DNA-binding Transcriptional Regulators Research Articles

Metabolic Engineering of Escherichia coli for High-Level Production of l-Phenylalanine.

l-Phenylalanine (l-Phe) is widely used in the food and pharmaceutical industries. However, the biosynthesis of l-Phe using Escherichia coli remains challenging due to its lower tolerance to high concentration of l-Phe. In this study, to efficiently synthesize l-Phe, the l-Phe biosynthetic pathway was reconstructed by expressing the heterologous genes aroK1, aroL1, and pheA1, along with the native genes aroA, aroC, and tyrB in the shikimate-producing strain E. coli SA09, resulting in the engineered strain E. coli PHE03. Subsequently, adaptive evolution was conducted on E. coli PHE03 to enhance its tolerance to high concentrations of l-Phe, resulting in the strain E. coli PHE04, which reduced the cell mortality to 36.2% after 48 h of fermentation. To elucidate the potential mechanisms, transcriptional profiling was conducted, revealing MarA, a DNA-binding transcriptional dual regulator, as playing a crucial role in enhancing cell membrane integrity and fluidity for improving cell tolerance to high concentrations of l-Phe. Finally, the titer, yield, and productivity of l-Phe with E. coli PHE05 overexpressing marA were increased to 80.48 g/L, 0.27 g/g glucose, and 1.68 g/L/h in a 5-L fed-batch fermentation, respectively.

Improving the production of recombinant L-Asparaginase-II in Escherichia coli by co-expressing catabolite repressor activator (cra) gene

Identification of a single genetic target for microbial strain improvement is difficult due to the complexity of the genetic regulatory network. Hence, a more practical approach is to identify bottlenecks in the regulatory networks that control critical metabolic pathways. The present work focuses on enhancing cellular physiology by increasing the metabolic flux through the central carbon metabolic pathway. Global regulator cra (catabolite repressor activator), a DNA-binding transcriptional dual regulator was selected for the study as it controls the expression of a large number of operons that modulate central carbon metabolism. To upregulate the activity of central carbon metabolism, the cra gene was co-expressed using a plasmid-based system. Co-expression of cra led to a 17% increase in the production of model recombinant protein L-Asparaginase-II. A pulse addition of 0.36% of glycerol every two hours post-induction, further increased the production of L-Asparaginase-II by 35% as compared to the control strain expressing only recombinant protein. This work exemplifies that upregulating the activity of central carbon metabolism by tuning the expression of regulatory genes like cra can relieve the host from cellular stress and thereby promote the growth as well as expression of recombinant hosts.

Characterization and Expression Analysis of HbC3H66: Implications for Transcriptional Regulation in Rubber Biosynthesis and Abiotic Stress Responses in Hevea brasiliensis

CCCH-type zinc-finger protein (ZnF_CCCH) family is one of the most important transcription factors (TFs) linked to various biotic and abiotic stressors and physiological and developmental processes in plants. This study aims to clone and characterise the latex gene expression patterns, evolution and characteristics of the Hevea brasiliensis ZnF_CCCH domain-containing protein 66 (HbC3H66) gene. These results showed that the open reading frame (ORF) of the HbC3H66 gene was 2,106 bp, encoding 701 aa and the calculated molecular weight of the encoded protein was 76.52 kDa. The N-terminal region of HbC3H66 contains 2 ANK repeats in, PfK_2/FBPase-2 and 2 type zinc finger motifs. Phylogenetic analysis showed that the C3H66 amino acid from Hevea and other plant C3H66 were clustered into 1 group and could be used for evolutionary analysis. Semi-quantitative RT-PCR (sqRT-PCR) revealed that the HbC3H66 mRNA was abundance in high-yielding Hevea clones (RRIT251 and RRIM600) and treatment with 2.5 % of ethephon (Eth) induced HbC3H66 mRNA expression in the latex of 15 years old Hevea trees. The HbC3H66 gene was induced by water deficit and 0.5 M NaCl in the latex of 3-month-old Hevea. In conclusion, the HbC3H66 protein may play a role in DNA-binding transcriptional regulation in NR pathways. Which would provide a theoretical basis for understanding the evolution and functions of the Hevea C3H66 gene in rubber biosynthesis. HIGHLIGHTS CCCH-type zinc-finger proteins (ZnF_CCCH) are essential transcription factors (TFs) for protein binding activity in plant growth and stress tolerance. Hevea brasiliensis zinc finger CCCH domain-containing protein 66 (HbC3H66) gene was clone and characterized. HbC3H66 contains 2 ANK repeat in, Pfk_2/FBPase-2 and 2 type zinc finger motifs. Semi-quantitative RT-PCR revealed that HbC3H66 gene was abundance in high-yielding Hevea clones (RRIT251 and RRIM600) and ethylene was induced HbC3H66 mRNA expression in the latex of Hevea The HbC3H66 gene was induced by water deficit and 0.5 M NaCl in the latex of seedling of Hevea trees. The HbC3H66 protein play a role in DNA-binding transcriptional regulation in NR pathway. GRAPHICAL ABSTRACT

