways, H2O2 treatment induced a dose-dependent increase in CXCR4 expression in both hepatocyte- and intrahepatic cholangiocyte-derived cells. Treatment of the cells with trichostatin and azacytidine modulated CXCR4 expression in hepatocytes by modifying the methylation status of CpG dinucleotides located in a pair of TA repeats adjacent to the TATA box of the CXCR4 gene promoter. Only MeCP2 bound to oligonucleotides representing the TATA box region when the cytosine residues within the sequence were methylated, as revealed by electrophoretic mobility shift assay (EMSA). Methylation-specific PCR analysis of microdissected samples revealed a correlation between the loss of CpG methylation and the upregulation of CXCR4 in injured hepatocytes, replicating the findings from the in vitro study. Besides the conventional MEK/ERK and NF-κB signaling pathways that activate CXCR4 in intrahepatic cholangiocytes, the unique epigenetic modifications observed in hepatocytes might also contribute to a shift in the CXCR4-CXCR7 balance towards CXCR4, leading to irreversible liver injury and fibrosis. This study highlights the importance of epigenetic modifications in regulating CXCR4 expression in liver injury and fibrosis.

Identification and promoter analysis of a GA-stimulated transcript 1 gene from Jatropha curcas.

Overexpression of JcGAST1 promotes plant growth but inhibits pistil development. The pyrimidine box and CGTCA motif of the JcGAST1 promoter were responsible for the GA and MeJA responses. Members of the gibberellic acid-stimulated Arabidopsis (GASA) gene family play roles in plant growth and development, particularly in flower induction and seed development. However, there is still relatively limited knowledge of GASA genes in Jatropha curcas. Herein, we identified a GASA family gene from Jatropha curcas, namely, JcGAST1, which encodes a protein containing a conserved GASA domain. Sequence alignment showed that the JcGAST1 protein shares 76% sequence identity and 80% sequence similarity with SlGAST1. JcGAST1 had higher expression and protein levels in the female flowers than in the male flowers. Overexpression of JcGAST1 in tobacco promotes plant growth but inhibits pistil development. JcGAST1 expression was upregulated by GA and downregulated by MeJA. Promoter analysis indicated that the pyrimidine box and CGTCA motif were the GA- and MeJA-responsive elements of the JcGAST1 promoter. Using a Y1H screen, six transcription factors were found to interact with the pyrimidine box, and three transcription factors were found to interact with the CGTCA motif. Overall, the results of this study improve our understanding of the JcGAST1 gene and provide useful information for further studies.

Investigating the sequence landscape in the Drosophila initiator core promoter element using an enhanced MARZ algorithm.

The core promoter elements are important DNA sequences for the regulation of RNA polymerase II transcription in eukaryotic cells. Despite the broad evolutionary conservation of these elements, there is extensive variation in the nucleotide composition of the actual sequences. In this study, we aim to improve our understanding of the complexity of this sequence variation in the TATA box and initiator core promoter elements in Drosophila melanogaster. Using computational approaches, including an enhanced version of our previously developed MARZ algorithm that utilizes gapped nucleotide matrices, several sequence landscape features are uncovered, including an interdependency between the nucleotides in position 2 and 5 in the initiator. Incorporating this information in an expanded MARZ algorithm improves predictive performance for the identification of the initiator element. Overall our results demonstrate the need to carefully consider detailed sequence composition features in core promoter elements in order to make more robust and accurate bioinformatic predictions.

