Global Transcriptional Regulator Research Articles

Systematic stress adaptation of Bacillus subtilis to tetracycline exposure.

To alleviate the harmful effects of antibiotics on the environment and human health, the stress response and molecular network of Bacillus under tetracycline stress were investigated using a proteomics approach. During the exposure process, Bacillus subtilis exhibited a strong adaptation mechanism. Cell membrane and intracellular reactive oxygen species (ROS) level returned to normal after 5 h. A total of 312 upregulated and 65 downregulated proteins were identified, mainly involved in metabolism and the synthesis of ribosomes, DNA, and RNA. After tetracycline exposure, the core metabolism network was accelerated to supply precursors for the synthesis of DNA, RNA, proteins, peptidoglycans, and saturated fatty acids that were involved in ribosome protection, and strengthened the cell wall and cell membrane. The signal transduction pathways involved were analyzed in association with the stress response of B. subtilis at 15 min of exposure to tetracycline. The primary damage to the ribosome by tetracycline activated a series of response proteins. Antitoxin and heat-shock proteins were activated for the global regulation of transcription and metabolism. Trigger factor Tig was upregulated to ensure proper initiation of transcription and aerobic respiration. Temperature-sensor protein VicR from the two-component system was used by the cell to regulate the composition of the cell wall and cell membrane. The over-consumption of metabolites, such as phosphoribosyl diphosphate (PRPP), purine nucleoside triphosphate (GTP), and acetyl-CoA forced the cells to assimilate more sugar for glycolysis. To this end, methyl-accepting chemotaxis proteins (MCPs) and sugar transportation protein PtsG were upregulated, simultaneously. Ultimately, peroxidase was activated to eliminate the redundant ROS, to minimize cell damage. These findings presented a system-level understanding of adaption processes of bacteria to antibiotic stress.

CDK6 Antagonizes TP53-Induced Responses during Tumorigenesis

Inhibitors directed against cyclin dependent kinases (CDKs) have raised much interest as anti-cancer therapeutics over the last years. In particular, inhibitors directed against CDK4/6 have been declared as a major breakthrough in cancer therapy by the FDA. CDK4 and CDK6 bind to D-type cyclins and subsequently phosphorylate the RB protein to allow cells to progress through the G1 phase of the cell cycle. The effectiveness of CDK4/6 inhibitors was primarily assigned to their ability to block cell cycle progression. In hematopoietic malignancies high levels of CDK6, but not CDK4, are frequently found. Over the last years we have assigned a novel and unexpected role for CDK6 as global transcriptional regulator. ChIP-Seq experiments identified more than 20.000 specific CDK6 binding sites in leukemic cells with the majority located in the promoter regions. CDK6 binding to chromatin does not require kinase activity whereas transcriptional control is regulated in a kinase- dependent as well as kinase-independent manner. Overlaying ChIP-Seq and RNA-Seq experiments showed that CDK6 contributes to the induction or repression of genes. Target genes of CDK6 which are important for leukemia progression include PIM1, c-MYC, AURKA, AURKB, AKT and VEGF-A. Murine leukemia models verified the importance of CDK6 for myeloid and lymphoid tumor formation downstream of a variety of oncogenes including FLT3-ITD, NPM/ALK, MLL/AF9, BCR/ABL or JAK2V617F. CDK6 contributes to disease development by regulating proliferation, cell survival, angiogenesis and cytokine production. In hematopoietic stem cells and leukemic stem cells kinase-independent functions dominate and CDK6 controls a network of transcription factors regulating stem cell quiescence and activation. The importance of kinase-dependent transcriptional effects is pronounced under conditions of stress and transformation. Upon oncogenic stress, CDK6 induces a set of genes that counteract pro-apoptotic TP53 responses including MDM4, PRMT5, PPM1D and BCL2. This response is induced by a CDK6 - dependent phosphorylation of the transcription factors SP1 and NFYA as verified by phospho-chromatome analysis. Murine Cdk6-deficient cells only survive oncogenic stress by mutating Tp53. The link between CDK6 and TP53 is conserved in human hematopoietic malignancies. Kollmann K, Heller G, Schneckenleithner C, et al. A kinase-independent function of CDK6 links the cell cycle to tumor angiogenesis. Cancer Cell. 2013;24(2):167-181.Scheicher R, Hoelbl-Kovacic A, Bellutti F, et al. CDK6 as a key regulator of hematopoietic and leukemic stem cell activation. Blood. 2015;125(1):90-101.Uras IZ, Walter GJ, Scheicher R, et al. Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6. Blood. 2016;127(23):2890-2902.Bellutti F, Tigan AS, Nebenfuehr S, et al. CDK6 antagonizes P53-induced responses during tumorigenesis. Cancer Discov. 2018;8(7):884-897.Uras IZ, Maurer B, Nivarthi H, et al. CDK6 coordinates JAK2V617F mutant MPN via NF-kB and apoptotic networks. Blood. 2019;133(15):1677-1690. Disclosures No relevant conflicts of interest to declare.

