Redox-responsive Transcription Factors Research Articles

A type III-Dv CRISPR-Cas system is controlled by the transcription factor RpaB and interacts with the DEAD-box RNA helicase CrhR

How CRISPR-Cas systems defend bacteria and archaea against invading genetic elements is well understood, but less is known about their regulation. In the cyanobacterium Synechocystis sp. PCC 6803, the expression of one of the three different CRISPR-Cas systems responds to changes in environmental conditions. The cas operon promoter of this system is controlled by the light- and redox-responsive transcription factor RpaB binding to an HLR1 motif, resulting in transcriptional activation at low light intensities. However, the strong promoter that drives transcription of the cognate repeat-spacer array is not controlled by RpaB. Instead, the leader transcript is bound by the redox-sensitive RNA helicase CrhR. Crosslinking coupled with mass spectrometry analysis and site-directed mutagenesis revealed six residues involved in the CrhR-RNA interaction, with C371 being critically important. Thus, the expression of a type III-Dv CRISPR-Cas system is linked to the redox status of the photosynthetic cell at the transcriptional and post-transcriptional levels.

Flavonoids Regulate Redox-Responsive Transcription Factors in Glioblastoma and Microglia.

The tumor microenvironment (TME) has emerged as a valuable therapeutic target in glioblastoma (GBM), as it promotes tumorigenesis via an increased production of reactive oxygen species (ROS). Immune cells such as microglia accumulate near the tumor and its hypoxic core, fostering tumor proliferation and angiogenesis. In this study, we explored the therapeutic potential of natural polyphenols with antioxidant and anti-inflammatory properties. Notably, flavonoids, including fisetin and quercetin, can protect non-cancerous cells while eliminating transformed cells (2D cultures and 3D tumoroids). We tested the hypothesis that fisetin and quercetin are modulators of redox-responsive transcription factors, for which subcellular location plays a critical role. To investigate the sites of interaction between natural compounds and stress-responsive transcription factors, we combined molecular docking with experimental methods employing proximity ligation assays. Our findings reveal that fisetin decreased cytosolic acetylated high mobility group box 1 (acHMGB1) and increased transcription factor EB (TFEB) abundance in microglia but not in GBM. Moreover, our results suggest that the most powerful modulator of the Nrf2-KEAP1 complex is fisetin. This finding is in line with molecular modeling and calculated binding properties between fisetin and Nrf2-KEAP1, which indicated more sites of interactions and stronger binding affinities than quercetin.

Far-Red Light Coordinates the Diurnal Changes in the Transcripts Related to Nitrate Reduction, Glutathione Metabolism and Antioxidant Enzymes in Barley.

Spectral quality, intensity and period of light modify many regulatory and stress signaling pathways in plants. Both nitrate and sulfate assimilations must be synchronized with photosynthesis, which ensures energy and reductants for these pathways. However, photosynthesis is also a source of reactive oxygen species, whose levels are controlled by glutathione and other antioxidants. In this study, we investigated the effect of supplemental far-red (735 nm) and blue (450 nm) lights on the diurnal expression of the genes related to photoreceptors, the circadian clock, nitrate reduction, glutathione metabolism and various antioxidants in barley. The maximum expression of the investigated four photoreceptor and three clock-associated genes during the light period was followed by the peaking of the transcripts of the three redox-responsive transcription factors during the dark phase, while most of the nitrate and sulfate reduction, glutathione metabolism and antioxidant-enzyme-related genes exhibited high expression during light exposure in plants grown in light/dark cycles for two days. These oscillations changed or disappeared in constant white light during the subsequent two days. Supplemental far-red light induced the activation of most of the studied genes, while supplemental blue light did not affect or inhibited them during light/dark cycles. However, in constant light, several genes exhibited greater expression in blue light than in white and far-red lights. Based on a correlation analysis of the gene expression data, we propose a major role of far-red light in the coordinated transcriptional adjustment of nitrate reduction, glutathione metabolism and antioxidant enzymes to changes of the light spectrum.

Meta-analysis of transcriptomic responses to cold stress in plants.

