CRNDE/ETS1/GPR17 Facilitates the Proliferation, Migration, and Invasion of Glioma

Background. Numerous lncRNAs were found as regulatory factors for occurrence and progression of various tumors, but there is still less research on the role of lncRNAs in malignant progression of glioma. Methods. Bioinformatics analysis analyzed differential genes (DEGs) in the TCGA database. MTT, flow cytometry, and Transwell assays were performed to test the proliferation, apoptosis, migration, and invasion of cells. qRT-PCR and western blot were conducted to detect RNA and protein expressions of each gene, respectively. CHIP assay verified the binding relationship between genes. FISH assayed subcellar location of CRNDE, and xenograft in nude mice was performed for in vivo verification. Results. CRNDE was upregulated in glioma cells, and overexpression of CRNDE facilitated malignant progression of glioma cells. CRNDE regulated occurrence and development of glioma through the CRNDE-ETS1-GPR17 axis. ETS1 was proved to target promoter region of GPR17. Overexpression of CRNDE promoted the binding between ETS1 and the promoter region of GPR17, thus, promoting the transcription of GPR17, while silencing of GPR17 inhibited promotion of CRNDE on proliferation, migration, and invasion of glioma cells. Conclusions. These results demonstrated that CRNDE regulated GPR17 expression by binding ETS1, a transcription factor, thereby affecting glioma development. The results also indicated that CRNDE could serve as a possible therapeutic target and prognostic biomarker for glioma.

Definition of the Binding Architecture to a Target Promoter of HP1043, the Essential Master Regulator of Helicobacter pylori

HP1043 is an essential orphan response regulator of Helicobacter pylori orchestrating multiple crucial cellular processes. Classified as a member of the OmpR/PhoB family of two-component systems, HP1043 exhibits a highly degenerate receiver domain and evolved to function independently of phosphorylation. Here, we investigated the HP1043 binding mode to a target sequence in the hp1227 promoter (Php1227). Scanning mutagenesis of HP1043 DNA-binding domain and consensus sequence led to the identification of residues relevant for the interaction of the protein with a target DNA. These determinants were used as restraints to guide a data-driven protein-DNA docking. Results suggested that, differently from most other response regulators of the same family, HP1043 binds in a head-to-head conformation to the Php1227 target promoter. HP1043 interacts with DNA largely through charged residues and contacts with both major and minor grooves of the DNA are required for a stable binding. Computational alanine scanning on molecular dynamics trajectory was performed to corroborate our findings. Additionally, in vitro transcription assays confirmed that HP1043 positively stimulates the activity of RNA polymerase.

Differential effect of homologous NicR2A/NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. JY-Q

Nicotine is a toxic environmental pollutant that widely exists in tobacco wastes. As a natural nicotine-degrading strain, Pseudomonas sp. JY-Q still has difficulties to degrade high-concentrations of nicotine. In this study, we investigated the effect of two homologous transcriptional regulators and endogenous ectopic strong promoters on the efficiency of nicotine degradation. Comparative genomics analysis showed that two homologous transcriptional regulators NicR2A and NicR2Bs can repress nicotine-degrading genes expression. When both of nicR2A and nicR2Bs were deleted, the resulting mutant QΔnicR2AΔnicR2B1ΔnicR2B2 exhibit 17% higher nicotine degradation efficiency than wide type JY-Q. The RNA-seq analysis showed that the transcription level (FPKM value) of six genes was particularly higher than the other genes in JY-Q. Based on the genetic organization of these genes, three putative promoters, PRS28250, PRS09985 and PRS24685, were identified. Their promoter activities were evaluated by comparing their expression levels using RT-qPCR. We found that the transcription levels of RS28250, RS09985 and RS24685 were 16.8-, 2.6-, and 1.6-times higher than that of hspB2, encoding 6-hydroxy-3-succinylpyridine hydroxylase involved in nicotine degradation. Thus, two strong endogenous promoters PRS28250 and PRS09985 were selected to replace the original promoters of Nic2 gene clusters. The effect of endogenous ectopic promoter was also related to the replaced position of target gene clusters. When the promoter PRS28250 replaced the promoter of hspB2, the resultant mutant, QΔABs-ΔPhspB2::PRS28250, exhibited 69% higher nicotine degrading efficiency than the JY-Q. This research suggests a feasible strategy to enhance strain ability by removal of repressing regulatory proteins and replacing target promoter with strong endogenous ectopic promoters. IMPORTANCE This study evaluated the differential effects of homologous NicR2A/NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. JY-Q. Based on our differential analysis, a feasible strategy is presented to modify wild type strain JY-Q by removing repressing regulatory proteins, NicR2A/NicR2Bs, and replacing the target promoter with strong endogenous ectopic promoters. The resulting mutants exhibited high tolerance and degradation of nicotine. These findings should be beneficial for improving pollutant-degrading capacity of naturally strains through genomic modification.