The PapB/FocB family protein TosR acts as a positive regulator of flagellar expression and is required for optimal virulence of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is a major causative agent of urinary tract infections. The bacteria internalize into the uroepithelial cells, where aggregate and form microcolonies. UPEC fimbriae and flagella are important for the formation of microcolonies in uroepithelial cells. PapB/FocB family proteins are small DNA-binding transcriptional regulators consisting of approximately 100 amino acids that have been reported to regulate the expression of various fimbriae, including P, F1C, and type 1 fimbriae, and adhesins. In this study, we show that TosR, a member of the PapB/FocB family is the activator of flagellar expression. The tosR mutant had similar expression levels of type 1, P and F1C fimbriae as the parent strain, but flagellar production was markedly lower than in the parent strain. Flagellin is a major component of flagella. The gene encoding flagellin, fliC, is transcriptionally activated by the sigma factor FliA. The fliA expression is induced by the flagellar master regulator FlhDC. The flhD and flhC genes form an operon. The promoter activity of fliC, fliA and flhD in the tosR mutant was significantly lower than in the parent strain. The purified recombinant TosR does not bind to fliC and fliA but to the upstream region of the flhD gene. TosR is known to bind to an AT-rich DNA sequence consisting of 29 nucleotides. The characteristic AT-rich sequence exists 550-578 bases upstream of the flhD gene. The DNA fragment lacking this sequence did not bind TosR. Furthermore, loss of the tosR gene reduced motility and the aggregation ability of UPEC in urothelial cells. These results indicate that TosR is a transcriptional activator that increases expression of the flhDC operon genes, contributing to flagellar expression and optimal virulence.

Based on network pharmacology to explore the effect and mechanism of Yipibushen decoction in improving obese type 2 diabetes mellitus with oligoasthenotspermia