Factors influencing bilirubin elevation and its correlation with UGT1A1 gene polymorphism in the early postoperative period of transjugular intrahepatic portosystemic shunt

Objective: To investigate the factors influencing total bilirubin elevation and its correlation with UGT1A1 gene polymorphism in the early postoperative period of transjugular intrahepatic portosystemic shunt (TIPS). Methods: 104 cases with portal hypertension and esophageal variceal hemorrhage (EVB) treated with elective TIPS treatment were selected as the study subjects and were divided into a bilirubin-elevated group and a normal bilirubin group according to the total bilirubin elevation level during the early postoperative period. Univariate analysis and logistic regression were used to analyze the factors influencing total bilirubin elevation in the early postoperative period. PCR amplification and first-generation sequencing technology were used to detect the polymorphic loci of the UGT1A1 gene promoter TATA box, enhancer c.-3279 T > G, c.211G > A, and c.686C > A. Logistic regression was used to analyze the correlation of four locus alleles and genotypes with elevated total bilirubin in the early postoperative period. Results: Among the 104 cases, 47 patients were in the bilirubin elevated group, including 35 males (74.5%) and 12 females (25.5%), aged (50.72 ± 12.56) years. There were 57 cases in the normal bilirubin group, including 42 males (73.7%) and 15 females (26.3%), aged (51.63 ± 11.10) years. There was no statistically significant difference in age (t = -0.391, P = 0.697) and gender (χ(2) = 0.008, P = 0.928) between the two groups of patients. Univariate analysis revealed that preoperative alanine transaminase (ALT) level (χ(2) = 5.954, P = 0.015), total bilirubin level (χ(2) = 16.638, P < 0.001), MELD score (χ(2) = 10.054, P = 0.018), Child-Pugh score (χ(2) = 6.844, P = 0.022), and postoperative portal vein branch development (χ(2) = 6.738, P = 0.034) were statistically significantly different between the two groups. Logistic regression analysis showed that preoperative ALT level, total bilirubin level, and portal vein branch development after TIPS were correlated with the elevated total bilirubin in the early postoperative period. The polymorphism of the c.211G > A locus of the UGT1A1 gene correlation had elevated total bilirubin in the early postoperative period of TIPS. The risk of elevated total bilirubin was increased in the population carrying allele A (P = 0.001, OR = 4.049) in the early postoperative period. Allelic polymorphisms in the TATA box promoter region and enhancer c.-3279 T > G and c.686C > A had no statistically significant difference between the bilirubin-elevated group and the normal bilirubin group. Conclusion: The preoperative ALT level, total bilirubin level, and portal vein branch development are correlated with the elevated total bilirubin in early postoperative patients. The polymorphisms of the UGT1A1 gene and enhancer c.211G > A are correlated with the occurrence of elevated total bilirubin in the early postoperative period of TIPS. Allele A carrier may have a higher risk of elevated total bilirubin in the early postoperative period.

Liraglutide on type 2 diabetes mellitus with nonalcoholic fatty liver disease: A systematic review and meta-analysis of 16 RCTs.

Nonalcoholic fatty liver disease (NAFLD) is a common comorbidity of type 2 diabetes mellitus (T2DM). Our aim is to investigate the effects of liraglutide on T2DM with NAFLD. Relevant articles published from the earliest publication to March 2022 were selected from several databases. The Cochrane Collaboration's RevMan software was used for the analysis. Sixteen studies are selected for this meta-analysis, which includes totally 634 patients in the treatment group and 630 patients in the control group. As a result, 14 studies show that fasting plasma glucose levels of the experimental group are lower than that of the control group; 15 studies show that glycosylated hemoglobin A1c levels of the experimental group are lower than that of the control group; 13 studies show that triglyceride levels of the experimental group are lower than that of the control group; twelve studies show that total cholesterol levels of the experimental group are lower than that of the control group; 10 studies show that alanine aminotransferase levels of the experimental group is lower than that of the control group; 10 studies show that no significant difference in changes in aspartate transaminase between 2 groups; 13 studies show that low density lipoprotein cholesterol levels of the experimental group is lower than that of the control group; 9 studies show that no significant difference in changes in high density lipoprotein cholesterol between 2 groups; 7 studies mentioned adverse effects and the difference is significant. Liraglutide is potentially curative for T2DM with NAFLD.