Horizontally acquired regulatory gene activates ancestral regulatory system to promote Salmonella virulence

Horizontally acquired genes are typically regulated by ancestral regulators. This regulation enables expression of horizontally acquired genes to be coordinated with that of preexisting genes. Here, we report a singular example of the opposite regulation: a horizontally acquired gene that controls an ancestral regulator, thereby promoting bacterial virulence. We establish that the horizontally acquired regulatory gene ssrB is necessary to activate the ancestral regulatory system PhoP/PhoQ of Salmonella enterica serovar Typhimurium (S. Typhimurium) in mildly acidic pH, which S. Typhimurium experiences inside macrophages. SsrB promotes phoP transcription by binding upstream of the phoP promoter. SsrB also increases ugtL transcription by binding to the ugtL promoter region, where it overcomes gene silencing by the heat-stable nucleoid structuring protein H-NS, enhancing virulence. The largely non-pathogenic species S. bongori failed to activate PhoP/PhoQ in mildly acidic pH because it lacks both the ssrB gene and the SsrB binding site in the target promoter. Low Mg2+ activated PhoP/PhoQ in both S. bongori and ssrB-lacking S. Typhimurium, indicating that the SsrB requirement for PhoP/PhoQ activation is signal-dependent. By controlling the ancestral genome, horizontally acquired genes are responsible for more crucial abilities, including virulence, than currently thought.

Structural basis of Rgg protein binding to their regulatory pheromones and target DNA promoters

Rgg family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in Streptococcus species, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg-SHP and Rgg-target DNA promoter specificity was unknown. To close this gap, we determined the cryo-EM structure of Streptococcus thermophilus Rgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures to that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of both S. dysgalactiae Rgg2 and S. thermophilus Rgg3 in complex with their target DNA promoters. The physiological importance of the observed Rgg-DNA interactions was dissected using in vivo genetic experiments and in vitro biochemical assays. Based on these structure-function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of the SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci.Significance StatementSecreted peptide pheromones regulate critical biological processes in Gram-positive bacteria. In streptococci such as the human pathogen S. pyogenes, oligopeptide pheromones, like the short hydrophobic peptides (SHPs), regulate virulence, antimicrobial resistance, and biofilm formation. SHPs directly regulate the activity of transcription factors called Rgg2 and Rgg3. We present the cryo-EM structure of Rgg3 in complex with SHP3, as well as X-ray crystal structures of Rgg2 bound to target promoter DNA, Rgg3 bound to target promoter DNA, and Rgg3 alone. Based on the cryo-EM, X-ray crystallographic, biochemical, and genetic studies presented here, we provide not only detailed mechanistic insight into the molecular basis of Rgg3-SHP3, Rgg2-DNA, and Rgg3-DNA binding specificity, but also a new model of transcription factor regulatory interplay.