Transcriptomic analyses are needful tools to gain insight into the molecular mechanisms underlying plant responses to abiotic stresses. The aim of this study was to identify key genes differentially regulated in response to chilling stress in various plant species with different levels of tolerance to low temperatures. A meta-analysis was performed using the RNA-Seq data of published studies whose experimental conditions were comparable. The results confirmed the importance of ethylene in the hormonal cross-talk modulating the defensive responses against chilling stress, especially in sensitive species. The transcriptomic activity of five Ethylene Response Factors genes and a REDOX Responsive Transcription Factor 1 involved in hormone-related pathways belonging to ethylene metabolism and signal transduction were induced. Transcription activity of two genes encoding for heat shock factors was enhanced, together with various genes associated with developmental processes. Several transcription factor families showed to be commonly induced between different plant species. Protein-protein interaction networks highlighted the role of the photosystems I and II, as well as genes encoding for HSF and WRKY transcription factors. A model of gene regulatory network underlying plant responses to chilling stress was developed, allowing the delivery of new candidate genes for genetic improvement of crops towards low temperatures tolerance.

Size and ligand effects of gold nanoclusters in alteration of organellar state and translocation of transcription factors in human primary astrocytes.

Ultra-small gold nanoclusters (AuNCs) with designed sizes and ligands are gaining popularity for biomedical purposes and ultimately for human imaging and therapeutic applications. Human non-tumor brain cells, astrocytes, are of particular interest because they are abundant and play a role in functional regulation of neurons under physiological and pathological conditions. Human primary astrocytes were treated with AuNCs of varying sizes (Au10, Au15, Au18, Au25) and ligand composition (glutathione, polyethylene glycol, N-acetyl cysteine). Concentration and time-dependent studies showed no significant cell loss with AuNC concentrations <10 μM. AuNC treatment caused marked differential astrocytic responses at the organellar and transcription factor level. The effects were exacerbated under severe oxidative stress induced by menadione. Size-dependent effects were most remarkable with the smallest and largest AuNCs (10, 15 Au atoms versus 25 Au atoms) and might be related to the accessibility of biological targets toward the AuNC core, as demonstrated by QM/MM simulations. In summary, these findings suggest that AuNCs are not inert in primary human astrocytes, and that their sizes play a critical role in modulation of organellar and redox-responsive transcription factor homeostasis.

RRFT1 (Redox Responsive Transcription Factor 1) is involved in extracellular ATP-regulated gene expression in Arabidopsis thaliana seedlings

ABSTRACTAs an apoplast signal molecule, extracellular ATP (eATP) is involved in the growth regulation of Arabidopsis thaliana seedlings. Recently, RRFT1 was revealed to be involved in eATP- regulated seedling growth. To further verify the role of RRTF1 in seedlings' eATP response, expression of 20 eATP-responsive genes in wild type (Col-0) and RRTF1 null mutant (rrtf1-1) seedlings were investigated by using realtime quantitative PCR. After 0.5 mM ATP stimulation, the response of these genes' expression in rrtf1-1 seedlings was significantly different from that in Col-0 seedlings. Proteins which are encoded by these genes include transcription factors, plasma membrane receptors like kinases, ion influx/efflux transporters and hormone signaling components. The results indicated that RRTF1 may be involved in eATP regulated physiological responses via regulating the expression of some functional genes.

Progressive Rotavirus Infection Downregulates Redox-Sensitive Transcription Factor Nrf2 and Nrf2-Driven Transcription Units

Eukaryotic cells adopt highly tuned stress response physiology under threats of exogenous stressors including viruses to maintain cellular homeostasis. Not surprisingly, avoidance of cellular stress response pathways is an essential facet of virus-induced obligatory host reprogramming to invoke a cellular environment conducive to viral perpetuation. Adaptive cellular responses to oxidative and electrophilic stress are usually taken care of by an antioxidant defense system, core to which lies the redox-responsive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2-driven transcriptional cascade. Deregulation of host redox balance and redox stress-sensitive Nrf2 antioxidant defense have been reported for many viruses. In the current study, we aimed to study the modulation of the Nrf2-based host cellular redox defense system in response to Rotavirus (RV) infection in vitro. Interestingly, we found that Nrf2 protein levels decline sharply with progression of RV infection beyond an initial upsurge. Moreover, Nrf2 decrease as a whole was found to be accompanied by active nuclear vacuity of Nrf2, resulting in lowered expression of stress-responsive Nrf2 target genes heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1, and superoxide dismutase 1 both in the presence and absence of Nrf2-driven transcriptional inducers. Initial induction of Nrf2 concurred with RV-induced early burst of oxidative stress and therefore was sensitive to treatments with antioxidants. Reduction of Nrf2 levels beyond initial hours, however, was found to be independent of the cellular redox status. Furthermore, increasing the half-life of Nrf2 through inhibition of the Kelch-like erythroid cell-derived protein with CNC homology- (ECH-) associated protein 1/Cullin3-RING Box1-based canonical Nrf2 turnover pathway could not restore Nrf2 levels post RV-SA11 infection. Depletion of the Nrf2/HO-1 axis was subsequently found to be sensitive to proteasome inhibition with concurrent observation of increased K48-linked ubiquitination associated with Nrf2. Together, the present study describes robust downregulation of Nrf2-dependent cellular redox defense beyond initial hours of RV infection, justifying our previous observation of potent antirotaviral implications of Nrf2 agonists.