DDX39B drives colorectal cancer progression by promoting the stability and nuclear translocation of PKM2

Metastasis is a major cause of colorectal cancer (CRC) mortality, but its molecular mechanisms are still not fully understood. Here, we show that upregulated DDX39B correlates with liver metastases and aggressive phenotypes in CRC. DDX39B is an independent prognostic factor associated with poor clinical outcome in CRC patients. We demonstrate that Sp1 potently activates DDX39B transcription by directly binding to the GC box of the DDX39B promoter in CRC cells. DDX39B overexpression augments the proliferation, migration, and invasion of CRC cells, while the opposite results are obtained in DDX39B-deficient CRC cells. Mechanistically, DDX39B interacts directly with and stabilizes PKM2 by competitively suppressing STUB1-mediated PKM2 ubiquitination and degradation. Importantly, DDX39B recruits importin α5 to accelerate the nuclear translocation of PKM2 independent of ERK1/2-mediated phosphorylation of PKM2, leading to the transactivation of oncogenes and glycolysis-related genes. Consequently, DDX39B enhances glucose uptake and lactate production to activate Warburg effect in CRC. We identify that Arg319 of DDX39B is required for PKM2 binding as well as PKM2 nuclear accumulation and for DDX39B to promote CRC growth and metastasis. In addition, blocking PKM2 nuclear translocation or treatment with glycolytic inhibitor 2-deoxy-D-glucose efficiently abolishes DDX39B-triggered malignant development in CRC. Taken together, our findings uncover a key role for DDX39B in modulating glycolytic reprogramming and aggressive progression, and implicate DDX39B as a potential therapeutic target in CRC.

RNA Polymerase II Transcription in Pneumocystis: TFIIB from Pneumocystis carinii Can Replace the Transcriptional Functions of Fission Yeast Schizosaccharomyces pombe TFIIB In Vivo and In Vitro.

The Pneumocystis genus is an opportunistic fungal pathogen that infects patients with AIDS and immunocompromised individuals. The study of this fungus has been hampered due to the inability to grow it in a (defined media/pure) culture. However, the use of modern molecular techniques and genomic analysis has helped researchers to understand its complex cell biology. The transcriptional process in the Pneumocystis genus has not been studied yet, although it is assumed that it has conventional transcriptional machinery. In this work, we have characterized the function of the RNA polymerase II (RNAPII) general transcription factor TFIIB from Pneumocystis carinii using the phylogenetically related biological model Schizosaccharomyces pombe. The results of this work show that Pneumocystis carinii TFIIB is able to replace the essential function of S. pombe TFIIB both in in vivo and in vitro assays. The S. pombe strain harboring the P carinii TFIIB grew slower than the parental wild-type S. pombe strain in complete media and in minimal media. The S. pombe cells carrying out the P. carinii TFIIB are larger than the wild-type cells, indicating that the TFIIB gene replacement confers a phenotype, most likely due to defects in transcription. P. carinii TFIIB forms very weak complexes with S. pombe TATA-binding protein on a TATA box promoter but it is able to form stable complexes in vitro when S. pombe TFIIF/RNAPII are added. P. carinii TFIIB can also replace the transcriptional function of S. pombe TFIIB in an in vitro assay. The transcription start sites (TSS) of the endogenous adh gene do not change when P. carinii TFIIB replaces S. pombe TFIIB, and neither does the TSS of the nmt1 gene, although this last gene is poorly transcribed in vivo in the presence of P. carinii TFIIB. Since transcription by RNA polymerase II in Pneumocystis is poorly understood, the results described in this study are promising and indicate that TFIIB from P. carinii can replace the transcriptional functions of S. pombe TFIIB, although the cells expressing the P. carini TFIIB show an altered phenotype. However, performing studies using a heterologous approach, like this one, could be relevant to understanding the basic molecular processes of Pneumocystis such as transcription and replication.

