Unraveling a Bacterial Starvation Response Through the Direct Targets of a Starvation-Induced Transcriptional Activator

Organisms frequently encounter environments with nutrient shortages and their survival depends on changes in physiology and the ability to conserve resources. In bacteria, many physiological changes associated with starvation have been identified, but the underlying genetic components and regulatory networks that direct these physiological changes are often poorly defined. Here, we aimed to better define the gene regulatory networks that mediate the starvation response in Myxococcus xanthus, a bacterium that copes with starvation by producing fruiting bodies filled with dormant and stress-resistant spores. We focused on the direct promoter/gene targets of Nla28, a transcriptional activator/enhancer binding protein (EBP) that is important for early rounds of gene expression following starvation. Using expression profiling to identify genes that are downregulated in nla28 mutant cells and bioinformatics to identify the putative promoters of these genes, 12 potential promoter targets (37 genes) of Nla28 were identified. The results of in vitro promoter binding assays, coupled with in vitro and in vivo mutational analyses, suggested that the 12 promoters are in vivo targets of Nla28 and that Nla28 dimers use tandem, imperfect repeats of an 8-bp sequence for binding. Interestingly, nine of the Nla28 target promoters are intragenic, located in the protein coding sequence of an upstream gene or in the protein coding sequence of one gene within an operon (internal promoters). Based on mutational analyses, we concluded that the 12 Nla28 target loci contain at least one gene important for production of stress-resistant spores following starvation. Most of these loci contain genes predicted to be involved in regulatory or defense-related functions. Using the consensus Nla28 binding sequence, followed by bioinformatics and expression profiling, 58 additional promoters and 102 genes were tagged as potential Nla28 targets. Among these putative Nla28 targets, functions such as regulatory, metabolic and cell envelope biogenesis were commonly assigned to genes.

Effects of P-Coumarate 3-Hydroxylase Downregulation on the Compositional and Structural Characteristics of Lignin and Hemicelluloses in Poplar Wood (Populus alba × Populus glandulosa)

Elucidating the chemical and structural characteristics of hemicelluloses and lignin in the p-coumarate 3-hydroxylase (C3H) down-regulated poplar wood will be beneficial to the upstream gene validation and downstream biomass conversion of this kind of transgenic poplar. Herein, the representative hemicelluloses and lignin with unaltered structures were prepared from control (CK) and C3H down-regulated 84K poplars. Modern analytical techniques, such as 13C NMR, 2D-HSQC NMR, and gel chromatography (GPC), were performed to better delineate the structural changes of hemicelluloses and lignin caused by transgenesis. Results showed that both the hemicelluloses (H-CK and H-C3H) extracted from control and C3H down-regulated poplar wood have a chain backbone of (1→4)-β-D-Xylan with 4-O-Me-α-D-GlcpA as side chain, and the branch degree of the H-C3H is higher than that of H-CK. With regarding to the lignin macromolecules, NMR results demonstrated that the syringyl/guaiacyl (S/G) ratio and dominant substructure β-O-4 linkages in C3H down-regulated poplar were lower than those of control poplar wood. By contrast, native lignin from C3H down-regulated poplar wood exhibited higher contents of p-hydroxybenzoate (PB) and p-hydroxyphenyl (H) units. In short, C3H down-regulation resulted in the chemical and structural changes of the hemicelluloses and lignin in these poplar wood. The identified structures will facilitate the downstream utilization and applications of lignocellulosic materials in the biorefinery strategy. Furthermore, this study could provide some illuminating results for genetic breeding on the improvement of wood properties and efficient utilization of poplar wood.

Dynamic spreading of chromatin-mediated gene silencing and reactivation between neighboring genes in single cells

In mammalian cells genes that are in close proximity are coupled transcriptionally: silencing or activating one gene can affect its neighbors. Understanding these dynamics is important for natural processes, such as heterochromatin spreading during development and aging, and when designing synthetic gene regulation. Here, we systematically dissect this process in single cells by recruiting and releasing repressive chromatin regulators at dual-gene synthetic reporters, and measuring how fast gene silencing and reactivation spread as a function of intergenic distance and configuration of insulator elements. We find that silencing by KRAB, associated with histone methylation, spreads between two genes within hours, with a time delay that increases with distance. This fast KRAB-mediated spreading is not blocked by the classical cHS4 insulators. Silencing by histone deacetylase HDAC4 of the upstream gene can also lead to downstream gene silencing, but with a days-long delay that does not change with distance. This slower silencing can sometimes be stopped by insulators. Gene reactivation of neighboring genes is also coupled, with strong promoters and insulators determining the order of reactivation. We propose a new model of multi-gene regulation, where both gene silencing and gene reactivation can act at a distance, allowing for coordinated dynamics via chromatin regulator recruitment.