Growth Phase-Dependent Chromosome Condensation and Heat-Stable Nucleoid-Structuring Protein Redistribution in Escherichia coli under Osmotic Stress.

The heat-stable nucleoid-structuring (H-NS) protein is a global transcriptional regulator implicated in coordinating the expression of over 200 genes in Escherichia coli, including many involved in adaptation to osmotic stress. We have applied superresolved microscopy to quantify the intracellular and spatial reorganization of H-NS in response to a rapid osmotic shift. We found that H-NS showed growth phase-dependent relocalization in response to hyperosmotic shock. In stationary phase, H-NS detached from a tightly compacted bacterial chromosome and was excluded from the nucleoid volume over an extended period of time. This behavior was absent during rapid growth but was induced by exposing the osmotically stressed culture to a DNA gyrase inhibitor, coumermycin. This chromosomal compaction/H-NS exclusion phenomenon occurred in the presence of either potassium or sodium ions and was independent of the presence of stress-responsive sigma factor σS and of the H-NS paralog StpA.IMPORTANCE The heat-stable nucleoid-structuring (H-NS) protein coordinates the expression of over 200 genes in E. coli, with a large number involved in both bacterial virulence and drug resistance. We report on the novel observation of a dynamic compaction of the bacterial chromosome in response to exposure to high levels of salt. This stress response results in the detachment of H-NS proteins and their subsequent expulsion to the periphery of the cells. We found that this behavior is related to mechanical properties of the bacterial chromosome, in particular, to how tightly twisted and coiled is the chromosomal DNA. This behavior might act as a biomechanical response to stress that coordinates the expression of genes involved in adapting bacteria to a salty environment.

Benchmarking two Saccharomyces cerevisiae laboratory strains for growth and transcriptional response to methanol

One-carbon compounds, such as methanol, are becoming potential alternatives to sugars as feedstocks for the biological production of chemicals, fuels, foods, and pharmaceuticals. Efficient biological production often requires extensive genetic manipulation of a microbial host strain, making well-characterised and genetically-tractable model organisms like the yeast Saccharomyces cerevisiae attractive targets for the engineering of methylotrophic metabolism. S. cerevisiae strains S288C and CEN.PK are the two best-characterised and most widely used hosts for yeast synthetic biology and metabolic engineering, yet they have unpredictable metabolic phenotypes related to their many genomic differences. We therefore sought to benchmark these two strains as potential hosts for engineered methylotrophic metabolism by comparing their growth and transcriptomic responses to methanol. CEN.PK had improved growth in the presence of methanol relative to the S288C derivative BY4741. The CEN.PK transcriptome also had a specific and relevant response to methanol that was either absent or less pronounced in the BY4741 strain. This response included up-regulation of genes associated with mitochondrial and peroxisomal metabolism, alcohol and formate dehydrogenation, glutathione metabolism, and the global transcriptional regulator of metabolism MIG3. Over-expression of MIG3 enabled improved growth in the presence of methanol, suggesting that MIG3 is a mediator of the superior CEN.PK strain growth. CEN.PK was therefore identified as a superior strain for the future development of synthetic methylotrophy in S. cerevisiae.

