MYC function and regulation in physiological perspective.

MYC, a key member of the Myc-proto-oncogene family, is a universal transcription amplifier that regulates almost every physiological process in a cell including cell cycle, proliferation, metabolism, differentiation, and apoptosis. MYC interacts with several cofactors, chromatin modifiers, and regulators to direct gene expression. MYC levels are tightly regulated, and deregulation of MYC has been associated with numerous diseases including cancer. Understanding the comprehensive biology of MYC under physiological conditions is an utmost necessity to demark biological functions of MYC from its pathological functions. Here we review the recent advances in biological mechanisms, functions, and regulation of MYC. We also emphasize the role of MYC as a global transcription amplifier.

Self-assembly of promoter DNA and RNA Pol II machinery into transcriptionally active biomolecular condensates.

Transcription in the nucleus occurs in a concentrated, dense environment, and no reasonable biochemical facsimile of this milieu exists. Such a biochemical environment would be important for further understanding transcriptional regulation. We describe here the formation of dense, transcriptionally active bodies in vitro with only nuclear extracts and promoter DNA. These biomolecular condensates (BMCs) are 0.5 to 1 µm in diameter, have a macromolecular density of approximately 100 mg/ml, and are a consequence of a phase transition between promoter DNA and nuclear extract proteins. BMCs are physically associated with transcription as any disruption of one compromised the other. The BMCs contain RNA polymerase II and elongation factors, as well as factors necessary for BMC formation in vivo. We suggest that BMCs are representative of the in vivo nuclear environment and a more physiologically relevant manifestation of the preinitiation complex/elongation machinery.

Discovery of Anthranilic Acid Derivatives as Difluoromethylornithine Adjunct Agents That Inhibit Far Upstream Element Binding Protein 1 (FUBP1) Function.

Polyamine biosynthesis is regulated by ornithine decarboxylase (ODC), which is transcriptionally activated by c-Myc. A large library was screened to find molecules that potentiate the ODC inhibitor, difluoromethylornithine (DFMO). Anthranilic acid derivatives were identified as DFMO adjunct agents. Further studies identified the far upstream binding protein 1 (FUBP1) as the target of lead compound 9. FUBP1 is a single-stranded DNA/RNA binding protein and a master controller of specific genes including c-Myc and p21. We showed that 9 does not inhibit 3H-spermidine uptake yet works synergistically with DFMO to limit cell growth in the presence of exogenous spermidine. Compound 9 was also shown to inhibit the KH4 FUBP1-FUSE interaction in a gel shift assay, bind to FUBP1 in a ChIP assay, reduce both c-Myc mRNA and protein expression, increase p21 mRNA and protein expression, and deplete intracellular polyamines. This promising hit opens the door to new FUBP1 inhibitors with increased potency.

Piperlongumine combined with vitamin C as a new adjuvant therapy against gastric cancer regulates the ROS-STAT3 pathway.

OBJECTIVE: To investigate the effects of piperlongumine (PL) and vitamin C (VC) on signal transducer and activator of transcription 3 (STAT3) signalling in gastric cancer cell lines. METHODS: In vivo tumour xenograft anticancer assays were undertaken to confirm the anticancer activity of PL. Cell viability, flow cytometry and Western blot assays were undertaken to evaluate the anticancer effects of PL, VC and combinations of PL and VC in AGS and KATO III cells. RESULTS: Both PL and VC induced apoptosis and inhibited cell proliferation in AGS and KATO III cells. These effects were dependent on reactive oxygen species (ROS). PL effectively suppressed STAT3 activation while VC caused abnormal activation of STAT3. The combination of PL and VC exhibited a stronger apoptotic effect compared with either agent alone. PL reversed the abnormal activation of STAT3 by VC, which could be a key to their synergistic effect. CONCLUSIONS: PL combined with VC exhibited a stronger anticancer effect by regulating the ROS-STAT3 pathway, suggesting that this combination might be a potential adjuvant therapy for gastric cancer.

Mechanical determinants of chromatin topology and gene expression.