Redox-Responsive Transcription Factor 1 (RRFT1) Is Involved in Extracellular ATP-Regulated Arabidopsis thaliana Seedling Growth.

Extracellular adenosine triphosphate (eATP) is an apoplastic signaling molecule that plays an essential role in the growth and development of plants. Arabidopsis seedlings have been reported to respond to eATP; however, the downstream signaling components are still not well understood. In this study, we report that an ethylene-responsive factor, Redox-Responsive Transcription Factor 1 (RRTF1), is involved in eATP-regulated Arabidopsis thaliana seedling growth. Exogenous adenosine triphosphate inhibited green seedling root growth and induced hypocotyl bending of etiolated seedlings. RRTF1 loss-of-function mutant (rrtf1) seedlings showed decreased responses to eATP, while its complementation or overexpression led to recovered or increased eATP responsiveness. RRTF1 was expressed rapidly after eATP stimulation and then migrated into the nuclei of root tip cells. eATP-induced auxin accumulation in root tip or hypocotyl cells was impaired in rrtf1. Chromatin immunoprecipitation and high-throughput sequencing results indicated that eATP induced some genes related to cell growth and development in wild type but not in rrtf1 cells. These results suggest that RRTF1 may be involved in eATP signaling by regulating functional gene expression and cell metabolism in Arabidopsis seedlings.

Catalase, glutathione, and protein phosphatase 2A-dependent organellar redox signalling regulate aphid fecundity under moderate and high irradiance.

Redox processes regulate plant/insect responses, but the precise roles of environmental triggers and specific molecular components remain poorly defined. Aphid fecundity and plant responses were therefore measured in Arabidopsis thaliana mutants deficient in either catalase 2 (cat2), different protein phosphatase 2A (PP2A) subunits or glutathione (cad2, pad2, and clt1) under either moderate (250 μmol m-2 s-1 ) or high (800 μmol m-2 s-1 ) light. Aphid fecundity was decreased in pp2a-b'γ, cat2 and the cat2 pp2a-b'γ double mutants relative to the wild type under moderate irradiance. High light decreased aphid numbers in all genotypes except for cat2. Aphid fecundity was similar in the cat2 and glutathione-, phytoalexin-, and glucosinolate-deficient cat2cad2 double mutants under both irradiances. Aphid-induced increases in transcripts encoding the abscisic acid-related ARABIDOPSIS ZINC-FINGER PROTEIN 1 transcription factor were observed only under moderate light. Conversely, aphid induced increases in transcripts encoding the jasmonate-synthesis enzyme ALLENE OXIDE CYCLASE 3 was observed in all genotypes only under high light. Aphid-induced increases in REDOX RESPONSIVE TRANSCRIPTION FACTOR 1 mRNAs were observed in all genotypes except pp2a-b'ζ1-1 under both irradiances. Aphid fecundity is therefore regulated by cellular redox signalling that is mediated, at least in part, through PP2A-dependent mitochondria to nucleus signalling pathways.

Transcriptional Regulation of the Glucose-6-Phosphate/Phosphate Translocator 2 Is Related to Carbon Exchange Across the Chloroplast Envelope.