A traditional Chinese medicine experience compound known as Yipibushen (YPBS) decoction stimulates qi and nourishes yin, stimulates the kidney and solid essence, dissolves phlegm and eliminates stasis. YPBS decoction has proven to be successful in treating obese type 2 diabetes mellitus with oligoasthenotspermia in clinical settings. Nevertheless, the pharmacological mechanism is not understood. Investigating the mechanism of action of YPBS decoction in enhancing the obese type 2 diabetes mellitus with oligoasthenotspermia involved network pharmacology and animal validation techniques. The YPBS Decoction' active components were found in the TCMSP database and their targets were identified using UniProtKB. Additionally, targets for the obese type 2 diabetes mellitus with oligoasthenotspermia were found in the GeneCard, DisGeNet, TTD and OMIM databases. The intersection of active ingredients, the obese type 2 diabetes mellitus with oligoasthenotspermia was chosen as the intersection target. The protein-protein interaction (PPI) network of the intersection target was built with the aid of Cytoscape 3.9.1, the core target of PPI was obtained through software analysis in R-project, GO enrichment and KEGG enrichment analysis was carried out on the core target. Finally, animal experiments were used to verify the intersection target. The research revealed 74 intersection targets of YPBS decoction active ingredients in the obese type 2 diabetes mellitus with oligoasthenotspermia. There were also 18 PPI core targets, GO enrichment analysis of PPI core targets involving response to oxidative stress, membrane raft, DNA-binding transcription regulator complex and other biological processes; KEGG involving endocrine resistance, PI3K/AKT signaling pathway, apoptosis and other signal pathways. In the obese type 2 diabetes mellitus with oligoasthenotspermia mice, animal studies have shown that YPBS decoction group could decrease blood glucose levels and improve insulin resistance; improve testicular function, enhance sperm count, sperm motility, sperm viability, and decrease the malformation rate. It could increase the levels of T-SOD and GSH-Px, and decrease the MDA level. In addition to this, it could improve the amount of testosterone hormone, and enhance the expression of PI3K, p-AKT and Bcl-2. By controlling the degree of oxidative stress and the PI3K/AKT/Bcl-2 pathway, YPBS decoction may enhance the obese type 2 diabetes mellitus with Oligoasthenotspermia, provide a scientific basis for clinical diagnosis and therapy.

Regulation of iron metabolism is critical for the survival of Salmonella Typhimurium in pasteurized milk

Salmonella is known to survive in raw/pasteurized milk and cause foodborne outbreaks. Lactoferrin, present in milk from all animal sources, is an iron-binding glycoprotein that limits the availability of iron to pathogenic bacteria. Despite the presence of lactoferrins, Salmonella can grow in milk obtained from different animal sources. However, the mechanism by which Salmonella overcomes iron scarcity induced by lactoferrin in milk is not evaluated yet. Salmonella employs the DNA binding transcriptional regulator Fur (ferric update regulator) to mediate iron uptake during survival in iron deplete conditions. To understand the importance of Fur in Salmonella milk growth, we profiled the growth of Salmonella Typhimurium Δfur (ST4/74Δfur) in both bovine and camel milk. ST4/74Δfur was highly inhibited in milk compared to wild-type ST4/74, confirming the importance of Fur mediated regulation of iron metabolism in Salmonella milk growth. We further studied the biology of ST4/74Δfur to understand the importance of iron metabolism in Salmonella milk survival. Using increasing concentrations of FeCl3, and the antibiotic streptonigrin we show that iron accumulates in the cytoplasm of ST4/74Δfur. We hypothesized that the accumulated iron could activate oxidative stress via Fenton’s reaction leading to growth inhibition. However, the inhibition of ST4/74Δfur in milk was not due to Fenton’s reaction, but due to the ‘iron scarce’ conditions of milk and microaerophilic incubation conditions which made the presence of the fur gene indispensable for Salmonella milk growth. Subsequently, survival studies of 14 other transcriptional mutants of ST4/74 in milk confirmed that RpoE-mediated response to extracytoplasmic stress is also important for the survival of Salmonella in milk. Though we have data only for fur and rpoE, many other Salmonella transcriptional factors could play important roles in the growth of Salmonella in milk, a theme for future research on Salmonella milk biology. Nevertheless, our data provide early insights into the biology of milk-associated Salmonella.

Krüppel-like factors in tumors: Key regulators and therapeutic avenues.

Krüppel-like factors (KLFs) are a group of DNA-binding transcriptional regulators with multiple essential functions in various cellular processes, including proliferation, migration, inflammation, and angiogenesis. The aberrant expression of KLFs is often found in tumor tissues and is essential for tumor development. At the molecular level, KLFs regulate multiple signaling pathways and mediate crosstalk among them. Some KLFs may also be molecular switches for specific biological signals, driving their transition from tumor suppressors to promoters. At the histological level, the abnormal expression of KLFs is closely associated with tumor cell stemness, proliferation, apoptosis, and alterations in the tumor microenvironment. Notably, the role of each KLF in tumors varies according to tumor type and different stages of tumor development rather than being invariant. In this review, we focus on the advances in the molecular biology of KLFs, particularly the regulations of several classical signaling pathways by these factors, and the critical role of KLFs in tumor development. We also highlight their strong potential as molecular targets in tumor therapy and suggest potential directions for clinical translational research.