The compaction of linear DNA into micrometer-sized nuclear boundaries involves the establishment of specific three-dimensional (3D) DNA structures complexed with histone proteins that form chromatin. The resulting structures modulate essential nuclear processes such as transcription, replication, and repair to facilitate or impede their multi-step progression and these contribute to dynamic modification of the 3D-genome organization. It is generally accepted that protein-protein and protein-DNA interactions form the basis of 3D-genome organization. However, the constant generation of mechanical forces, torques, and other stresses produced by various proteins translocating along DNA could be playing a larger role in genome organization than currently appreciated. Clearly, a thorough understanding of the mechanical determinants imposed by DNA transactions on the 3D organization of the genome is required. We provide here an overview of our current knowledge and highlight the importance of DNA and chromatin mechanics in gene expression.

MYC assembles and stimulates topoisomerases 1 and 2 in a "topoisome".

High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.

Preparation of Near-Infrared/Photoacoustic Dual-Mode Imaging and Photothermal/Chemo Synergistic Theranostic Nanoparticles and Their Imaging and Treating of Hepatic Carcinoma.

At present, the clinical diagnosis of and treatment methods for hepatic carcinoma still fail to fully meet the needs of patients. The integrated theranostic system, in which functional materials are used to load different active molecules, created a new developmental direction for the combination treatment of hepatic carcinoma, realizing the synchronization of diagnosis and treatment. In this study, polydopamine (PDA), which has the functions of self-assembly, encapsulation, photothermal conversion, and photoacoustic interaction, was used as the carrier material. The IR780, a near-infrared fluorescence imaging (NIFI), photoacoustic imaging (PAI), and photothermal therapy (PTT) agent, and paclitaxel (PTX), a broad-spectrum chemotherapy drug, were selected to build the NIF/PA dual-mode imaging and PTT/chemo synergistic theranostic nanoparticles (DIST NPs). The DIST NPs have a 103.4 ± 13.3 nm particle size, a weak negative charge on the surface, good colloidal stability, slow and controlled drug release, and high photothermal conversion ability. The experiments results showed that the DIST NPs have a long circulation in vivo, high bioavailability, high biocompatibility, and low effective dose. DIST NPs showed an excellent NIFI/PAI dual-mode imaging and significant synergistic antitumor effect in hepatic carcinoma models. DIST NPs met the initial design requirements. A set of fast and low-cost preparation methods was established. This study provides an experimental basis for the development of new clinical theranostic methods for hepatic carcinoma.

Mycobacterium tuberculosis SufR responds to nitric oxide via its 4Fe-4S cluster and regulates Fe-S cluster biogenesis for persistence in mice.

The persistence of Mycobacterium tuberculosis (Mtb) is a major problem in managing tuberculosis (TB). Host-generated nitric oxide (NO) is perceived as one of the signals by Mtb to reprogram metabolism and respiration for persistence. However, the mechanisms involved in NO sensing and reorganizing Mtb's physiology are not fully understood. Since NO damages iron-sulfur (Fe-S) clusters of essential enzymes, the mechanism(s) involved in regulating Fe-S cluster biogenesis could help Mtb persist in host tissues. Here, we show that a transcription factor SufR (Rv1460) senses NO via its 4Fe-4S cluster and promotes persistence of Mtb by mobilizing the Fe-S cluster biogenesis system; suf operon (Rv1460-Rv1466). Analysis of anaerobically purified SufR by UV-visible spectroscopy, circular dichroism, and iron-sulfide estimation confirms the presence of a 4Fe-4S cluster. Atmospheric O2 and H2O2 gradually degrade the 4Fe-4S cluster of SufR. Furthermore, electron paramagnetic resonance (EPR) analysis demonstrates that NO directly targets SufR 4Fe-4S cluster by forming a protein-bound dinitrosyl-iron-dithiol complex. DNase I footprinting, gel-shift, and in vitro transcription assays confirm that SufR directly regulates the expression of the suf operon in response to NO. Consistent with this, RNA-sequencing of MtbΔsufR demonstrates deregulation of the suf operon under NO stress. Strikingly, NO inflicted irreversible damage upon Fe-S clusters to exhaust respiratory and redox buffering capacity of MtbΔsufR. Lastly, MtbΔsufR failed to recover from a NO-induced non-growing state and displayed persistence defect inside immune-activated macrophages and murine lungs in a NO-dependent manner. Data suggest that SufR is a sensor of NO that supports persistence by reprogramming Fe-S cluster metabolism and bioenergetics.

Long-Noncoding RNA CASC9 Promotes Progression of Non-Small Cell Lung Cancer by Promoting the Expression of CDC6 Through Binding to HuR.