The exchange of reduced carbon across the inner chloroplast envelope has a large impact on photosynthesis and growth. Under steady-state conditions it is thought that glucose 6-phosphate (G6P) does not cross the chloroplast membrane. However, growth at high CO2, or disruption of starch metabolism can result in the GPT2 gene for a G6P/Pi translocator to be expressed presumably allowing G6P exchange across the chloroplast envelope. We found that after an increase in light, the transcript for GPT2 transiently increases several 100-fold within 2 h in both the Col-0 and WS ecotypes of Arabidopsis thaliana. The increase in transcript for GPT2 is preceded by an increase in transcript for many transcription factors including Redox Responsive Transcription Factor 1 (RRTF1). The increase in GPT2 transcript after exposure to high light is suppressed in a mutant lacking the RRTF1 transcription factor. The GPT2 response was also suppressed in a mutant with a T-DNA insert in the gene for the triose-phosphate/Pi translocator (TPT). However, plants lacking TPT still had a robust rise in RRTF1 transcript in response to high light. From this, we conclude that both RRTF1 (and possibly other transcription factors) and high amounts of cytosolic triose phosphate are required for induction of the expression of GPT2. We hypothesize that transient GPT2 expression and subsequent translation is adaptive, allowing G6P to move into the chloroplast from the cytosol. The imported G6P can be used for starch synthesis or may flow directly into the Calvin-Benson cycle via an alternative pathway (the G6P shunt), which could be important for regulating and stabilizing photosynthetic electron transport and carbon metabolism.

Staphylococcus aureus adaptation to aerobic low-redox-potential environments: implications for an intracellular lifestyle.

Methicillin-resistant Staphylococcus aureus is a 'superbug' that is responsible for extensive death and morbidity. Chronic S. aureus infections are associated with the presence of intracellular bacteria and the host cytosol is an aerobic low-redox-potential (Eh) environment. How S. aureus adapts to aerobic low-Eh environments is understudied. A low external Eh, imposed by the non-metabolizable reductant dithiothreitol, resulted in transcriptional reprogramming mediated by the redox-responsive transcription factors AgrA, Rex and SrrBA, resulting in a shift towards fermentative metabolism. Accordingly, in the presence of the host cytoplasmic reductant glutathione, the aerobic respiration of S. aureus was impaired, the intracellular NADH:NAD+ ratio increased, lactate dehydrogenase was induced, resistance to the aminoglycoside antibiotic gentamicin was enhanced and greater numbers of small-colony variants (SCVs) were detected. These observations suggest that entry of S. aureus into the aerobic low-Eh environment of the host cytosol could result in adaptive responses that promote the formation of SCVs.

Biocomputational Analyses and Experimental Validation Identify the Regulon Controlled by the Redox-Responsive Transcription Factor RpaB

SummaryOxygenic photosynthesis requires the coordination of environmental stimuli with the regulation of transcription. The transcription factor RpaB is conserved from the simplest unicellular cyanobacteria to complex eukaryotic algae, representing more than 1 billion years of evolution. To predict the RpaB-controlled regulon in the cyanobacterium Synechocystis, we analyzed the positional distribution of binding sites together with high-resolution mapping data of transcriptional start sites (TSSs). We describe more than 150 target promoters whose activity responds to fluctuating light conditions. Binding sites close to the TSS mediate repression, whereas sites centered ∼50 nt upstream mediate activation. Using complementary experimental approaches, we found that RpaB controls genes involved in photoprotection, cyclic electron flow and state transitions, photorespiration, and nirA and isiA for which we suggest cross-regulation with the transcription factors NtcA or FurA. The deep integration of RpaB with diverse photosynthetic gene functions makes it one of the most important and versatile transcriptional regulators.

Responsiveness and Adaptation to Salt Stress of the REDOX-RESPONSIVE TRANSCRIPTION FACTOR 1 (RRTF1) Gene are Controlled by its Promoter.

The REDOX-RESPONSIVE TRANSCRIPTION FACTOR 1 (RRTF1) gene encodes a member of the ERF/AP2 transcription factor family involved in redox homeostasis. The RRTF1 gene shows tissue-specific responsiveness to various abiotic stress treatments including a response to salt stress in roots. An interesting feature of this response is an adaptation phase that follows its activation, when promoter levels revert to a base line level, even if salt stress is maintained. It is unclear if adaption is controlled by a switch in promoter activity or by changes in transcript levels. Here we show that the RRTF1 promoter is sufficient for the control of both activation and adaptation to salt stress. As constitutive expression of RRTF1 turned out to be detrimental to the plant, we propose that promoter-regulated adaptation evolved as a protection mechanism to balance the beneficial effects of short-term gene activation and the detrimental effects of long-term gene expression.