Topological Constraints on Noise Propagation in Gene Regulatory Networks

Gene expression, the production of protein from DNA and mRNA in the biological cell, is inherently stochastic. Cells with identical DNA exhibit fluctuations or 'noise' in gene expression. This noise propagates over gene regulatory networks (GRNs), which encode gene-gene interactions. The propagated 'extrinsic' noise interacts and combines with 'intrinsic' noise to affect biological decisions. Consequently, it is essential to understand how GRN topology affects total noise. Recently, uncertainty principles were established for noise propagation over GRN. In particular, in ring GRNs, exactly one node can have noise reduction below the intrinsic limit. We establish necessary and sufficient conditions for noise reduction in ring GRN. Specifically, for two- and three-node rings, an odd number of negative regulations is necessary for noise reduction. Further, sufficiency is ensured if sensitivities to input for feedforward and feedback regulations are bounded from below and above, respectively. These constraints are valid even if the ring GRN are regulated by an upstream gene. Finally, we use graph theory to decompose noise propagation in a general directed network over its strongly connected components. The combination of graph theory and stochastic processes may be a general framework for studying noise propagation.

Processive translocation of cohesive and non-cohesive cohesin in vivo

Cohesin is a central architectural element of chromosome structure that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops. In vitro, cohesin translocates along DNA and extrudes loops in an ATP-dependent fashion. In vivo, cohesin redistributes in response to transcription as if pushed by RNA polymerase. Direct evidence of processive genomic translocation by the complex, however, is lacking. Here, obstacles of increasing size were tethered to DNA in yeast to detect translocation. The obstacles were built from a GFP-lacI core fused to one or more mCherries. Cohesin translocation was initiated from an upstream gene. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier to passage depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by only three mCherries. That synthetic barriers alter cohesin redistribution demonstrates that the complex translocates processively on chromatin in vivo. The approach provides a relative measure of the maximum size of the protein chamber(s) that embraces DNA during cohesin translocation. The data indicate that the cohesive embrace is more restrictive than the embrace of non-cohesive complexes.

A Unique Gene Module in Thermococcales Archaea Centered on a Hypervariable Protein Containing Immunoglobulin Domains

Molecular mechanisms involved in biological conflicts and self vs nonself recognition in archaea remain poorly characterized. We apply phylogenomic analysis to identify a hypervariable gene module that is widespread among Thermococcales. These loci consist of an upstream gene coding for a large protein containing several immunoglobulin (Ig) domains and unique combinations of downstream genes, some of which also contain Ig domains. In the large Ig domain containing protein, the C-terminal Ig domain sequence is hypervariable, apparently, as a result of recombination between genes from different Thermococcales. To reflect the hypervariability, we denote this gene module VARTIG (VARiable Thermococcales IG). The overall organization of the VARTIG modules is similar to the organization of Polymorphic Toxin Systems (PTS). Archaeal genomes outside Thermococcales encode a variety of Ig domain proteins, but no counterparts to VARTIG and no Ig domains with comparable levels of variability. The specific functions of VARTIG remain unknown but the identified features of this system imply three testable hypotheses: (i) involvement in inter-microbial conflicts analogous to PTS, (ii) role in innate immunity analogous to the vertebrate complement system, and (iii) function in self vs nonself discrimination analogous to the vertebrate Major Histocompatibility Complex. The latter two hypotheses seem to be of particular interest given the apparent analogy to the vertebrate immunity.

Characterizing chloroplast genomes and inferring maternal divergence of the Triticum–Aegilops complex