OBJECTIVE: The long-noncoding RNAs (lncRNAs) have been identified as key players in diverse cellular processes in non-small cell lung cancer (NSCLC). However, the understanding of biological functions and detailed mechanisms of lncRNAs is still limited. Herein, the lncRNA cancer susceptibility candidate 9 (CASC9) on NSCLC progression is investigated. MATERIALS AND METHODS: Expressions of CASC9, HuR and cell division cycle 6 (CDC6) in NSCLC tissues were detected with quantitative real-time polymerase chain reaction (qRT-PCR). The cell counting kit-8, transwell assays, and flow cytometry were used to examine cell proliferation, migration, and the cell cycle. Tumor growth in vivo was evaluated by xenograft tumor experiments and immunohistochemistry. RNA-binding protein immunoprecipitation (RIP) was used to identify the interaction between HuR and CDC6, and CASC9 and HuR. RESULTS: CASC9, CDC6 and HuR expression were found significantly upregulated in NSCLC tissues, which predicted poorer 5-year overall survival in NSCLC patients. Inhibition of CASC9 significantly reduced the malignancy of NSCLC cells, such as proliferation, migration and cell cycle. In vivo experiments further demonstrated that CASC9 knockdown reduced the tumor growth and the Ki-67 expression. Moreover, CASC9 knockdown inhibited the expression of CDC6 which was detected overexpressed in NSCLC tumor tissues. Then, up-regulation of CDC6 could partly reverse the negative effects of CASC9 on cell proliferation, migration and cell cycle. RIP assay and rescue experiment showed that CASC9 regulated CDC via binding to HuR. CONCLUSION: Our results indicate that CASC9 conferred an aggressive phenotype in NSCLC and might be a pivotal target for this disease.

A Type IA DNA/RNA Topoisomerase with RNA Hydrolysis Activity Participates in Ribosomal RNA Processing.

Topoisomerases maintain topological homeostasis of bacterial chromosomes by catalysing changes in DNA linking number. The resolution of RNA entanglements occurring in the cell would also require catalytic action of topoisomerases. We describe RNA topoisomerase and hydrolysis activities in DNA topoisomerase I (topo I) from mycobacteria. The interaction of topo I with mRNA, tRNA and rRNA suggested its role in some aspect of RNA metabolism; the enzyme participates in rRNA maturation via its RNA hydrolysis activity. Accumulation of rRNA precursors in a topo I knockdown strain and the rescue of rRNA processing deficiency in RNaseE knockdown cells by topo I expression indicated the enzyme's back-up support to RNases involved in rRNA processing. We demonstrate that the active-site tyrosine of the enzyme mediates catalytic reactions with both DNA/RNA substrates, and RNA topoisomerase activity can follow two reaction paths in contrast to its DNA topoisomerase activity. Mutation in the canonical proton relay pathway impacts DNA topoisomerase activity whilst retaining activity on RNA substrates. The mycobacterial topo I thus exemplifies the resourcefulness and parsimony of biological catalysis in harnessing the limited chemical repertoire at its disposal to find common solutions to mechanistically related challenges of phosphodiester breakage/exchange reactions in DNA and RNA that are essential for cell survival.

DNA flowerstructure co-localizes with human pathogens in infected macrophages.

Herein, we characterize the cellular uptake of a DNA structure generated by rolling circle DNA amplification. The structure, termed nanoflower, was fluorescently labeled by incorporation of ATTO488-dUTP allowing the intracellular localization to be followed. The nanoflower had a hydrodynamic diameter of approximately 300 nanometer and was non-toxic for all mammalian cell lines tested. It was internalized specifically by mammalian macrophages by phagocytosis within a few hours resulting in specific compartmentalization in phagolysosomes. Maximum uptake was observed after eight hours and the nanoflower remained stable in the phagolysosomes with a half-life of 12 h. Interestingly, the nanoflower co-localized with both Mycobacterium tuberculosis and Leishmania infantum within infected macrophages although these pathogens escape lysosomal degradation by affecting the phagocytotic pathway in very different manners. These results suggest an intriguing and overlooked potential application of DNA structures in targeted treatment of infectious diseases such as tuberculosis and leishmaniasis that are caused by pathogens that escape the human immune system by modifying macrophage biology.

Conditional down-regulation of GreA impacts expression of rRNA and transcription factors, affecting Mycobacterium smegmatis survival.

