Specific Gene Expression Responses Research Articles

Interaction of acetamiprid, Varroa destructor, and Nosema ceranae in honey bees

Health of honey bees is threatened by a variety of stressors, including pesticides and parasites. Here, we investigated effects of acetamiprid, Varroa destructor, and Nosema ceranae, which act either alone or in combination. Our results suggested that interaction between the three factors was additive, with survival risk increasing as the number of stressors increased. Although exposure to 150 μg/L acetamiprid alone did not negatively impact honey bee survival, it caused severe damage to midgut tissue. Among the three stressors, V. destructor posed the greatest threat to honey bee survival, and N. ceranae exacerbated intestinal damage and increased thickness of the midgut wall. Transcriptomic analysis indicated that different combinations of stressors elicited specific gene expression responses in honey bees, and genes involved in energy metabolism, immunity, and detoxification were altered in response to multiple stressor combinations. Additionally, genes associated with Toll and Imd signalling, tyrosine metabolism, and phototransduction pathway were significantly suppressed in response to different combinations of multiple stressors. This study enhances our understanding of the adaptation mechanisms to multiple stressors and aids in development of suitable protective measures for honey bees. Environmental ImplicationWe believe our study is environmentally relevant for the following reasons: This study investigates combined effects of pesticide, Varroa destructor, and Nosema ceranae. These stressors are known to pose a threat to long-term survival of honey bees (Apis mellifera) and stability of the ecosystems. The research provides valuable insights into the adaptive mechanisms of honey bees in response to multiple stressors and developing effective conservation strategies. Further research can identify traits that promote honey bee survival in the face of future challenges from multiple stressors to maintain the overall stability of environment.

AP-1 subunits converge promiscuously at enhancers to potentiate transcription.

The AP-1 transcription factor (TF) dimer contributes to many biological processes and environmental responses. AP-1 can be composed of many interchangeable subunits. Unambiguously determining the binding locations of these subunits in the human genome is challenging because of variable antibody specificity and affinity. Here, we definitively establish the genome-wide binding patterns of five AP-1 subunits by using CRISPR to introduce a common antibody tag on each subunit. We find limited evidence for strong dimerization preferences between subunits at steady state and find that, under a stimulus, dimerization patterns reflect changes in the transcriptome. Further, our analysis suggests that canonical AP-1 motifs indiscriminately recruit all AP-1 subunits to genomic sites, which we term AP-1 hotspots. We find that AP-1 hotspots are predictive of cell type–specific gene expression and of genomic responses to glucocorticoid signaling (more so than super-enhancers) and are significantly enriched in disease-associated genetic variants. Together, these results support a model where promiscuous binding of many AP-1 subunits to the same genomic location play a key role in regulating cell type–specific gene expression and environmental responses.

Quantification and comparison of gene expression associated with iron regulation and metabolism in a virulent and attenuated strain of Flavobacterium psychrophilum.

Iron is essential for growth and virulence in most pathogenic bacterial strains. In some cases, the hosts for these pathogenic bacteria develop specialized strategies to sequester iron and limit infectivity. This in turn may result in the invading pathogens utilizing high-affinity iron transport mechanisms, such as the use of iron-chelating siderophores, to extend beyond the host defences. Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD) in salmonids, relies on iron metabolism for infectivity, and the genome of the model CSF-259-93 strain has recently been made available. Further, this strain serves as a parent strain for a live-attenuated vaccine strain, B.17, which has been shown to provide rainbow trout with protection against BCWD. To elucidate specific gene expression responses to iron metabolism and compare strain differences, both F.psychrophilum strains were grown under iron-limiting conditions and 26 genes related to iron metabolism were mapped for 96hr in culture via qPCR analyses. Results indicate increased production of the ferrous iron transport protein B (FITB; p=.008), and ferric receptor CfrA (FR 1; p=.012) in the wild-type CSF-259-93 strain at 72hr and 96hr post-exposure to iron-limiting media. In the B.17 vaccine strain, siderophore synthase (SS) expression was found to be downregulated at 72hr, in comparison with 0h (p=.018). When strains were compared, FITB (p=.021), FR1 (p=.009) and SS (p=.016) were also elevated in B.17 at 0hr and TonB outer protein membrane receptor 1 (TBomr1; p=.005) had a lower expression at 96hr. Overall, this study demonstrated strain-related gene expression changes in only a fraction of the iron metabolism genes tested; however, results provide insight on potential virulence mechanisms and clarification on iron-related gene expression for F.psychrophilum.