Roles and regulation of Spx family transcription factors in Bacillus subtilis and related species.

Bacillus subtilis Spx is the prototype for a large family of redox-responsive transcription factors found in many bacteria, most notably those from the phylum Firmicutes. Unusually for a transcription factor, B. subtilis Spx protein modulates gene expression by binding as a monomer to the αCTD domain of RNA polymerase (RNAP), and only interacts with DNA during subsequent promoter engagement. B. subtilis Spx drives the expression of a large regulon in response to proteotoxic conditions, such as heat and disulfide stress, as well as cell wall stress. Here, we review the detailed mechanisms that control the expression, stability, and activity of Spx in response to a variety of stress conditions. We also summarize current knowledge regarding Spx homologs in other Firmicutes, the environmental conditions in which those homologs are activated, and their biological role.

A distinct class of antioxidant response elements is consistently activated in tumors with NRF2 mutations

NRF2 is a redox-responsive transcription factor that regulates expression of cytoprotective genes via its interaction with DNA sequences known as antioxidant response elements (AREs). NRF2 activity is induced by oxidative stress, but oxidative stress is not the only context in which NRF2 can be activated. Mutations that disrupt the interaction between NRF2 and KEAP1, an inhibitor of NRF2, lead to NRF2 hyperactivation and promote oncogenesis. The mechanisms underlying NRF2's oncogenic properties remain unclear, but likely involve aberrant expression of select NRF2 target genes. We tested this possibility using an integrative genomics approach to get a precise view of the direct NRF2 target genes dysregulated in tumors with NRF2 hyperactivating mutations. This approach revealed a core set of 32 direct NRF2 targets that are consistently upregulated in NRF2 hyperactivated tumors. This set of NRF2 “cancer target genes” includes canonical redox-related NRF2 targets, as well as target genes that have not been previously linked to NRF2 activation. Importantly, NRF2-driven upregulation of this gene set is largely independent of the organ system where the tumor developed. One key distinguishing feature of these NRF2 cancer target genes is that they are regulated by high affinity AREs that fall within genomic regions possessing a ubiquitously permissive chromatin signature. This implies that these NRF2 cancer target genes are responsive to oncogenic NRF2 in most tissues because they lack the regulatory constraints that restrict expression of most other NRF2 target genes. This NRF2 cancer target gene set also serves as a reliable proxy for NRF2 activity, and high NRF2 activity is associated with significant decreases in survival in multiple cancer types. Overall, the pervasive upregulation of these NRF2 cancer targets across multiple cancers, and their association with negative outcomes, suggests that these will be central to dissecting the functional implications of NRF2 hyperactivation in several cancer contexts.

YAP1 homologue‐mediated redox sensing is crucial for a successful infection by Monilinia fructicola

Monilinia fructicola (G. Winter) Honey is a devastating pathogen on Rosaceae which causes blossom blight and fruit rot. Only a few studies related to the plant-pathogen interaction have been published and there is limited knowledge on the relationship between oxidative stress and successful infection in M. fructicola. In this study, we cloned and characterized a redox-responsive transcription factor MFAP1, a YAP1 homologue. MfAP1-silenced strains were generated by polyethylene glycol-mediated protoplast transformation or Agrobacterium T-DNA-mediated transformation. Pathogenicity assay demonstrated that MfAP1-silenced strains caused smaller lesions on rose and peach petals. Transformants carrying extra copies of MfAP1, driven by the native promoter, were generated for MfAP1 overexpression. Interestingly, MfAP1-overexpressing strains also caused smaller lesions on rose petals. Strains carrying two copies of MfAP1 accumulated reactive oxygen species (ROS) at higher levels and exhibited delayed accumulation of MfAP1 transcripts compared with the wild-type during pathogenesis. By the analysis of ROS production and the expression patterns of redox- and virulence-related genes in the wild-type strain and an MfAP1-overexpressing strain, we found that the M. fructicola wild-type strain responded to oxidative stress at the infection site, activated the expression of MfAP1 and up-regulated the genes required for ROS detoxification and fungal virulence. In contrast, MfAP1 expression in the MfAP1-overexpressing strain was suppressed after the induction of a strong oxidative burst at the infection site, altering the expression of ROS detoxification and virulence-related genes. Our results highlight the importance of MfAP1 and ROS accumulation in the successful infection of M. fructicola.