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the transcriptional regulator Lsr2 at threonine 112.

Summary Mycobacterium tuberculosis (Mtb) is able to persist in the body through months of multi‐drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the protein kinase B (PknB) which controls peptidoglycan biosynthesis during growth. Here, we observed that depletion of PknB resulted in specific transcriptional changes that are likely caused by reduced phosphorylation of the H‐NS‐like regulator Lsr2 at threonine 112. The activity of PknB towards this phosphosite was confirmed with purified proteins, and this site was required for adaptation of Mtb to hypoxic conditions, and growth on solid media. Like H‐NS, Lsr2 binds DNA in sequence‐dependent and non‐specific modes. PknB phosphorylation of Lsr2 reduced DNA binding, measured by fluorescence anisotropy and electrophoretic mobility shift assays, and our NMR structure of phosphomimetic T112D Lsr2 suggests that this may be due to increased dynamics of the DNA‐binding domain. Conversely, the phosphoablative T112A Lsr2 had increased binding to certain DNA sites in ChIP‐sequencing, and Mtb containing this variant showed transcriptional changes that correspond with the change in DNA binding. In summary, PknB controls Mtb growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.

Epigenetic Mechanisms of Ischemic Stroke

Stroke is one of the main causes of human death and disability. Occlusion of the cerebral vessels in minutes leads to oxygen and glucose depletion and the infarction of nervous tissue. However, neuroprotectors that can rescue neurons have not been found yet. This review describes the biochemical processes leading to cell death in the ischemic brain and the molecular factors that spread the damage to neighboring tissues and form the transition zone (penumbra). Even though ischemic damage generally suppresses biosynthetic processes, some proteins are expressed in the ischemic penumbra. Epigenetic processes are the mechanisms of the global regulation of transcription and protein synthesis as well as the functional state of cells. The review considers main epigenetic processes that regulate gene expression and protein synthesis: DNA methylation, methylation and acetylation of histones. The main attention is paid to epigenetic regulatory proteins: DNA methyltransferases, histone acetyltransferases, histone deacetylases and their isoforms. Their role in the brain responses to ischemic damage is discussed. The inhibition of some epigenetic proteins is one of the promising strategies for the treatment of ischemic stroke. The capabilities of some inhibitors of histone deacetylases and DNA methyltransferases as potential neuroprotectors in ischemic stroke are discussed.

The regulation of Moco biosynthesis and molybdoenzyme gene expression by molybdenum and iron in bacteria.

Bacterial molybdoenzymes are key enzymes involved in the global sulphur, nitrogen and carbon cycles. These enzymes require the insertion of the molybdenum cofactor (Moco) into their active sites and are able to catalyse a large range of redox-reactions. Escherichia coli harbours nineteen different molybdoenzymes that require a tight regulation of their synthesis according to substrate availability, oxygen availability and the cellular concentration of molybdenum and iron. The synthesis and assembly of active molybdoenzymes are regulated at the level of transcription of the structural genes and of translation in addition to the genes involved in Moco biosynthesis. The action of global transcriptional regulators like FNR, NarXL/QP, Fur and ArcA and their roles on the expression of these genes is described in detail. In this review we focus on what is known about the molybdenum- and iron-dependent regulation of molybdoenzyme and Moco biosynthesis genes in the model organism E. coli. The gene regulation in E. coli is compared to two other well studied model organisms Rhodobacter capsulatus and Shewanella oneidensis.

The Role of SATB1 in Tumour Progression and Metastasis.

Carcinogenesis is a long-drawn, multistep process, in which metastatic spread is an unequivocal hallmark of a poor prognosis. The progression and dissemination of epithelial cancers is commonly thought to rely on the epidermal-mesenchymal transition (EMT) process. During EMT, epithelial cells lose their junctions and apical-basal polarity, and they acquire a mesenchymal phenotype with its migratory and invasive capabilities. One of the proteins involved in cancer progression and EMT may be SATB1 (Special AT-Rich Binding Protein 1)—a chromatin organiser and a global transcriptional regulator. SATB1 organizes chromatin into spatial loops, providing a “docking site” necessary for the binding of further transcription factors and chromatin modifying enzymes. SATB1 has the ability to regulate whole sets of genes, even those located on distant chromosomes. SATB1 was found to be overexpressed in numerous malignancies, including lymphomas, breast, colorectal, prostate, liver, bladder and ovarian cancers. In the solid tumours, an elevated SATB1 level was observed to be associated with an aggressive phenotype, presence of lymph node, distant metastases, and a poor prognosis. In this review, we briefly describe the prognostic significance of SATB1 expression in most common human cancers, and analyse its impact on EMT and metastasis.