Cultivation techniques to study lanthanide metal interactions in the haloalkaliphilic Type I methanotroph "Methylotuvimicrobium buryatense" 5GB1C.

Lanthanide metals are commonly used in technological devices including batteries, computers, catalysts and magnets. Despite their important properties, mining difficulties and pollution concerns limit the number of mines worldwide. Because of these concerns, biometallurgy is an attractive possibility for lanthanide extraction from recycled materials or from contaminated sites. Methylotrophs, bacteria that grow on reduced carbon substrates like methane and methanol, utilize lanthanides for a central reaction in their metabolisms. They must have some mechanism for uptake or trafficking, and are therefore excellent candidates for applying small molecules or proteins for selective lanthanide metal recycling. The haloalkaliphilic methanotroph "Methylotuvimicrobium buryatense" 5GB1C is the fastest growing methanotroph isolated to date, and thus has great industrial potential. The MxaFI enzyme complex uses calcium as a Lewis acid in conjunction with the pyroquinoline quinone cofactor to oxidize methanol, while the alternative enzyme XoxF uses lanthanide metals (e.g. lanthanum and cerium) for the same function. Lanthanide metals, abundant in the earth's crust, strongly repress the transcription of mxaF yet activate the transcription of xoxF, implying that XoxF may be the predominant methanol dehydrogenase in the bacterium's native environment. It may be that lanthanum interaction mechanisms are different from those in other microorganisms. In addition, the facile genetics in this strain and existing background information make it a good study organism for biological lanthanum uptake. The interesting physiology of this organism required empirical work to develop cultivation methods that allow robust assays of gene expression and measurement of lanthanum associated with cell biomass. In this chapter, we show that altering the metal chelator increased the availability of lanthanum to the cell as measured by the specific gene expression response. We also made further alterations to prevent lanthanum precipitation in medium for the growth of haloalkaliphiles.

Design Principle for Decoding Calcium Signals to Generate Specific Gene Expression Via Transcription.

The second messenger calcium plays a key role in conveying specificity of signaling pathways in plant cells. Specific calcium signatures are decoded to generate correct gene expression responses and amplification of calcium signatures is vital to this process. (1) It is not known if this amplification is an intrinsic property of all calcium-regulated gene expression responses and whether all calcium signatures have the potential to be amplified, or (2) how a given calcium signature maintains specificity in cells containing a great number of transcription factors (TFs) and other proteins with the potential to be calcium-regulated. The work presented here uncovers the design principle by which it is possible to decode calcium signals into specific changes in gene transcription in plant cells. Regarding the first question, we found that the binding mechanism between protein components possesses an intrinsic property that will nonlinearly amplify any calcium signal. This nonlinear amplification allows plant cells to effectively distinguish the kinetics of different calcium signatures to produce specific and appropriate changes in gene expression. Regarding the second question, we found that the large number of calmodulin (CaM)-binding TFs or proteins in plant cells form a buffering system such that the concentration of an active CaM-binding TF is insensitive to the concentration of any other CaM-binding protein, thus maintaining specificity. The design principle revealed by this work can be used to explain how any CaM-binding TF decodes calcium signals to generate specific gene expression responses in plant cells via transcription.

Plant-parasitic nematodes respond to root exudate signals with host-specific gene expression patterns.