Gre, one of the conserved transcription factors in bacteria, modulates RNA polymerase (RNAP) activity to ensure processivity and fidelity of RNA synthesis. Gre factors regulate transcription by inducing the intrinsic-endonucleolytic activity of RNAP, allowing the enzyme to resume transcription from the paused and arrested sites. While Escherichia coli and a number of eubacteria harbor GreA and GreB, genus mycobacteria has a single Gre (GreA). To address the importance of the GreA in growth, physiology and gene expression of Mycobacterium smegmatis, we have constructed a conditional knock-down strain of GreA. The GreA depleted strain exhibited slow growth, drastic changes in cell surface phenotype, cell death, and increased susceptibility to front-line anti-tubercular drugs. Transcripts and 2D-gel electrophoresis (2D-PAGE) analysis of the GreA conditional knock-down strain showed altered expression of the genes involved in transcription regulation. Among the genes analysed, expression of RNAP subunits (ß, ß' and ω), carD, hupB, lsr2, and nusA were affected to a large extent. Severe reduction in the expression of genes of rRNA operon in the knock-down strain reveal a role for GreA in regulating the core components of the translation process.

Dual Loading of Nanoparticles with Doxorubicin and Icotinib for the Synergistic Suppression of Non-Small Cell Lung Cancer.

Background: Combination chemotherapy plays an important role in the clinical therapy of non-small cell lung cancer (NSCLC). However, the pharmacokinetic differences between drugs are an insurmountable barrier in traditional treatment. For the synergistic therapy of NSCLC, synergistic nanoparticles (EDS NPs) loaded with both an EGFR inhibitor and doxorubicin (DOX) were designed and prepared. Methods: Erlotinib, apatinib and icotinib were evaluated for optimal combination with DOX in treatment of NSCLC via CCK-8 assay. Then the cationic amphipathic starch (CSaSt) and hyaluronic acid (HA) were applied to coencapsulate DOX and EGFR inhibitor to form the EDS NPs. EDS NPs were evaluated in NSCLC cell lines (A549, NCI-H1975 and PC9) and NSCLC xenograft mouse models. Results: Icotinib was found to be the optimal synergistic drug in combination with DOX in the tested. Subsequently, icotinib and DOX were coencapsulated in the NPs. EDS NPs were roughly spherical with an average size of 65.7±6.2 nm and possessed stable loading and releasing properties. In the in vitro investigation, EDS NPs could efficiently deliver payloads into cells, exhibited cytotoxicity and produced strong anti-migration properties. In vivo hypotoxicity was confirmed by acute toxicity and hemolytic assays. The in vivo distribution showed that EDS NPs could enhance accumulation in tumors and decrease nonspecific accumulation in normal organs. EDS NPs significantly promoted the in vivo synergistic effects of icotinib and DOX in the mouse model. Conclusions: The study suggests that EDS NPs possess noteworthy potential for development as therapeutics for NSCLC clinical chemotherapy.

Vestibular Schwanomma: An Experience in a Developing World.

BACKGROUND: Tumors related to the acoustic nerves represent 90% of cerebellopontine angle diseases and have been in the picture for at least 200 years. Famous as acoustic neuromas and vestibular neuromas, these are usually benign, slow-growing tumors of Schwann cells of the myelin sheath. Surgery is the treatment of choice though some authors have suggested "wait and watch" policy. The aims of our study were to study the clinical presentation and management of the tumors, and to evaluate the perioperative outcomes of the surgery. METHODS: A retrospective review of the datasheet of 33 patients diagnosed with vestibular schwanomma who had undergone surgery from January 2014 to January 2017 was performed in National Academy of Medical Sciences, Bir Hospital, Kathmandu, Nepal. Analysis of the demographic data and perioperative outcomes was performed. RESULTS: Hearing loss was the main presenting symptom in 72% cases followed by tinnitus, dizziness, facial numbness and sudden sensorineural hearing loss. Mean tumur size was 39.7 ± 3 mm. The mean age of the patients was 46 ± 3 years with a female preponderance (1.2:1). In particular, the retrosigmoid route was preferred in all the cases since it was the most employed approach at our center and 63% of the tumors presented to us were grade 5. The surgical techniques allowed safe preservation of the facial function which was 93%. The hearing loss did not improve after the surgery in 94% while it worsened in 6% of cases. We did not find any significant relation between outcome and size, age, gender or laterality of the tumor (P > 0.05). There was no perioperative mortality. CONCLUSIONS: The benign and slow-growing nature of vestibular schwanomma usually poses problems for the early diagnosis and treatment especially in a poor resource setting like ours. Likewise, there are very few studies so far done in the country regarding the incidence and management of the disease. Thus, this study might be helpful in providing insight into the occurrence of the disease in the present scenario and the need for much more studies in the future.