Identification of Promoter Region Markers Associated With Altered Expression of Resistance-Nodulation-Division Antibiotic Efflux Pumps in Acinetobacter baumannii.

Genetic alterations leading to the constitutive upregulation of specific efflux pumps contribute to antibacterial resistance in multidrug resistant bacteria. The identification of such resistance markers remains one of the most challenging tasks of genome-level resistance predictors. In this study, 487 non-redundant genetic events were identified in upstream zones of three operons coding for resistance-nodulation-division (RND) efflux pumps of 4,130 Acinetobacter baumannii isolates. These events included insertion sequences, small indels, and single nucleotide polymorphisms. In some cases, alterations explicitly modified the expression motifs described for these operons, such as the promoter boxes, operators, and Shine-Dalgarno sequences. In addition, changes in DNA curvature and mRNA secondary structures, which are structural elements that regulate expression, were also calculated. According to their influence on RND upregulation, the catalog of upstream modifications were associated with “experimentally verified,” “presumed,” and “probably irrelevant” degrees of certainty. For experimental verification, DNA of upstream sequences independently carrying selected markers, three for each RND operon, were fused to a luciferase reporter plasmid system. Five out of the nine selected markers tested showed significant increases in expression with respect to the wild-type sequence control. In particular, a 25-fold expression increase was observed with the ISAba1 insertion sequence upstream the adeABC pump. Next, overexpression of each of the three multi-specific RND pumps was linked to their respective antibacterial substrates by a deep A. baumannii literature screen. Consequently, a data flow framework was then developed to link genomic upregulatory RND determinants to potential antibiotic resistance. Assignment of potential increases in minimal inhibitory concentrations at the “experimentally verified” level was permitted for 42 isolates to 7–8 unrelated antibacterial agents including tigecycline, which is overlooked by conventional resistome predictors. Thus, our protocol may represent a time-saving filter step prior to laborious confirmation experiments for efflux-driven resistance. Altogether, a computational-experimental pipeline containing all components required for identifying the upstream regulatory resistome is proposed. This schema may provide the foundational stone for the elaboration of tools approaching antibiotic efflux that complement routine resistome predictors for preventing antimicrobial therapy failure against difficult-to-threat bacteria.

An experimental study of the effects of SNPs in the TATA boxes of the GRIN1, ASCL3 and NOS1 genes on interactions with the TATA-binding protein.

The GRIN1, ASCL3, and NOS1 genes are associated with various phenotypes of neuropsychiatric disorders. For instance, these genes contribute to the development of schizophrenia, Alzheimer’s and Parkinson’s diseases, and epilepsy. These genes are also associated with various cancers. For example, ASCL3 is overexpressed in breast cancer, and NOS1, in ovarian cancer cell lines. Based on our findings and literature data, we had previously obtained results suggesting that the single-nucleotide polymorphisms (SNPs) that disrupt erythropoiesis are highly likely to be associated with cognitive and neuropsychiatric disorders in humans. In the present work, using SNP_TATA_Z-tester, we investigated the influence of unannotated SNPs in the TATA boxes of the promoters of the GRIN1, ASCL3, and NOS1 genes (which are involved in neuropsychiatric disorders and cancers) on the interaction of the TATA boxes with the TATA-binding protein (TBP). Double-stranded oligodeoxyribonucleotides identical to the TATA-containing promoter regions of the GRIN1, ASCL3, and NOS1 genes (reference and minor alleles) and recombinant human TBP were employed to study in vitro (by an electrophoretic mobility shift assay) kinetic characteristics of the formation of TBP–TATA complexes and their affinity. It was found, for example, that allele A of rs1402667001 in the GRIN1 promoter increases TBP–TATA affinity 1.4-fold, whereas allele C in the TATA box of the ASCL3 promoter decreases the affinity 1.4-fold. The lifetime of the complexes in both cases decreased by ~20 % due to changes in the rates of association and dissociation of the complexes (ka and kd, respectively). Our experimental results are consistent with the literature showing GRIN1 underexpression in schizophrenic disorders as well as an increased risk of cervical, bladder, and kidney cancers and lymphoma during ASCL3 underexpression. The effect of allele A of the –27G>A SNP (rs1195040887) in the NOS1 promoter is suggestive of an increased risk of ischemic damage to the brain in carriers. A comparison of experimental TBP–TATA affinity values (KD) of wild-type and minor alleles with predicted ones showed that the data correlate well (linear correlation coefficient r = 0.94, p <0.01).