Plant parasitic nematodes must be able to locate and feed from their host in order to survive. Here we show that Pratylenchus coffeae regulates the expression of selected cell-wall degrading enzyme genes relative to the abundance of substrate in root exudates, thereby tailoring gene expression for root entry of the immediate host. The concentration of cellulose or xylan within the exudate determined the level of β-1,4-endoglucanase (Pc-eng-1) and β-1,4-endoxylanase (Pc-xyl) upregulation respectively. Treatment of P. coffeae with cellulose or xylan or with root exudates deficient in cellulose or xylan conferred a specific gene expression response of Pc-eng-1 or Pc-xyl respectively with no effect on expression of another cell wall degrading enzyme gene, a pectate lyase (Pc-pel). RNA interference confirmed the importance of regulating these genes as lowered transcript levels reduced root penetration by the nematode. Gene expression in this plant parasitic nematode is therefore influenced, in a host-specific manner, by cell wall components that are either secreted by the plant or released by degradation of root tissue. Transcriptional plasticity may have evolved as an adaptation for host recognition and increased root invasion by this polyphagous species.

Exposure to 4100 K fluorescent light elicits sex specific transcriptional responses in Xiphophorus maculatus skin

It has been reported that exposure to artificial light may affect oxygen intake, heart rate, absorption of vitamins and minerals, and behavioral responses in humans. We have reported specific gene expression responses in the skin of Xiphophorus fish after exposure to ultraviolet light (UV), as well as, both broad spectrum and narrow waveband visible light. In regard to fluorescent light (FL), we have shown that male X. maculatus exposed to 4100K FL (i.e. “cool white”) rapidly suppress transcription of many genes involved with DNA replication and repair, chromosomal segregation, and cell cycle progression in skin. We have also detailed sex specific transcriptional responses of Xiphophorus skin after exposure to UVB. However, investigation of gender differences in global gene expression response after exposure to 4100K FL has not been reported, despite common use of this FL source for residential, commercial, and animal facility illumination.Here, we compare RNA-Seq results analyzed to assess changes in the global transcription profiles of female and male X. maculatus skin in response to 4100K FL exposure. Our results suggest 4100K FL exposure incites a sex-biased genetic response including up-modulation of inflammation in females and down modulation of DNA repair/replication in males. In addition, we identify clusters of genes that become oppositely modulated in males and females after FL exposure that are principally involved in cell death and cell proliferation.

Exposure to CO2 influences metabolism, calcification and gene expression of the thecosome pteropod Limacina retroversa.

Thecosomatous pteropods, a group of aragonite shell-bearing zooplankton, are becoming an important sentinel organism for understanding the influence of ocean acidification on pelagic organisms. These animals show vulnerability to changing carbonate chemistry conditions, are geographically widespread, and are both biogeochemically and trophically important. The objective of this study was to determine how increasing duration and severity of CO2 treatment influence the physiology of the thecosome Limacina retroversa, integrating both gene expression and organism-level (respiration and calcification) metrics. We exposed pteropods to over-saturated, near-saturated or under-saturated conditions and sampled individuals at 1, 3, 7, 14 and 21 days of exposure to test for the effect of duration. We found that calcification was affected by borderline and under-saturated conditions by week two, while respiration appeared to be more strongly influenced by an interaction between severity and duration of exposure, showing complex changes by one week of exposure. The organismal metrics were corroborated by specific gene expression responses, with increased expression of biomineralization-associated genes in the medium and high treatments throughout and complex changes in metabolic genes corresponding to both captivity and CO2 treatment. Genes associated with other physiological processes such as lipid metabolism, neural function and ion pumping had complex responses, influenced by both duration and severity. Beyond these responses, our findings detail the captivity effects for these pelagic organisms, providing information to contextualize the conclusions of previous studies, and emphasizing a need for better culturing protocols.

A Period of Controlled Elevation of IOP (CEI) Produces the Specific Gene Expression Responses and Focal Injury Pattern of Experimental Rat Glaucoma.