Salmonella enterica serovar Typhimurium STM1266 encodes a regulator of curli biofilm formation: the brfS gene.

The major biofilm pathway in Salmonella enterica serovar Typhimurium involves specific growth conditions that induce the csgA gene whose product forms surface curli fibers that mediate biofilm formation. We have found that the previously uncharacterized STM1266 gene in S. Typhimurium plays a role in regulating biofilm formation via the curli pathway. S. Typhimurium ΔSTM1266 strains display a biofilm defect, and overexpression of STM1266 results in enhanced biofilm formation. STM1266 deletion resulted in lowered csgA expression using promoter-reporter β-galactosidase assays, and csgA and csgD deletions abrogate the effects of STM1266 overexpression on biofilm formation while deletion of bcsA (encoding an essential enzyme for cellulose formation) has no effect. In a mouse infection model, the ΔSTM1266 strain displayed results similar to those seen for previously reported ΔcsgA strains. The STM1266 gene is predicted to encode a DNA-binding transcriptional regulator of the MerR family and is homologous to the Escherichia coli BluR regulator protein. We respectfully propose to ascribe the name brfS (biofilm regulator for Salmonella Typhimurium) to the STM1266 gene.

Antibiotic Susceptibility, Resistance Gene Determinants and Corresponding Genomic Regions in Lactobacillus amylovorus Isolates Derived from Wild Boars and Domestic Pigs.

Restrictions on the use of antibiotics in pigs lead to the continuous search for new probiotics serving as an alternative to antibiotics. One of the key parameters for probiotic bacteria selection is the absence of horizontally transmissible resistance genes. The aim of our study was to determine antibiotic susceptibility profiles in 28 Lactobacillus amylovorus isolates derived from the digestive tract of wild boars and farm pigs by means of the broth microdilution method and whole genome sequencing (WGS). We revealed genetic resistance determinants and examined sequences flanking resistance genes in these strains. Our findings indicate that L. amylovorus strains from domestic pigs are predominantly resistant to tetracycline, erythromycin and ampicillin. WGS analysis of horizontally transmissible genes revealed only three genetic determinants (tetW, ermB and aadE) of which all tetW and ermB genes were present only in strains derived from domestic pigs. Sequence analysis of coding sequences (CDS) in the neighborhood of the tetW gene revealed the presence of site-specific recombinase (xerC/D), site-specific DNA recombinase (spoIVCA) or DNA-binding transcriptional regulator (xre), usually directly downstream of the tetW gene. In the case of ermB, CDS for omega transcriptional repressor or mobilization protein were detected upstream of the ermB gene.

Time-resolved emission spectroscopy to elucidate the functional nature of heat-stable transcription factor.

A 22 kDa protein from Thermus thermophilus is characterised as a DNAbinding transcription regulator and its function is established using the fluorescence spectroscopy technique. The steady-state fluorescence spectroscopy result shows significant binding of calf thymus DNA and protein molecule. To confirm, the DNA quenching effect in real-time, a time-resolved emission spectroscopy study was performed and the result shows good agreement with steady-state quenching analysis.

Molecular Regulatory Mechanisms Drive Emergent Pathogenetic Properties of Neisseria gonorrhoeae.

Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection (STI) gonorrhea, with an estimated 87 million annual cases worldwide. N. gonorrhoeae predominantly colonizes the male and female genital tract (FGT). In the FGT, N. gonorrhoeae confronts fluctuating levels of nutrients and oxidative and non-oxidative antimicrobial defenses of the immune system, as well as the resident microbiome. One mechanism utilized by N. gonorrhoeae to adapt to this dynamic FGT niche is to modulate gene expression primarily through DNA-binding transcriptional regulators. Here, we describe the major N. gonorrhoeae transcriptional regulators, genes under their control, and how these regulatory processes lead to pathogenic properties of N. gonorrhoeae during natural infection. We also discuss the current knowledge of the structure, function, and diversity of the FGT microbiome and its influence on gonococcal survival and transcriptional responses orchestrated by its DNA-binding regulators. We conclude with recent multi-omics data and modeling tools and their application to FGT microbiome dynamics. Understanding the strategies utilized by N. gonorrhoeae to regulate gene expression and their impact on the emergent characteristics of this pathogen during infection has the potential to identify new effective strategies to both treat and prevent gonorrhea.

HMGA2 Supports Cancer Hallmarks in Triple-Negative Breast Cancer.

Simple SummaryTriple-negative breast cancer grows and spreads faster compared to other types of invasive breast cancer and has limited treatment options and subsequently worse prognosis. High mobility group A 2 (HMGA2) protein is widely expressed in undifferentiated cells including cancer cells. HMGA2 is a DNA binding protein involved in DNA architecture and the functional regulation of DNA. It was reported that HMGA2 is aberrantly regulated malignant signaling in different types of cancers. In this study, we aim to reveal the role of HMGA2 in TNBC biology and demonstrate a link between HMGA2-mediated cancer phenotypes and the regulation of NF-kB/IL-6/STAT3 signaling.Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that exhibits a high proliferation rate and early metastasis leading to a poor prognosis. HMGA2 is a DNA binding transcriptional regulator implicated in tumorigenesis. Here, we demonstrate that the HMGA2 promoter is demethylated in TNBC tumors, leading to increased expression of HMGA2 at both mRNA and protein levels. Importantly, high HMGA2 levels in TNBC tumors are correlated with poor prognosis. To detail the role of HMGA2 in TNBC development and progression, we studied its effect on core cancer phenotypes. Stable knockdown of HMGA2 in TNBC cells revealed that HMGA2 may support cell proliferation, cell migration and invasion. In addition, HMGA2 knockdown decreased cancer stem cell (CSC) features. Importantly, we found that silencing HMGA2 inhibited NF-kB signaling and lead to decreased expression of the downstream molecules IL-6 and IL-8 and reduced STAT3 pathway activation. Our results demonstrate that HMGA2 supports cancer hallmarks in TNBC and may represent a promising target for TNBC treatment.

Whole-Genome Assessment of Clinical Acinetobacter baumannii Isolates Uncovers Potentially Novel Factors Influencing Carbapenem Resistance.

Carbapenems—one of the important last-line antibiotics for the treatment of gram-negative infections—are becoming ineffective for treating Acinetobacter baumannii infections. Studies have identified multiple genes (and mechanisms) responsible for carbapenem resistance. In some A. baumannii strains, the presence/absence of putative resistance genes is not consistent with their resistance phenotype—indicating the genomic factors underlying carbapenem resistance in A. baumannii are not fully understood. Here, we describe a large-scale whole-genome genotype-phenotype association study with 349 A. baumannii isolates that extends beyond the presence/absence of individual antimicrobial resistance genes and includes the genomic positions and pairwise interactions of genes. Ten known resistance genes exhibited statistically significant associations with resistance to imipenem, a type of carbapenem: blaOXA-23, qacEdelta1, sul1, mphE, msrE, ant(3”)-II, aacC1, yafP, aphA6, and xerD. A review of the strains without any of these 10 genes uncovered a clade of isolates with diverse imipenem resistance phenotypes. Finer resolution evaluation of this clade revealed the presence of a 38.6 kbp conserved chromosomal region found exclusively in imipenem-susceptible isolates. This region appears to host several HTH-type DNA binding transcriptional regulators and transporter genes. Imipenem-susceptible isolates from this clade also carried two mutually exclusive plasmids that contain genes previously known to be specific to imipenem-susceptible isolates. Our analysis demonstrates the utility of using whole genomes for genotype-phenotype correlations in the context of antibiotic resistance and provides several new hypotheses for future research.

Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations.

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B–BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.

Comparative proteomics unravelled the hexachlorocyclohexane (HCH) isomers specific responses in an archetypical HCH degrading bacterium Sphingobium indicum B90A.

Hexachlorocyclohexane (HCH) is a persistent organochlorine pesticide that poses threat to different life forms. Sphingobium indicum B90A that belong to sphingomonad is well-known for its ability to degrade HCH isomers (α-, β-, γ-, δ-), but effects of HCH isomers and adaptive mechanisms of strain B90A under HCH load remain obscure. To investigate the responses of strain B90A to HCH isomers, we followed the proteomics approach as this technique is considered as the powerful tool to study the microbial response to environmental stress. Strain B90A culture was exposed to α-, β-, γ-, δ-HCH (5 mgL-1) and control (without HCH) taken for comparison and changes in whole cell proteome were analyzed. In β- and δ-HCH-treated cultures growth decreased significantly when compared to control, α-, and γ-HCH-treated cultures. HCH residue analysis corroborated previous observations depicting the complete depletion of α- and γ-HCH, while only 66% β-HCH and 34% δ-HCH were depleted from culture broth. Comparative proteome analyses showed that β- and δ-HCH induced utmost systemic changes in strain B90A proteome, wherein stress-alleviating proteins such as histidine kinases, molecular chaperons, DNA binding proteins, ABC transporters, TonB proteins, antioxidant enzymes, and transcriptional regulators were significantly affected. Besides study confirmed constitutive expression of linA, linB, and linC genes that are crucial for the initiation of HCH isomers degradation, while increased abundance of LinM and LinN in presence of β- and δ-HCH suggested the important role of ABC transporter in depletion of these isomers. These results will help to understand the HCH-induced damages and adaptive strategies of strain B90A under HCH load which remained unravelled to date.

Genomic Analysis of Mesorhizobium loti Strain TONO Reveals Dehalogenases for Bioremediation

Halogenated compounds are extensively utilized in different industrial applications such as pesticides and herbicides and cause severe environmental problems because of their toxicity and persistence. Degradation of these compounds by the biological method is a significant method to reduce these recalcitrant. Mesorhizobium loti is important for nitrogen fixation in legume roots. Up to now, there is no report to indicate M. loti can produce dehalogenase enzymes. Thus, a total of twenty-five genomes of M. loti strains from the National Center for Biotechnology Information (NCBI) were analyzed. These strains notably carry dehalogenase genes and were further investigated. The relative ratio of haloalkane and haloacid dehalogenase type II or L-type from all twenty-five genomes was 26% and 74%, respectively, suggesting type II dehalogenase is common. Surprisingly, only M. loti strain TONO carries four dehalogenases and therefore it was further characterized. The chromosome of M. loti strain TONO contains four haloacid dehalogenase type II genes namely, dehLt1 (MLTONO_2099), dehLt2 (MLTONO_3660), dehLt3 (MLTONO_4143), and dehLt4 (MLTONO_6945), and their corresponding enzymes were designated as DehLt1, DehLt2, DehLt3, and DehLt4, respectively. The only haloalkane dehalogenase gene (MLTONO_4828) was located upstream of the dehLt3 gene and its amino acid share 88% identity with DmlA of Mesorhizobium japonicum MAFF 303099. The putative haloacid permease gene designated as dehrPt (MLTONO_0284) was located downstream of the dehLt1 and its amino acids show 69% identity with haloacid permease of Rhizobium sp. RC1. The gene encoding helix-turn-helix (HTH) motif family DNA-binding protein regulator and LysR family transcriptional regulator genes were also identified, possibly for regulatory functions. The genomic studies as such, have good potential to be screened for new type of dehalogenases based on basic molecular structure and functions analysis

Crosstalk Between MYC and lncRNAs in Hematological Malignancies.