PTEN interacts with RNA polymerase II to dephosphorylate polymerase II C-terminal domain.

Gene transcription is a highly complex and strictly regulated process. RNA polymerase II (Pol II) C-terminal domain (CTD) undergoes massive cycles of phosphorylation and dephosphorylation during the process of gene transcription. These post-translational modifications of CTD provide an interactive platform for various factors required for transcription initiation, elongation, termination, and co-transcriptional RNA processing. Pol II CTD kinases and phosphatases are key regulators and any deviation may cause genome-wide transcriptional dysregulation leading to various pathological conditions including cancer. PTEN, a well known tumor suppressor, is one of the most commonly somatically altered in diverse malignancies. When mutated in the germline, PTEN causes cancer predisposition. Numerous studies have demonstrated that PTEN regulates the expression of hundreds of genes, however, no mechanism is known so far. PTEN is a dual specificity phosphatase, using both lipid and protein as substrates. In the present study, we demonstrate that PTEN interacts with the RNA Pol II and that PTEN expression is inversely correlated with global phosphorylation of Pol II CTD. Furthermore, PTEN dephosphorylates Pol II CTD in vitro with a significant specificity for Ser5p. Interestingly, ChIP-seq data analysis revealed that PTEN globally binds to promoter proximal regions, and PTEN loss increases genome-wide Pol II Ser5p occupancy, suggest that PTEN is a Pol II CTD phosphatase. Our observations demonstrate an unexplored function of PTEN with the potential of global transcriptional regulation, adding a new dimension to somatic carcinogenesis and germline cancer predisposition.

Enhancement of l-arginine production by increasing ammonium uptake in an AmtR-deficient Corynebacterium crenatum mutant

L-Arginine is an important amino acid with extensive application in the food and pharmaceutical industries. The efficiency of nitrogen uptake and assimilation by organisms is extremely important for L-arginine production. In this study, a strain engineering strategy focusing on upregulate intracellular nitrogen metabolism in Corynebacterium crenatum for L-arginine production was conducted. Firstly, the nitrogen metabolism global transcriptional regulator AmtR was deleted, which has demonstrated the beneficial effect on L-arginine production. Subsequently, this strain was engineered by overexpressing the ammonium transporter AmtB to increase the uptake of NH4+ and L-arginine production. To overcome the drawbacks of using a plasmid to express amtB, Ptac, a strong promoter with amtB gene fragment, was integrated into the amtR region on the chromosome in the Corynebacterium crenatum/ΔamtR. The final strain results in L-arginine production at a titer of 60.9g/L, which was 35.14% higher than that produced by C. crenatum SYPA5-5.

A MYST family histone acetyltransferase, MoSAS3, is required for development and pathogenicity in the rice blast fungus.