Abstract 15948: Inhibition of NF-kB Signaling Attenuates Age-related Aggravation of Myocardial Ischemia/Reperfusion Injury

Introduction: Emerging evidence indicates that myocardial ischemia/reperfusion (I/R) injury is more severe in aged hearts. Although nuclear factor-kappaB (NF-kB) activity increases with aging, whether this redox-responsive transcription factor plays any role in age-related worsening of I/R injury remains unknown. Hypothesis: We hypothesized that activation of myocardial NF-kB exerts deleterious effects during I/R injury in young as well as aged hearts; and that NF-kB signaling contributes to age-related worsening of myocardial I/R injury. Methods: We used transgenic mice overexpressing a mutant IkBa that prevents NF-kB activation only in the heart. Age-matched young (10-week-old) and aged (86-week-old) male non-transgenic littermates (NTg) and IkBa transgenic (Tg) mice underwent a 30-min coronary occlusion followed by 24 h of reperfusion. Following sacrifice, infarct size was measured and molecular assays were performed. Results: The infarct size was significantly smaller in young Tg mice compared with young NTg mice (Fig), indicating a deleterious role of NF-kB signaling during myocardial I/R injury even in the young. The infarct size in aged NTg hearts was greater compared with young NTg hearts, consistent with an age-related worsening of I/R injury effects. However, infarct size in aged Tg hearts was significantly smaller than aged NTg hearts, and similar to young Tg hearts, indicating that NF-kB signaling contributes toward age-related aggravation of I/R injury. Interestingly, the levels of molecular markers of senescence, such as p16, were lower in aged Tg hearts (Fig), indicating a deleterious role of NF-kB in cardiac aging and susceptibility to I/R injury. Conclusions: We conclude that inhibition of cardiac NF-kB signaling protects against age-related aggravation of acute myocardial I/R injury. These findings suggest that modulation of NF-kB signaling may be potentially used to achieve therapeutic cardioprotection in the elderly.

Abstract 960: Targeting Egr-1 is an effective strategy for overcoming kinase inhibitor resistance in CML

Abstract The primary oncogene associated with CML is BCR/ABL which controls proliferative and survival signaling and is a potent inducer of reactive oxygen species (ROS). ROS play both positive and negative roles in proliferation and survival; this dual nature has been exploited by leukemia cells to promote growth, genomic instability, and drug resistance. However, the distinct molecular alterations that occur as a result of BCR/ABL-induced ROS are not well described. BCR/ABL-targeted therapeutics have improved clinical response rates, therefore the number of CML patients living with detectable disease burden is rising. Complete hematologic responses to tyrosine kinase inhibitor (TKI) therapy are seen in ∼10-30% of CML patients, so acquired drug resistance and relapse remain major issues. Thus, novel targets for combined therapeutics are needed. We have shown that early growth response 1 (Egr-1) is a BCR/ABL-dependent, redox-responsive transcription factor that modulates expression of the non-receptor tyrosine kinase Fyn in CML leading to proliferation and survival. Tissue microarray analysis of 26 CML patients (10 chronic phase, 6 accelerated phase, and 10 blast crisis), corroborated by western blotting on independent patient samples, showed that Egr-1 protein expression increased as CML progresses from chronic phase to the more treatment resistant accelerated phase and blast crisis. Egr-1 protein expression was also elevated 2.5 fold in a model of acquired pan-TKI resistance (K562-STI) when compared with parental K562 cells. When Egr-1 was genetically inhibited, proliferation of K562-STI cells decreased by 56%. Egr-1 knockdown was also sufficient to sensitize K562-STI cells to growth inhibition caused by first and second generation BCR/ABL-directed TKI, further implicating Egr-1 in acquired TKI resistance. Egr-1 is well known as a redox-responsive transcription factor, and we found that ROS were elevated in K562-STI vs. K562 cells. While analysis of mitochondrial respiration using a Seahorse Bioanalyzer showed no increase in mitochondrial respiration, spare respiratory capacity, nor proton leak in K562-STI vs. K562, there remained a basal level of oxygen consumption from non-mitochondrial sources in both cell lines. Interestingly, fluorigenic and western blotting assays showed increases in NADPH oxidase (NOX) activity (1.35 fold) and p47phox (2 fold), an essential component of the NOX complex, respectively in K562-STI cells, suggesting NOX as a source of ROS in these cells. To this end, inhibition of the NOX complex with diphenyleneiodonium decreased ROS levels and Egr-1 expression by 50% in K562-STI but not K562 cells. These data suggest that K562-STI have altered regulation of Egr-1 controlled, in part, by NOX. Together, our findings suggest that targeting the transcription factor Egr-1 directly, or through the NOX complex, may be beneficial for improving outcomes for CML patients. Citation Format: Mary E. Irwin, Roxsan Manshouri, Blake Johnson, Hesham M. Amin, Joya Chandra. Targeting Egr-1 is an effective strategy for overcoming kinase inhibitor resistance in CML. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 960. doi:10.1158/1538-7445.AM2014-960