Characterizing methanol metabolism-related promoters for metabolic engineering of Ogataea polymorpha.

Promoters play an important role in regulating gene expression, and construction of microbial cell factories requires multiple promoters for balancing the metabolic pathways. However, there are only a limited number of characterized promoters for gene expression in the methylotrophic yeast Ogataea polymorpha, which hampers the extensive harnessing of this important yeast toward a cell factory. Here we characterized the promoters of methanol utilization pathway, precursor supply pathway, and reactive oxygen species (ROS) defense system, by using a green fluorescence protein variant (GFPUV) as a quantification signal. Finally, the characterized promoters were used for tuning a fatty alcohol biosynthetic pathway in O. polymorpha and realized fatty alcohol production from methanol. This promoter box should be helpful for gene expression and pathway optimization in the methylotrophic yeast O. polymorpha. KEY POINTS : • 22 promoters related to methanol metabolism were characterized in O. polymorpha. • Promoter truncation resulted shorter and compact promoters. • Promoters with various strengths were used for regulating a fatty alcohol biosynthesis from methanol.

Mutations in adaptively evolved Escherichia coli LGE2 facilitated the cost-effective upgrading of undetoxified bio-oil to bioethanol fuel

Levoglucosan is a promising sugar present in the lignocellulose pyrolysis bio-oil, which is a renewable and environment-friendly source for various value-added productions. Although many microbial catalysts have been engineered to produce biofuels and chemicals from levoglucosan, the demerits that these biocatalysts can only utilize pure levoglucosan while inhibited by the inhibitors co-existing with levoglucosan in the bio-oil have greatly limited the industrial-scale application of these biocatalysts in lignocellulose biorefinery. In this study, the previously engineered Escherichia coli LGE2 was evolved for enhanced inhibitor tolerance using long-term adaptive evolution under the stress of multiple inhibitors and finally, a stable mutant E. coli-H was obtained after ~ 374 generations’ evolution. In the bio-oil media with an extremely acidic pH of 3.1, E. coli-H with high inhibitor tolerance exhibited remarkable levoglucosan consumption and ethanol production abilities comparable to the control, while the growth of the non-evolved strain was completely blocked even when the pH was adjusted to 7.0. Finally, 8.4 g/L ethanol was achieved by E. coli-H in the undetoxified bio-oil media with ~ 2.0% (w/v) levoglucosan, reaching 82% of the theoretical yield. Whole-genome re-sequencing to monitor the acquisition of mutations identified 4 new mutations within the globally regulatory genes rssB, yqhA, and basR, and the − 10 box of the putative promoter of yqhD-dgkA operon. Especially, yqhA was the first time to be revealed as a gene responsible for inhibitor tolerance. The mutations were all responsible for improved fitness, while basR mutation greatly contributed to the fitness improvement of E. coli-H. This study, for the first time, generated an inhibitor-tolerant levoglucosan-utilizing strain that could produce cost-effective bioethanol from the toxic bio-oil without detoxification process, and provided important experimental evidence and valuable genetic/proteinic information for the development of other robust microbial platforms involved in lignocellulose biorefining processes.

Transcription factor MaMADS36 plays a central role in regulating banana fruit ripening.