SummaryHistone acetylation has been established as a principal epigenetic regulatory mechanism in eukaryotes. Sas3, a histone acetyltransferase belonging to the largest family of acetyltransferase, MYST, is the catalytic subunit of a conserved histone acetyltransferase complex. To date, the functions of Sas3 and its orthologues have been extensively studied in yeast, humans and flies in relation to global acetylation and transcriptional regulation. However, its precise impact on development and pathogenicity in fungal plant pathogens has yet to be elucidated. Considering the importance of Sas3 in H3K14 acetylation, here we investigate the roles of its orthologue in the rice blast fungus, Magnaporthe oryzae (Pyricularia oryzae). Unlike a previously reported Sas3 deletion in yeast, which led to no remarkable phenotypic changes, we found that MoSAS3 deletion alone had a profound effect on fungal growth and development, including asexual reproduction, germination and appressorium formation in M. oryzae. Such defects in pre‐penetration development resulted in complete loss of pathogenicity in the deletion mutant. Furthermore, genetic analysis of MoSAS3 and MoGCN5 encoding a Gcn5‐related N‐acetyltransferase family histone acetyltransferase suggested that two conserved components of histone acetylation are integrated differently into epigenetic regulatory mechanisms in the yeast and a filamentous fungus. RNA‐seq analysis of ΔMosas3 showed two general trends: many DNA repair and DNA damage response genes are up‐regulated, while carbon and nitrogen metabolism genes are down‐regulated in ΔMosas3. Our work demonstrates the importance of MYST family histone acetyltransferase as a developmental regulator and illuminates a degree of functional variation in conserved catalytic subunits among different fungal species.

Phz1 contributes much more to phenazine-1-carboxylic acid biosynthesis than phz2 in Pseudomonas aeruginosa rpoS mutant.

Pseudomonas aeruginosa PAO1, a common opportunistic bacterial pathogen, contains two phenazine-biosynthetic operons, phz1 (phzA1 B1 C1 D1 E1 F1 G1 ) and phz2 (phzA2 B2 C2 D2 E2 F2 G2 ). Each of two operons can independently encode a set of enzymes involving in the biosynthesis of phenazine-1-carboxylic acid. As a global transcriptional regulator, RpoS mediates a lot of genes involving secondary metabolites biosynthesis in many bacteria. In an other previous study, it was reported that RpoS deficiency caused overproduction of pyocyanin, a derivative of phenazine-1-carboxylic acid in P. aeruginosa PAO1. But it is not known how RpoS mediates the expression of each of two phz operons and modulates phenazine-1-carboxylic acid biosynthesis in detail. In this study, by deleting the rpoS gene in the mutant PNΔphz1 and the mutant PNΔphz2, we found that the phz1 operon contributes much more to phenazine-1-carboxylic acid biosynthesis than the phz2 operon in the absence of RpoS. With the construction of the translational and transcriptional fusion vectors with the truncated lacZ reporter gene, we demonstrated that RpoS negatively regulates the expression of phz1 and positively controls the expression of phz2, and the regulation of phenazine-1-carboxylic acid biosynthesis mediated by RopS occurs at the posttranscriptional level, not at the transcriptional level. Obviously, two copies of phz operons and their differential expression mediated by RpoS might help P. aeruginosa adapt to its diverse environments and establish infection in its hosts.

Quantitative modelling predicts the impact of DNA methylation on RNA polymerase II traffic

Patterns of gene expression are primarily determined by proteins that locally enhance or repress transcription. While many transcription factors target a restricted number of genes, others appear to modulate transcription levels globally. An example is MeCP2, an abundant methylated-DNA binding protein that is mutated in the neurological disorder Rett syndrome. Despite much research, the molecular mechanism by which MeCP2 regulates gene expression is not fully resolved. Here, we integrate quantitative, multidimensional experimental analysis and mathematical modeling to indicate that MeCP2 is a global transcriptional regulator whose binding to DNA creates "slow sites" in gene bodies. We hypothesize that waves of slowed-down RNA polymerase II formed behind these sites travel backward and indirectly affect initiation, reminiscent of defect-induced shockwaves in nonequilibrium physics transport models. This mechanism differs from conventional gene-regulation mechanisms, which often involve direct modulation of transcription initiation. Our findings point to a genome-wide function of DNA methylation that may account for the reversibility of Rett syndrome in mice. Moreover, our combined theoretical and experimental approach provides a general method for understanding how global gene-expression patterns are choreographed.