PurposeWe determine if several hours of controlled elevation of IOP (CEI) will produce the optic nerve head (ONH) gene expression changes and optic nerve (ON) damage pattern associated with early experimental glaucoma in rats.MethodsThe anterior chambers of anesthetized rats were cannulated and connected to a reservoir to elevate IOP. Physiologic parameters were monitored. Following CEI at various recovery times, ON cross-sections were graded for axonal injury. Anterior ONHs were collected at 0 hours to 10 days following CEI and RNA extracted for quantitative PCR measurement of selected messages. The functional impact of CEI was assessed by electroretinography (ERG).ResultsDuring CEI, mean arterial pressure (99 ± 6 mm Hg) and other physiologic parameters remained stable. An 8-hour CEI at 60 mm Hg produced significant focal axonal degeneration 10 days after exposure, with superior lesions in 83% of ON. Message analysis in CEI ONH demonstrated expression responses previously identified in minimally injured ONH following chronic IOP elevation, as well as their sequential patterns. Anesthesia with cannulation at 20 mm Hg did not alter these message levels. Electroretinographic A- and B-waves, following a significant reduction at 2 days after CEI, were fully recovered at 2 weeks, while peak scotopic threshold response (pSTR) remained mildly but significantly depressed.ConclusionsA single CEI reproduces ONH message changes and patterns of ON injury previously observed with chronic IOP elevation. Controlled elevation of IOP can allow detailed determination of ONH cellular and functional responses to an injurious IOP insult and provide a platform for developing future therapeutic interventions.

Combining modelling and experimental approaches to explain how calcium signatures are decoded by calmodulin-binding transcription activators (CAMTAs) to produce specific gene expression responses.

Summary Experimental data show that Arabidopsis thaliana is able to decode different calcium signatures to produce specific gene expression responses. It is also known that calmodulin‐binding transcription activators (CAMTAs) have calmodulin (CaM)‐binding domains. Therefore, the gene expression responses regulated by CAMTAs respond to calcium signals. However, little is known about how different calcium signatures are decoded by CAMTAs to produce specific gene expression responses.A dynamic model of Ca2+–CaM–CAMTA binding and gene expression responses is developed following thermodynamic and kinetic principles. The model is parameterized using experimental data. Then it is used to analyse how different calcium signatures are decoded by CAMTAs to produce specific gene expression responses.Modelling analysis reveals that: calcium signals in the form of cytosolic calcium concentration elevations are nonlinearly amplified by binding of Ca2+, CaM and CAMTAs; amplification of Ca2+ signals enables calcium signatures to be decoded to give specific CAMTA‐regulated gene expression responses; gene expression responses to a calcium signature depend upon its history and accumulate all the information during the lifetime of the calcium signature.Information flow from calcium signatures to CAMTA‐regulated gene expression responses has been established by combining experimental data with mathematical modelling.

Specific gene expression responses to parasite genotypes reveal redundancy of innate immunity in vertebrates.

Vertebrate innate immunity is the first line of defense against an invading pathogen and has long been assumed to be largely unspecific with respect to parasite/pathogen species. However, recent phenotypic evidence suggests that immunogenetic variation, i.e. allelic variability in genes associated with the immune system, results in host-parasite genotype-by-genotype interactions and thus specific innate immune responses. Immunogenetic variation is common in all vertebrate taxa and this reflects an effective immunological function in complex environments. However, the underlying variability in host gene expression patterns as response of innate immunity to within-species genetic diversity of macroparasites in vertebrates is unknown. We hypothesized that intra-specific variation among parasite genotypes must be reflected in host gene expression patterns. Here we used high-throughput RNA-sequencing to examine the effect of parasite genotypes on gene expression patterns of a vertebrate host, the three-spined stickleback (Gasterosteus aculeatus). By infecting naïve fish with distinct trematode genotypes of the species Diplostomum pseudospathaceum we show that gene activity of innate immunity in three-spined sticklebacks depended on the identity of an infecting macroparasite genotype. In addition to a suite of genes indicative for a general response against the trematode we also find parasite-strain specific gene expression, in particular in the complement system genes, despite similar infection rates of single clone treatments. The observed discrepancy between infection rates and gene expression indicates the presence of alternative pathways which execute similar functions. This suggests that the innate immune system can induce redundant responses specific to parasite genotypes.

Identification of structural features in chemicals associated with cancer drug response: a systematic data-driven analysis.