The human genome project revealed the existence of many thousands of long non-coding RNAs (lncRNAs). These transcripts that are over 200 nucleotides long were soon recognized for their importance in regulating gene expression. However, their poor conservation among species and their still controversial annotation has limited their study to some extent. Moreover, a generally lower expression of lncRNAs as compared to protein coding genes and their enigmatic biochemical mechanisms have impeded progress in the understanding of their biological roles. It is, however, known that lncRNAs engage in various kinds of interactions and can form complexes with other RNAs, with genomic DNA or proteins rendering their functional regulatory network quite complex. It has emerged from recent studies that lncRNAs exert important roles in gene expression that affect many cellular processes underlying development, cellular differentiation, but also the pathogenesis of blood cancers like leukemia and lymphoma. A number of lncRNAs have been found to be regulated by several well-known transcription factors including Myelocytomatosis viral oncogene homolog (MYC). The c-MYC gene is known to be one of the most frequently deregulated oncogenes and a driver for many human cancers. The c-MYC gene is very frequently activated by chromosomal translocations in hematopoietic cancers most prominently in B- or T-cell lymphoma or leukemia and much is already known about its role as a DNA binding transcriptional regulator. Although the understanding of MYC’s regulatory role controlling lncRNA expression and how MYC itself is controlled by lncRNA in blood cancers is still at the beginning, an intriguing picture emerges indicating that c-MYC may execute part of its oncogenic function through lncRNAs. Several studies have identified lncRNAs regulating c-MYC expression and c-MYC regulated lncRNAs in different blood cancers and have unveiled new mechanisms how these RNA molecules act. In this review, we give an overview of lncRNAs that have been recognized as critical in the context of activated c-MYC in leukemia and lymphoma, describe their mechanism of action and their effect on transcriptional reprogramming in cancer cells. Finally, we discuss possible ways how an interference with their molecular function could be exploited for new cancer therapies.

RRE-Finder: a Genome-Mining Tool for Class-Independent RiPP Discovery.

Many ribosomally synthesized and posttranslationally modified peptide classes (RiPPs) are reliant on a domain called the RiPP recognition element (RRE). The RRE binds specifically to a precursor peptide and directs the posttranslational modification enzymes to their substrates. Given its prevalence across various types of RiPP biosynthetic gene clusters (BGCs), the RRE could theoretically be used as a bioinformatic handle to identify novel classes of RiPPs. In addition, due to the high affinity and specificity of most RRE-precursor peptide complexes, a thorough understanding of the RRE domain could be exploited for biotechnological applications. However, sequence divergence of RREs across RiPP classes has precluded automated identification based solely on sequence similarity. Here, we introduce RRE-Finder, a new tool for identifying RRE domains with high sensitivity. RRE-Finder can be used in precision mode to confidently identify RREs in a class-specific manner or in exploratory mode to assist in the discovery of novel RiPP classes. RRE-Finder operating in precision mode on the UniProtKB protein database retrieved ∼25,000 high-confidence RREs spanning all characterized RRE-dependent RiPP classes, as well as several yet-uncharacterized RiPP classes that require future experimental confirmation. Finally, RRE-Finder was used in precision mode to explore a possible evolutionary origin of the RRE domain. The results suggest RREs originated from a co-opted DNA-binding transcriptional regulator domain. Altogether, RRE-Finder provides a powerful new method to probe RiPP biosynthetic diversity and delivers a rich data set of RRE sequences that will provide a foundation for deeper biochemical studies into this intriguing and versatile protein domain.IMPORTANCE Bioinformatics-powered discovery of novel ribosomal natural products (RiPPs) has historically been hindered by the lack of a common genetic feature across RiPP classes. Herein, we introduce RRE-Finder, a method for identifying RRE domains, which are present in a majority of prokaryotic RiPP biosynthetic gene clusters (BGCs). RRE-Finder identifies RRE domains 3,000 times faster than current methods, which rely on time-consuming secondary structure prediction. Depending on user goals, RRE-Finder can operate in precision mode to accurately identify RREs present in known RiPP classes or in exploratory mode to assist with novel RiPP discovery. Employing RRE-Finder on the UniProtKB database revealed several high-confidence RREs in novel RiPP-like clusters, suggesting that many new RiPP classes remain to be discovered.