LuxR Solos in the Plant Endophyte Kosakonia sp. Strain KO348

ABSTRACT Endophytes are microorganisms that live inside plants and are often beneficial for the host. Kosakonia is a novel bacterial genus that includes several species that are diazotrophic and plant associated. This study revealed two quorum sensing-related LuxR solos, designated LoxR and PsrR, in the plant endophyte Kosakonia sp. strain KO348. LoxR modeling and biochemical studies demonstrated that LoxR binds N-acyl homoserine lactones (AHLs) in a promiscuous way. PsrR, on the other hand, belongs to the subfamily of plant-associated-bacterium (PAB) LuxR solos that respond to plant compounds. Target promoter studies as well as modeling and phylogenetic comparisons suggest that PAB LuxR solos are likely to respond to different plant compounds. Finally, LoxR is involved in the regulation of T6SS and PsrR plays a role in root endosphere colonization. IMPORTANCE Cell-cell signaling in bacteria allows a synchronized and coordinated behavior of a microbial community. LuxR solos represent a subfamily of proteins in proteobacteria which most commonly detect and respond to signals produced exogenously by other microbes or eukaryotic hosts. Here, we report that a plant-beneficial bacterial endophyte belonging to the novel genus of Kosakonia possesses two LuxR solos; one is involved in the detection of exogenous N-acyl homoserine lactone quorum sensing signals and the other in detecting a compound(s) produced by the host plant. These two Kosakonia LuxR solos are therefore most likely involved in interspecies and interkingdom signaling.

Modulating FOXO3 transcriptional activity by small, DBD-binding molecules

FOXO transcription factors are critical regulators of cell homeostasis and steer cell death, differentiation and longevity in mammalian cells. By combined pharmacophore-modeling-based in silico and fluorescence polarization-based screening we identified small molecules that physically interact with the DNA-binding domain (DBD) of FOXO3 and modulate the FOXO3 transcriptional program in human cells. The mode of interaction between compounds and the FOXO3-DBD was assessed via NMR spectroscopy and docking studies. We demonstrate that compounds S9 and its oxalate salt S9OX interfere with FOXO3 target promoter binding, gene transcription and modulate the physiologic program activated by FOXO3 in cancer cells. These small molecules prove the druggability of the FOXO-DBD and provide a structural basis for modulating these important homeostasis regulators in normal and malignant cells.

The first enhancer in an enhancer chain safeguards subsequent enhancer-promoter contacts from a distance

Background Robustness and evolutionary stability of gene expression in the human genome are established by an array of redundant enhancers. Results Using Hi-C data in multiple cell lines, we report a comprehensive map of promoters and active enhancers connected by chromatin contacts, spanning 9000 enhancer chains in 4 human cell lines associated with 2600 human genes. We find that the first enhancer in a chain that directly contacts the target promoter is commonly located at a greater genomic distance from the promoter than the second enhancer in a chain, 96 kb vs. 45 kb, respectively. The first enhancer also features higher similarity to the promoter in terms of tissue specificity and higher enrichment of loop factors, suggestive of a stable primary contact with the promoter. In contrast, a chain of enhancers which connects to the target promoter through a neutral DNA segment instead of an enhancer is associated with a significant decrease in target gene expression, suggesting an important role of the first enhancer in initiating transcription using the target promoter and bridging the promoter with other regulatory elements in the locus. Conclusions The widespread chained structure of gene enhancers in humans reveals that the primary, critical enhancer is distal, commonly located further away than other enhancers. This first, distal enhancer establishes contacts with multiple regulatory elements and safeguards a complex regulatory program of its target gene.

Chromatin conformation remains stable upon extensive transcriptional changes driven by heat shock

Heat shock (HS) initiates rapid, extensive, and evolutionarily conserved changes in transcription that are accompanied by chromatin decondensation and nucleosome loss at HS loci. Here we have employed in situ Hi-C to determine how heat stress affects long-range chromatin conformation in human and Drosophila cells. We found that compartments and topologically associating domains (TADs) remain unchanged by an acute HS. Knockdown of Heat Shock Factor 1 (HSF1), the master transcriptional regulator of the HS response, identified HSF1-dependent genes and revealed that up-regulation is often mediated by distal HSF1 bound enhancers. HSF1-dependent genes were usually found in the same TAD as the nearest HSF1 binding site. Although most interactions between HSF1 binding sites and target promoters were established in the nonheat shock (NHS) condition, a subset increased contact frequency following HS. Integrating information about HSF1 binding strength, RNA polymerase abundance at the HSF1 bound sites (putative enhancers), and contact frequency with a target promoter accurately predicted which up-regulated genes were direct targets of HSF1 during HS. Our results suggest that the chromatin conformation necessary for a robust HS response is preestablished in NHS cells of diverse metazoan species.