Motivation: Analysis of relationships of drug structure to biological response is key to understanding off-target and unexpected drug effects, and for developing hypotheses on how to tailor drug therapies. New methods are required for integrated analyses of a large number of chemical features of drugs against the corresponding genome-wide responses of multiple cell models.Results: In this article, we present the first comprehensive multi-set analysis on how the chemical structure of drugs impacts on genome-wide gene expression across several cancer cell lines [Connectivity Map (CMap) database]. The task is formulated as searching for drug response components across multiple cancers to reveal shared effects of drugs and the chemical features that may be responsible. The components can be computed with an extension of a recent approach called Group Factor Analysis. We identify 11 components that link the structural descriptors of drugs with specific gene expression responses observed in the three cell lines and identify structural groups that may be responsible for the responses. Our method quantitatively outperforms the limited earlier methods on CMap and identifies both the previously reported associations and several interesting novel findings, by taking into account multiple cell lines and advanced 3D structural descriptors. The novel observations include: previously unknown similarities in the effects induced by 15-delta prostaglandin J2 and HSP90 inhibitors, which are linked to the 3D descriptors of the drugs; and the induction by simvastatin of leukemia-specific response, resembling the effects of corticosteroids.Availability and implementation: Source Code implementing the method is available at: http://research.ics.aalto.fi/mi/software/GFAsparseContact: suleiman.khan@aalto.fi or samuel.kaski@aalto.fiSupplementary Information: Supplementary data are available at Bioinformatics online.

Parent-of-origin genetic background affects the transcriptional levels of circadian and neuronal plasticity genes following sleep loss

Sleep homoeostasis refers to a process in which the propensity to sleep increases as wakefulness progresses and decreases as sleep progresses. Sleep is tightly organized around the circadian clock and is regulated by genetic and epigenetic mechanisms. The homoeostatic response of sleep, which is classically triggered by sleep deprivation, is generally measured as a rebound effect of electrophysiological measures, for example delta sleep. However, more recently, gene expression changes following sleep loss have been investigated as biomarkers of sleep homoeostasis. The genetic background of an individual may affect this sleep-dependent gene expression phenotype. In this study, we investigated whether parental genetic background differentially modulates the expression of genes following sleep loss. We tested the progeny of reciprocal crosses of AKR/J and DBA/2J mouse strains and we show a parent-of-origin effect on the expression of circadian, sleep and neuronal plasticity genes following sleep deprivation. Thus, we further explored, by in silico, specific functions or upstream mechanisms of regulation and we observed that several upstream mechanisms involving signalling pathways (i.e. DICER1, PKA), growth factors (CSF3 and BDNF) and transcriptional regulators (EGR2 and ELK4) may be differentially modulated by parental effects. This is the first report showing that a behavioural manipulation (e.g. sleep deprivation) in adult animals triggers specific gene expression responses according to parent-of-origin genomic mechanisms. Our study suggests that the same mechanism may be extended to other behavioural domains and that the investigation of gene expression following experimental manipulations should take seriously into account parent-of-origin effects.

Production, Detection, and Signaling of Singlet Oxygen in Photosynthetic Organisms

In photosynthetic organisms, excited chlorophylls (Chl) can stimulate the formation of singlet oxygen ((1)O(2)), a highly toxic molecule that acts in addition to its damaging nature as an important signaling molecule. Thus, due to this dual role of (1)O(2), its production and detoxification have to be strictly controlled. Regulation of pigment synthesis is essential to control (1)O(2) production, and several components of the Chl synthesis and pigment insertion machineries to assemble and disassemble protein/pigment complexes have recently been identified. Once produced, (1)O(2) activates a signaling cascade from the chloroplast to the nucleus that can involve multiple mechanisms and stimulate a specific gene expression response. Further, (1)O(2) signaling was shown to interact with signal cascades of other reactive oxygen species, oxidized carotenoids, and lipid hydroperoxide-derived reactive electrophile species. Despite recent progresses, hardly anything is known about how and where the (1)O(2) signal is sensed and transmitted to the cytoplasm. One reason for that is the limitation of available detection methods challenging the reliable quantification and localization of (1)O(2) in plant cells. In addition, the process of Chl insertion into the reaction centers and antenna complexes is still unclear. Unraveling the mechanisms controlling (1)O(2) production and signaling would help clarifying the specific role of (1)O(2) in cellular stress responses. It would further enable to investigate the interaction and sensitivity to other abiotic and biotic stress signals and thus allow to better understand why some stressors activate an acclimation, while others provoke a programmed cell death response.