The Triticum (wheat)–Aegilops (goatgrass) complex has been extensively studied, but the evolutionary history of polyploid wheats has not been fully elucidated. The chloroplast (cp) with maternal inheritance and homoplasy can simplify the sequence-based evolutionary inferences, but informative inferences would require a complete and accurate cp genome sequence. In this study, 16 cp genomes representing five Aegilops and 11 Triticum species and subspecies were sequenced, assembled and annotated, yielding five novel circular cp genome sequences. Analyzing the assembled cp genomes revealed no marked differences in genome structure and gene arrangement across the assayed species. A polymorphism analysis of 72 published cp genome sequences representing 10 Aegilops and 15 Triticum species and subspecies detected 1183 SNPs and 1881 SSRs. More than 80% SNPs detected resided on the downstream and upstream gene regions and only 2.78% or less SNPs were predicted to be deleterious. The largest nucleotide diversity was observed in the short single-copy genomic region. Relatively weak selection pressure on cp coding genes was detected. Different phylogenetic analyses confirmed that the maternal divergence of the Triticum–Aegilops complex had three deep lineages each representing a diploid species with nuclear A, B, or D genome. Dating the maternal divergence yielded age estimates of divergence that matched well with those reported previously. The divergence between emmer and bread wheats occurred at 8200–11,200 years ago. These findings are useful for further genomic studies, provide insight into cp genome evolvability and allow for better understanding of the maternal divergence of the Triticum–Aegilops complex.

LDHA-Mediated Glycolytic Metabolism in Nucleus Pulposus Cells Is a Potential Therapeutic Target for Intervertebral Disc Degeneration

The intervertebral disc degeneration (IDD) is considered to be an initiator of a series of spinal diseases, among which changes in the nucleus pulposus (NP) are the most significant. NP cells reside in a microenvironment with a lack of blood vessels, hypoxia, and low glucose within the intervertebral disc. Due to the strong activity of HIF-1α, glycolysis is the main pathway for energy metabolism in NP cells. Our previous study found that higher SIRT1 expression is beneficial to delay the degeneration of NP cells. In order to find the downstream genes by which SIRT1 acts on NP cells, we used iTRAQ sequencing to detect the differences between degenerated NP cells overexpressing SIRT1 and a control group (human NP cells were derived from surgery) and found that the expression of LDHA changed in the same direction with SIRT1. This suggests that SIRT1 may delay the degeneration of NP cells by regulating glycolysis. We then used a Seahorse XFe24 analyzer to measure the bioenergetic parameters of NP cells and obtained three findings: (a) glycolysis is the main energy metabolism pathway in NP cells, (b) there is a large difference in ATP production between senescent cells and young cells, and (c) SIRT1 can regulate the production of ATP from glycolysis by regulating LDHA. We also found that SIRT1 in NP cells has a positive regulatory effect on c-Myc which is an upstream gene of LDHA. Through observing IDD-related indicators such as apoptosis, proliferation, senescence, and extracellular matrix, we found that SIRT1 can delay degeneration, and interference with c-Myc and LDHA, respectively, weakens the protective effect of SIRT1. Interfering with LDHA alone can also inhibit glycolysis and accelerate degeneration. Overall, we found that the inhibition of glycolysis in Np cells significantly affects their normal physiological functions and determined that LDHA is a potential therapeutic target for the treatment of IDD.

Ropivacaine induces cell cycle arrest in the G0/G1 phase and apoptosis of PC12 cells via inhibiting mitochondrial STAT3 translocation

STAT3 has neuroprotective effect via non-canonical activation and mitochondrial translocation, but its effects on ropivacaine-induced neurotoxicity remain unclear. Our previous study revealed that apoptosis was an important mechanism of ropivacaine-induced neurotoxicity, this study is to illustrate the relationship between STAT3 with ropivacaine-induced apoptosis. Those results showed that ropivacaine treatment decreased cell viability, induced cell cycle arrest in the G0/G1 phase, apoptosis, oxidative stress, and mitochondrial dysfunction in PC12 cells. Besides, ropivacaine decreased the phosphorylated levels of STAT3 at Ser727 and downregulated the expression of STAT3 upstream gene IL-6. The mitochondrial translocation of STAT3 was also hindered by ropivacaine. To further illustrate the connection of STAT3 protein structure with ropivacaine, the autodock-vina was used to examine the interaction between STAT3 and ropivacaine, and the results showed that ropivacaine could bind to STAT3’s proline site and other sites. In addition, the activator and inhibitor of mitoSTAT3 translocation were used to demonstrate it was involved in ropivacaine-induced apoptosis, the results showed that enhancing the mitochondrial STAT3 translocation could prevent ropivacaine-induced apoptosis. Finally, the expression of p-STAT3 and the levels of apoptosis in the spinal cord were also detected, the results were consistent with the cell experiment, ropivacaine decreased the expression of p-STAT3 protein and increased the levels of apoptosis in the spinal cord. We demonstrated that ropivacaine induced apoptosis by inhibiting the phosphorylation of STAT3 at Ser727 and the mitochondrial STAT3 translocation. This effect was reversed by the activation of the mitochondrial STAT3 translocation.