NapA (Rv0430), a Novel Nucleoid-Associated Protein that Regulates a Virulence Operon in Mycobacterium tuberculosis in a Supercoiling-Dependent Manner.

Comparison of Mycobacterium tuberculosis with Escherichia coli reveals a reduction in the diversity of DNA-managing proteins, such as DNA topoisomerases, although genome sizes are similar for the two species. The same is true for nucleoid-associated proteins (NAPs), important factors in bacterial chromosome compaction, chromosome remodeling, and regulation of gene expression. In a search for still uncharacterized NAPs, we found that M. tuberculosis protein Rv0430 has NAP-like features: it binds to DNA in a length- and supercoil-dependent fashion, prefers A/T-rich DNA sequences, protects DNA from damaging agents, and modulates DNA supercoiling. At a ratio of 1 dimer/40 bps of DNA, Rv0430 bridges distant DNA segments; at 1 dimer/20 bps, it coats DNA, forming inflexible rods. Rv0430 also stimulates the DNA relaxation activity of topoisomerase I. Remarkably, Rv0430 stimulates its own promoter in a supercoil-dependent manner. It is the first gene of an operon harboring two regulators of M. tuberculosis virulence (virR and sodC), and controls the expression of these downstream virulence regulators and therefore itself is a virulence regulator. The sensitivity of rv0430 expression to supercoiling is consistent with supercoiling being important for infection by M. tuberculosis. Thus, Rv0430 is a novel NAP, doubling up as a topology modulator of M. tuberculosis.

Physical and functional interaction between nucleoid-associated proteins HU and Lsr2 of Mycobacterium tuberculosis: altered DNA binding and gene regulation.

Nucleoid-associated proteins (NAPs) in bacteria contribute to key activities such as DNA compaction, chromosome organization and regulation of gene expression. HU and Lsr2 are two principal NAPs in Mycobacterium tuberculosis (Mtb). HU is essential for Mtb survival and is one of the most abundant NAPs. It differs from other eubacterial HU proteins in having a long, flexible lysine- and arginine-rich carboxy-terminal domain. Lsr2 of Mtb is the functional analogue of the bacterial NAP commonly called H-NS. Lsr2 binds to and regulates expression of A/T-rich portions of the otherwise G/C-rich mycobacterial chromosome. Here, we demonstrate that HU and Lsr2 interact to form a complex. The interaction occurs primarily through the flexible carboxy-terminal domain of HU and the acidic amino-terminal domain of Lsr2. The resulting complex, upon binding to DNA, forms thick nucleoprotein rods, in contrast to the DNA bridging seen with Lsr2 and the DNA compaction seen with HU. Furthermore, transcription assays indicate that the HU-Lsr2 complex is a regulator of gene expression. This physical and functional interaction between two NAPs, which has not been reported previously, is likely to be important for DNA organization and gene expression in Mtb and perhaps other bacterial species.

Docetaxel and Doxorubicin Codelivery by Nanocarriers for Synergistic Treatment of Prostate Cancer.