A gene expression profile indicative of early stage HER2 tyrosine kinase inhibitor response.

e11536 Background: Lapatinib, afatinib and neratinib are tyrosine kinase inhibitors (TKIs) of HER2 and EGFR growth receptors. A panel of breast cancer cell lines was treated with these agents and gefintib (EGFR inhibitor) and the expression pattern of a specific panel of genes investigated as a potential marker of early drug response. Methods: RNA was extracted from breast cancer cell lines (BT474, SKBR3 and MDAMB453) with differing HER2 expression patterns and sensitivity to lapatinib before and 12hrs after treatment with 1 µM of lapatinib, 150nM of afatinib, 150nM of neratinib or 1µM of gefitinib. Gene expression changes were measured by Taqman RT-PCR and RQ values were determined using the comparative cycle threshold (Ct) method. Results: The expression of RB1CC1, ERBB3, FOXO3a, NR3C1 was directly correlated with the degree of sensitivity of the cell line to lapatinib and was observed to “switch” from up-regulated to down-regulated in the HER2 expressing lapatinib insensitive cell line (MDAMD453). The CCND1 gene (functionally linked to the other four genes) demonstrated an inversely proportional response to drug exposure; showing a trend of strong down-regulation in lapatinib-sensitive lines. A similar expression pattern was observed following the treatment with both neratinib and afatinib. In contrast, gefitinib treatment, resulted in a completely different expression pattern change. Conclusions: In these HER2-expressing cell models, lapatinib, neratinib and afatinib treatment generated a common, characteristic and specific gene expression response, proportionate to the sensitivity of the cell lines to the HER2 inhibitor. Characterisation of changes in these genes shortly after drug treatment may therefore give a valuable predictor of the likely extent and specificity of tumour HER2 inhibitor response in patients, potentially guiding more specific use of these agents.

A gene expression profile indicative of early stage HER2 targeted therapy response

BackgroundEfficacious application of HER2-targetting agents requires the identification of novel predictive biomarkers. Lapatinib, afatinib and neratinib are tyrosine kinase inhibitors (TKIs) of HER2 and EGFR growth factor receptors. A panel of breast cancer cell lines was treated with these agents, trastuzumab, gefitinib and cytotoxic therapies and the expression pattern of a specific panel of genes using RT-PCR was investigated as a potential marker of early drug response to HER2-targeting therapies.ResultsTreatment of HER2 TKI-sensitive SKBR3 and BT474 cell lines with lapatinib, afatinib and neratinib induced an increase in the expression of RB1CC1, ERBB3, FOXO3a and NR3C1. The response directly correlated with the degree of sensitivity. This expression pattern switched from up-regulated to down-regulated in the HER2 expressing, HER2-TKI insensitive cell line MDAMB453. Expression of the CCND1 gene demonstrated an inversely proportional response to drug exposure. A similar expression pattern was observed following the treatment with both neratinib and afatinib. These patterns were retained following exposure to traztuzumab and lapatinib plus capecitabine. In contrast, gefitinib, dasatinib and epirubicin treatment resulted in a completely different expression pattern change.ConclusionsIn these HER2-expressing cell line models, lapatinib, neratinib, afatinib and trastuzumab treatment generated a characteristic and specific gene expression response, proportionate to the sensitivity of the cell lines to the HER2 inhibitor.Characterisation of the induced changes in expression levels of these genes may therefore give a valuable, very early predictor of the likely extent and specificity of tumour HER2 inhibitor response in patients, potentially guiding more specific use of these agents.

Elevated Cyclin G2 Expression Intersects with DNA Damage Checkpoint Signaling and Is Required for a Potent G2/M Checkpoint Arrest Response to Doxorubicin

To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G(1)/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G(2)/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.