The functions of WHIRLY1 and REDOX-RESPONSIVE TRANSCRIPTION FACTOR 1 in cross tolerance responses in plants: a hypothesis

Chloroplasts are important sensors of environment change, fulfilling key roles in the regulation of plant growth and development in relation to environmental cues. Photosynthesis produces a repertoire of reductive and oxidative (redox) signals that provide information to the nucleus facilitating appropriate acclimation to a changing light environment. Redox signals are also recognized by the cellular innate immune system allowing activation of non-specific, stress-responsive pathways that underpin cross tolerance to biotic-abiotic stresses. While these pathways have been intensively studied in recent years, little is known about the different components that mediate chloroplast-to-nucleus signalling and facilitate cross tolerance phenomena. Here, we consider the properties of the WHIRLY family of proteins and the REDOX-RESPONSIVE TRANSCRIPTION FACTOR 1 (RRTF1) in relation to chloroplast redox signals that facilitate the synergistic co-activation of gene expression pathways and confer cross tolerance to abiotic and biotic stresses. We propose a new hypothesis for the role of WHIRLY1 as a redox sensor in chloroplast-to-nucleus retrograde signalling leading to cross tolerance, including acclimation and immunity responses. By virtue of its association with chloroplast nucleoids and with nuclear DNA, WHIRLY1 is an attractive candidate coordinator of the expression of photosynthetic genes in the nucleus and chloroplasts. We propose that the redox state of the photosynthetic electron transport chain triggers the movement of WHIRLY1 from the chloroplasts to the nucleus, and draw parallels with the regulation of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1).

NF‐κB activation in hindlimb muscles from adult and old mice at rest and following contractile activity (LB814)

Skeletal muscle adapts following contractions by increases in reactive oxygen species (ROS) leading to the detection of activation of redox‐responsive transcription factors (TFs) such as nuclear factor kappa B (NF‐κB) by electrophoresis mobility shift assay (EMSA). This TF activation is reduced in muscles from old mice and humans1,2. Additional studies from our laboratory have demonstrated that NF‐κB binding activity of quiescent muscles from old wild‐type (WT) mice was significantly increased compared with adult WT mice1. However, the significance of these findings to changes in transcriptional processes is unclear due to the ex‐vivo nature of EMSA analyses. This study used NF‐κB1 luciferase reporter mice to determine the transcriptional effect of this NF‐κB activation in hindlimb muscles from adult and old mice at rest or following an isometric contraction protocol 1 using immunofluorescence antibody staining of luciferase in transverse frozen sections of tibialis anterior (TA) muscles. Muscles were also examined for evidence of changes in the expression of cytokines known to be under the transcriptional control of NF‐κB. Luciferase staining was observed in all muscle sections with a patchwork distribution between fibres, showing as more intense overall staining in the quiescent muscles from old compared with adult mice. Immediately following the contraction protocol, the luciferase staining was increased in muscles from adult NF‐κB1 mice. In contrast, no increase was seen in the muscles from old mice following contractions compared with quiescent muscle from old mice. The chronic activation of NF‐κB was associated with increased muscle mRNA levels of a number of cytokines. These data confirm previous EMSA observations and suggest that the chronic activation of NF‐κB in muscle from old mice is associated with the generation of a pro‐inflammatory environment and this may restrict the ability in muscles of old mice to further adapt following contractile activity.Grant Funding Source: Supported by BBSRC and NIA/NIH (AG‐020591)