Tumor-derived neomorphic mutations in ASXL1 impairs the BAP1-ASXL1-FOXK1/K2 transcription network

Additional sex combs-like 1 (ASXL1) interacts with BRCA1-associated protein 1 (BAP1) deubiquitinase to oppose the polycomb repressive complex 1 (PRC1)-mediated histone H2A ubiquitylation. Germline BAP1 mutations are found in a spectrum of human malignancies, while ASXL1 mutations recurrently occur in myeloid neoplasm and are associated with poor prognosis. Nearly all ASXL1 mutations are heterozygous frameshift or nonsense mutations in the middle or to a less extent the C-terminal region, resulting in the production of C-terminally truncated mutant ASXL1 proteins. How ASXL1 regulates specific target genes and how the C-terminal truncation of ASXL1 promotes leukemogenesis are unclear. Here, we report that ASXL1 interacts with forkhead transcription factors FOXK1 and FOXK2 to regulate a subset of FOXK1/K2 target genes. We show that the C-terminally truncated mutant ASXL1 proteins are expressed at much higher levels than the wild-type protein in ASXL1 heterozygous leukemia cells, and lose the ability to interact with FOXK1/K2. Specific deletion of the mutant allele eliminates the expression of C-terminally truncated ASXL1 and increases the association of wild-type ASXL1 with BAP1, thereby restoring the expression of BAP1-ASXL1-FOXK1/K2 target genes, particularly those involved in glucose metabolism, oxygen sensing, and JAK-STAT3 signaling pathways. In addition to FOXK1/K2, we also identify other DNA-binding transcription regulators including transcription factors (TFs) which interact with wild-type ASXL1, but not C-terminally truncated mutant. Our results suggest that ASXL1 mutations result in neomorphic alleles that contribute to leukemogenesis at least in part through dominantly inhibiting the wild-type ASXL1 from interacting with BAP1 and thereby impairing the function of ASXL1-BAP1-TF in regulating target genes and leukemia cell growth.

Pseudomonas syringae dual-function protein Lon switches between virulence and metabolism by acting as both DNA-binding transcriptional regulator and protease in different environments.

Lon, a member of the AAA+ protease family, plays vital roles in Type III secretion systems (T3SS), agglutination and colony shape in the model plant pathogen Pseudomonas syringae. Lon also functions as a transcriptional regulator in other bacterial species such as Escherichia coli and Brevibacillus thermoruber. To reveal the molecular mechanisms of Lon as a dual-function protein in P. syringae, we studied Lon-regulated genes by using RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and liquid chromatography-tandem mass spectrometry. As a transcriptional regulator, Lon directly regulated a group of genes (PSPPH_4788, gacA, fur, gntR, clpS, lon and glyA) and consequently regulated their functions, such as 1-dodecanol oxidation activity, motility, pyoverdine production, glucokinase activity, N-end rule pathway, lon expression and serine hydroxymethyltransferase activity. Mass spectrometry results revealed that the expression levels of five T3SS proteins (such as HrcV, HrpW1) were higher in the ∆lon strain than the wild-type (WT) strain in KB. In MM, 12 metabolic proteins (such as AcdS and NuoI) showed lower levels in the ∆lon strain than the WT strain. Taken together, these data demonstrate that the dual-function protein Lon sophisticatedly regulates virulence and metabolism in P. syringae.