Uncariitannin, a polyphenolic polymer from Uncaria gambier, attenuates Staphylococcus aureus virulence through an MgrA-mediated regulation of α-hemolysin

A global transcriptional regulator, MgrA, was previously identified as a key determinant of virulence in Staphylococcus aureus. An 80% EtOH extract of Uncaria gambier was found to attenuate the virulence of S. aureus via its effects on MgrA. Using bioassay-guided fractionation, a polyphenolic polymer, uncariitannin, was found to be the main bioactive constituent of the extract, and its structure was characterized using spectral and chemical analysis. The molecular weight and polydispersity of uncariitannin were determined by gel permeation chromatography-refractive index-light scattering analysis. An electrophoretic mobility shift assay showed that uncariitannin could effectively inhibit the interaction of MgrA with DNA in a dose-dependent manner. Treatment with uncariitannin could decrease the mRNA and protein levels of Hla in both the S. aureus Newman and USA300 LAC strains. Further analysis of Hla expression levels in the Newman ΔmgrA and Newman ΔmgrA/pYJ335-mgrA strains indicated that uncariitannin altered Hla expression primarily in an MgrA-dependent manner. A mouse model of infection indicated that uncariitannin could attenuate MRSA virulence. In conclusion, uncariitannin may be a potential candidate for further development as an antivirulence agent for the treatment of S. aureus infection.

Transcriptional regulation of the Pseudomonas aeruginosa iron-sulfur cluster assembly pathway by binding of IscR to multiple sites.

Iron-sulfur ([Fe-S]) cluster proteins have essential functions in many biological processes. [Fe-S] homeostasis is crucial for bacterial survival under a wide range of environmental conditions. IscR is a global transcriptional regulator in Pseudomonas aeruginosa; it has been shown to regulate genes involved in [Fe-S] cluster biosynthesis, iron homeostasis, resistance to oxidants, and pathogenicity. Many aspects of the IscR transcriptional regulatory mechanism differ from those of other well-studied systems. This study demonstrates the mechanisms of IscR Type-1 binding to its target sites that mediate the repression of gene expression at the isc operon, nfuA, and tpx. The analysis of IscR binding to multiple binding sites in the promoter region of the isc operon reveals that IscR first binds to the high-affinity site B followed by binding to the low-affinity site A. The results of in vitro IscR binding assays and in vivo analysis of IscR-mediated repression of gene expression support the role of site B as the primary site, while site A has only a minor role in the efficiency of IscR repression of gene expression. Ligation of an [Fe-S] cluster to IscR is required for the binding of IscR to target sites and in vivo repression and stress-induced gene expression. Analysis of Type-1 sites in many bacteria, including P. aeruginosa, indicates that the first and the last three AT-rich bases were among the most highly conserved bases within all analyzed Type-1 sites. Herein, we first propose the putative sequence of P. aeruginosa IscR Type-1 binding motif as 5’AWWSSYRMNNWWWTNNNWSGGNYWW3’. This can benefit further studies in the identification of novel genes under the IscR regulon and the regulatory mechanism model of P. aeruginosa IscR as it contributes to the roles of an [Fe-S] cluster in several biologically important cellular activities.

Ralstonia solanacearum virulence in eggplant seedlings by the leaf-clip inoculation

Ralstonia solanacearum causes a lethal bacterial wilt disease in numerous plants including important vegetable crops such as eggplant and tomato. One of the difficulties in studying virulence of this bacterium in different host plants is the development of an easy and stable pathogenicity assay. Recently we described a leaf-clip inoculation method to study its pathogenicity at the cotyledon stage of tomato seedlings. Hereafter, we demonstrated the leaf-clip inoculation method to be equally efficient for studying R. solanacearum pathogenicity in the cotyledon stage of eggplant seedlings. Our study revealed eggplant seedlings to be highly susceptible to R. solanacearum as compared to tomato seedlings, illustrated by appearance of disease symptoms in significantly higher number of seedlings. We also tested the virulence of several global transcription regulator mutants of R. solanacearum including hrpB, hrpG and phcA in eggplant seedlings. The phcA mutant was found to be only moderately virulence deficient in eggplant seedlings but was significantly reduced in virulence in tomato. This is indicative of some host specific responses towards certain pathogenicity functions of R. solanacearum, which are markedly different in tomato and eggplant seedlings. Apart from being economical in requiring less labor, time and space, this simple gnotobiotic leaf-clip inoculation method is anticipated to be helpful in further exploring the interaction between R. solanacearum and eggplant seedlings at the cotyledon stage.