Combination chemotherapy has been proven to be an efficient strategy for the treatment of prostate cancer (PCA). However, the pharmacokinetic distinction between the relevant drugs is an insurmountable barrier to the realization of their synergistic use against cancer. To overcome the disadvantages of combination chemotherapy in the treatment of PCA, targeted nanoparticles (NPs), which can codeliver docetaxel (DOC) and doxorubicin (DOX) at optimal synergistic proportions, have been designed. In this study, the DOC and DOX codelivery nanoparticles (DDC NPs) were constructed by hyaluronic acid (HA) and cationic amphipathic starch (CSaSt) through a self-assembly process. Human PCA cell lines (PC-3, DU-145, and LNCap) and mouse models were then used for evaluation in vitro and in vivo, respectively, of delivery and antitumor effects. The DDC NPs were spherical with rough surfaces, and the size and zeta potential were 68.4 ± 7.1 nm and -22.8 ± 2.2 mV, respectively. The encapsulation efficiencies of DOC and DOX in the NPs were 96.1 ± 2.3% and 91.4 ± 3.7%, respectively, while the total drug loading was 9.1 ± 1.7%. Moreover, the ratio of DOC to DOX in the DDC NPs was approximately 1:400, which aligned with the optimal synergistic proportions of the drugs. The DDC NPs exhibited excellent loading capacities, performed sustained and enzymatic release, and were stable in PBS, medium, and serum. After investigations in vitro, the DDC NPs were as effective as the dual drug combination in terms of cytotoxicity, antimigration, and apoptosis. Internalization results indicated that the DDC NPs could effectively deliver and fully release the payloads into PCA cells, and the process was mediated by the ligand-receptor interaction of HA with the CD44 protein. Low toxicity in vivo was confirmed by acute toxicity and hemolytic assays. The distribution in vivo showed that DDC NPs could enhance the accumulation of drugs in tumors and decrease nonspecific accumulation in normal organs. More importantly, DDC NPs significantly promoted the curative effect of the DOC and DOX combination in the PCA cell xenograft mouse model, indicating that the drugs with NPs did indeed act synergistically. This study suggests that the DDC NPs possess noteworthy potential as prospects for the development of PCA clinical chemotherapy.

Regulation of the gyr operon of Mycobacterium tuberculosis by overlapping promoters, DNA topology, and reiterative transcription.

DNA gyrase introduces negative supercoils into DNA to maintain topological homeostasis. The genes encoding gyrase, gyrB and gyrA, form a dicistronic operon in Mycobacterium tuberculosis (Mtb) and other actinobacteria. Earlier work indicated that DNA relaxation stimulates the expression of the gyr genes, a phenomenon termed relaxation-stimulated transcription (RST). The present study addresses the underlying mechanism of gyr operon regulation. The operon is regulated by overlapping and divergently oriented promoters located upstream of gyrB. The principal promoter, PgyrB1, drives transcription of the operon, while a weak "reverse" promoter, PgyrR, transcribes in opposite direction. We demonstrate that PgyrR plays a role in fine tuning gyr gene expression by reiterative transcription (RT), a regulatory mechanism hitherto not found in Mtb. In vitro transcription assays showed that RT at PgyrR depended on the negatively supercoiled state of the DNA template. The principal promoter, PgyrB1, was also sensitive to DNA supercoiling, but it was stimulated by DNA relaxation. Moreover, RNA polymerase binding to the promoter was efficient at PgyrB1 when template DNA was relaxed, whereas binding to PgyrR was preferred when DNA was supercoiled. Thus, a collaboration between RST and RT governs the regulation of the gyr operon; the differing sensitivity of the two overlapping promoters to superhelix density explains how gyrase expression responds to changes in supercoiling to determine the efficiency of transcription initiation.

Efficacy of ß-lactam/ß-lactamase inhibitor combination is linked to WhiB4-mediated changes in redox physiology of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) expresses a broad-spectrum ß-lactamase (BlaC) that mediates resistance to one of the highly effective antibacterials, ß-lactams. Nonetheless, ß-lactams showed mycobactericidal activity in combination with ß-lactamase inhibitor, clavulanate (Clav). However, the mechanistic aspects of how Mtb responds to ß-lactams such as Amoxicillin in combination with Clav (referred as Augmentin [AG]) are not clear. Here, we identified cytoplasmic redox potential and intracellular redox sensor, WhiB4, as key determinants of mycobacterial resistance against AG. Using computer-based, biochemical, redox-biosensor, and genetic strategies, we uncovered a functional linkage between specific determinants of ß-lactam resistance (e.g. ß-lactamase) and redox potential in Mtb. We also describe the role of WhiB4 in coordinating the activity of ß-lactamase in a redox-dependent manner to tolerate AG. Disruption of WhiB4 enhances AG tolerance, whereas overexpression potentiates AG activity against drug-resistant Mtb. Our findings suggest that AG can be exploited to diminish drug-resistance in Mtb through redox-based interventions.

Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase.

Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the in vivo interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in Mycobacterium tuberculosis taking advantage of minimal topoisomerase representation in the organism. The study unveils the first in vivo genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in Mtb, near the Ter domain of the Mtb chromosome validates its function as a decatenase.