Speed, Sensitivity, and Bistability in Auto-activating Signaling Circuits

Cells employ a myriad of signaling circuits to detect environmental signals and drive specific gene expression responses. A common motif in these circuits is inducible auto-activation: a transcription factor that activates its own transcription upon activation by a ligand or by post-transcriptional modification. Examples range from the two-component signaling systems in bacteria and plants to the genetic circuits of animal viruses such as HIV. We here present a theoretical study of such circuits, based on analytical calculations, numerical computations, and simulation. Our results reveal several surprising characteristics. They show that auto-activation can drastically enhance the sensitivity of the circuit's response to input signals: even without molecular cooperativity, an ultra-sensitive threshold response can be obtained. However, the increased sensitivity comes at a cost: auto-activation tends to severely slow down the speed of induction, a stochastic effect that was strongly underestimated by earlier deterministic models. This slow-induction effect again requires no molecular cooperativity and is intimately related to the bimodality recently observed in non-cooperative auto-activation circuits. These phenomena pose strong constraints on the use of auto-activation in signaling networks. To achieve both a high sensitivity and a rapid induction, an inducible auto-activation circuit is predicted to acquire low cooperativity and low fold-induction. Examples from Escherichia coli's two-component signaling systems support these predictions.

BZL101, a phytochemical extract from the Scutellaria barbata plant, disrupts proliferation of human breast and prostate cancer cells through distinct mechanisms dependent on the cancer cell phenotype

BZL101 is an aqueous extract from the Scutellaria barbata plant shown to have anticancer properties in a variety of human cancers. In order to determine its efficacy on human reproductive cancers, we assessed the responses of two human breast cancer cell lines, estrogen sensitive MCF7 and estrogen insensitive MDA-MB-231, and of two human prostate cancer cell lines, androgen sensitive LNCaP and androgen insensitive PC3 which are human cell lines that represent early and late stage reproductive cancers. BZL101 inhibited reproductive cancer growth in all cell lines by regulating expression levels of key cell cycle components that differ with respect to the cancer cell phenotypes. In early stage estrogen sensitive MCF7 cells, BZL101 induced a G1 cell cycle arrest and ablated expression of key G1 cell cycle regulators Cyclin D1, CDK2, and CDK4, as well as growth factor stimulatory pathways and estrogen receptor-α expression. Transfection of luciferase reporter plasmids revealed that the loss of CDK2, CDK4 and estrogen receptor-α transcript expression resulted from the BZL-dependent ablation of promoter activities. BZL101 growth arrests early stage androgen sensitive LNCaP cells in the G2/M phase with corresponding decreases in Cyclin B1, CDK1, and androgen receptor expression. In late stage hormone insensitive breast (MDA-MB-231) and prostate (PC3) cancer cells, BZL101 induced an S phase arrest with corresponding ablations in Cyclin A2 and CDK2 expression. Our results demonstrate that BZL101 exerts phenotype specific anti-proliferative gene expression responses in human breast and prostate cancer cells, which will be valuable in the potential development of BZL-based therapeutic strategies for human reproductive cancers.

Abstract SY15-04: Systematic reconstruction of regulatory networks in mammalian cells: Lessons from the pathogen response

Abstract Deciphering the regulatory networks that control dynamic and specific gene expression responses in mammalian cells remains a major challenge. While models inferred from genomic data have identified candidate regulatory mechanisms, such models remain largely unvalidated. Here, we present an unbiased strategy based on systematic gene perturbation and innovative multiplex detection to derive regulatory networks in mammalian cells. We apply this approach to decipher the network that controls the transcriptional response to pathogens in primary dendritic cells (DCs), testing the regulatory function of 125 transcription factors, chromatin modifiers, and RNA binding proteins. Our approach accurately assigned 32 known regulators (e.g. NFκB, IRFs, and STATs) to their target genes and discovered 68 additional functional regulators that were not previously implicated in this response. We quantify the contribution of each regulator to two major transcriptional programs (inflammatory and antiviral), identifying a core network of two dozen key regulators and 76 fine-tuners, that uses a combination of coherent feed-forward circuits, dominant activation, and cross-inhibition to control response specificity. Among these we discover a tier of chromatin modifiers that specifically repress interferon beta 1 (IFNβ1) expression upon bacterial but not viral stimulation, and a large circuit of cell cycle regulators that was co-opted to regulate the viral response. Our work establishes a broadly-applicable, comprehensive and unbiased approach to identifying the wiring and function of a regulatory network controlling a major transcriptional response in primary mammalian cells. Citation Format: Aviv Regev. Systematic reconstruction of regulatory networks in mammalian cells: Lessons from the pathogen response [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY15-04