The Bacteriophages of Streptococcus pyogenes.

The bacteriophages of Streptococcus pyogenes (group A streptococcus) play a key role in population shaping, genetic transfer, and virulence of this bacterial pathogen. Lytic phages like A25 can alter population distributions through elimination of susceptible serotypes but also serve as key mediators for genetic transfer of virulence genes and antibiotic resistance via generalized transduction. The sequencing of multiple S. pyogenes genomes has uncovered a large and diverse population of endogenous prophages that are vectors for toxins and other virulence factors and occupy multiple attachment sites in the bacterial genomes. Some of these sites for integration appear to have the potential to alter the bacterial phenotype through gene disruption. Remarkably, the phage-like chromosomal islands (SpyCI), which share many characteristics with endogenous prophages, have evolved to mediate a growth-dependent mutator phenotype while acting as global transcriptional regulators. The diverse population of prophages appears to share a large pool of genetic modules that promotes novel combinations that may help disseminate virulence factors to different subpopulations of S. pyogenes. The study of the bacteriophages of this pathogen, both lytic and lysogenic, will continue to be an important endeavor for our understanding of how S. pyogenes continues to be a significant cause of human disease.

Acute depletion of CTCF directly affects MYC regulation through loss of enhancer-promoter looping.

Numerous pieces of evidence support the complex, 3D spatial organization of the genome dictates gene expression. CTCF is essential to define topologically associated domain boundaries and to facilitate the formation of insulated chromatin loop structures. To understand CTCF’s direct role in global transcriptional regulation, we integrated the miniAID-mClover3 cassette to the endogenous CTCF locus in a human pediatric B-ALL cell line, SEM, and an immortal erythroid precursor cell line, HUDEP-2, to allow for acute depletion of CTCF protein by the auxin-inducible degron system. In SEM cells, CTCF loss notably disrupted intra-TAD loops and TAD integrity in concurrence with a reduction in CTCF-binding affinity, while showing no perturbation to nuclear compartment integrity. Strikingly, the overall effect of CTCF’s loss on transcription was minimal. Whole transcriptome analysis showed hundreds of genes differentially expressed in CTCF-depleted cells, among which MYC and a number of MYC target genes were specifically downregulated. Mechanically, acute depletion of CTCF disrupted the direct interaction between the MYC promoter and its distal enhancer cluster residing ∼1.8 Mb downstream. Notably, MYC expression was not profoundly affected upon CTCF loss in HUDEP-2 cells suggesting that CTCF could play a B-ALL cell line specific role in maintaining MYC expression.

Cytokeratin 18 regulates the transcription and alternative splicing of apoptotic‑related genes and pathways in HeLa cells.

Cytokeratin 18 (CK18), one of the major components of intermediate filaments (IF) in simple epithelial cells, undergoes caspase-mediated cleavage upon epithelial cell necrosis and apoptosis. CK18 has been used as a biomarker of several cancers and has been reported to be dysregulated in cervical cancers. The effects of dysregulated expression of CK18 at a molecular level are, however, unclear. In the present study, the function of CK18 in HeLa cells, a cell line derived from a cervical cancer cells, was investigated using shRNA knockdown. Reduced levels of CK18 led to a significant decrease in cell apoptosis, compared with control cells. Notably, RNA-seq analysis of the transcriptomes of HeLa cells, with or without CK18 knockdown, revealed that genes in the NF-κB pathway, and certain apoptosis pathways, were under global transcriptional and alternative splicing regulation. Quantitative RT-PCR confirmed the CK18-regulated transcription of apoptotic genes FAS and FADD, as well as immune genes CXCL2 and CD79B, in addition to alternative splicing of FAS and CTNNB1. Western blot analysis further revealed that CK18 knockdown led to reduced expression of CASP8. In conclusion, the present study indicated that CK18 played a role in apoptosis, which may be mediated via a feed-back regulation loop and may involve regulation of transcription and alternative splicing of a number of genes in apoptotic pathways.

Bacterial genome architecture shapes global transcriptional regulation by DNA supercoiling.

DNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes’ local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.