Bananas are model fruits for studying starch conversion and climactericity. Starch degradation and ripening are two important biological processes that occur concomitantly in banana fruit. Ethylene biosynthesis and postharvest fruit ripening processes, i.e. starch degradation, fruit softening, and sugar accumulation, are highly correlated and thus could be controlled by a common regulatory switch. However, this switch has not been identified. In this study, we transformed red banana (Musa acuminata L.) with sense and anti-sense constructs of the MaMADS36 transcription factor gene (also MuMADS1, Ma05_g18560.1). Analysis of these lines showed that MaMADS36 interacts with 74 other proteins to form a co-expression network and could act as an important switch to regulate ethylene biosynthesis, starch degradation, softening, and sugar accumulation. Among these target genes, musa acuminata beta-amylase 9b (MaBAM9b, Ma05_t07800.1), which encodes a starch degradation enzyme, was selected to further investigate the regulatory mechanism of MaMADS36. Our findings revealed that MaMADS36 directly binds to the CA/T(r)G box of the MaBAM9b promoter to increase MaBAM9b transcription and, in turn, enzyme activity and starch degradation during ripening. These results will further our understanding of the fine regulatory mechanisms of MADS-box transcription factors in regulating fruit ripening, which can be applied to breeding programs to improve fruit shelf-life.

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

BackgroundIn biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research.ResultsIn this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica.ConclusionWe envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

TaqMan real time PCR for the Detection of the Gilbert's Syndrome Markers UGT1A1*28; UGT1A1*36 and UGT1A1*37.

Gilbert's syndrome is characterized by mild unconjugated hyperbilirubinemia. The key of this disease is a diminished activity of UDP-glucuronosyltransferase 1A1 (UGT1A1). TA insertion into the TATA box promoter region of the UGT1A1 gene on chromosome 2 is the genetic basis of Gilbert's syndrome (UGT1A1*28). An extra TA insert leads to eight (TA)8 repeats (UGT1A1*37) resulting in a further reduction of glucuronidation activity. A variant lacking one TA repeat (TA)5 (UGT1A1*36) has been identified. (TA)8 repeats (UGT1A1*37) and (TA)5 (UGT1A1*36) have been detected in Africans (frequency up to 0.07 and 0.08 respectively). We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR on 7500 and cfx96 Real-Time PCR System. We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR. About clinical validation, all 53 samples collected from patients referred for suspected Gilbert's syndrome were analyzed. We found 21 on the 53 patients (39.6%) were homozygotes (UGT1A1-TATA (TA)6) and referred as wild-type, 13 on the 53 patients (24.5%) were homozygotes (UGT1A1-TATA (TA)7) and referred as mutated, 1 on the 53 patients (1.9%) were homozygotes (UGT1A1-TATA (TA)8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)7/8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)5/6) and referred as mutated, and 16 on the 53 patients (30.2%) were heterozygotes (UGT1A1-TATA (TA)6/7). None were homozygotes UGT1A1-TATA (TA)5, homozygotes UGT1A1-TATA (TA)8, or heterozygotes with (TA)5 or (TA)8 alleles. The newly described technique represents a valid alternative method to sequencing, mainly due to its rapidity, easiness, and minor costs.

Assembling for transcription initiation

Transcription Eukaryotic transcription initiation by RNA polymerase II (Pol II) requires the assembly of a preinitiation complex (PIC) on core promoters. The binding of TATA box–binding protein (TBP) to the TATA box promoter has been thought to be a general rule in PIC assembly and transcription initiation. However, most coding genes lack a TATA box, and nearly all Pol II–mediated gene transcription requires the TBP-containing multisubunit complex transcription factor IID (TFIID). Chen et al. determined the structures of human TFIID-based PIC in sequential assembly states and revealed that TFIID supports distinct PIC assembly on TATA-containing and TATA-lacking promoters. The finding resolves the long-standing mystery of how one set of general transcription machinery initiates transcription on diverse promoters. Science , this issue p. [eaba8490][1] [1]: /lookup/doi/10.1126/science.aba8490