Methylmercury Activates Enhancer-of-Split and Bearded Complex Genes Independent of the Notch Receptor

Toxicological Sciences, Jul 2008

Methylmercury (MeHg) is a persistent environmental toxin that has targeted effects on fetal neural development. Although a number of cytotoxic mechanisms of MeHg have been characterized in cultured cells, its mode of action in the developing nervous system in vivo is less clear. Studies of MeHg-affected rodent and human brains show disrupted cortical and cerebellar architecture suggestive of mechanisms that augment cell signaling pathways potentially affecting cell migration and proliferation. We previously identified the Notch receptor pathway, a highly conserved signaling mechanism fundamental for neural development, as a target for MeHg-induced signaling in Drosophila neural cell lines. Here we have expanded our use of the Drosophila model to resolve a broader spectrum of transcriptional changes resulting from MeHg exposure in vivo and in vitro. Several Notch target genes within the Enhancer-of-split (E(spl)C) and Bearded (BrdC) complexes are upregulated with MeHg exposure in the embryo and in cultured neural cells. However, the profile of MeHg-induced E(spl)C and BrdC gene expression differs significantly from that seen with activation of the Notch receptor. Targeted knockdown of Notch and of the downstream coactivator Suppressor of Hairless (Su(H)), shows no effect on MeHg-induced transcription, indicating a novel Notch-independent mechanism of action for MeHg. MeHg transcriptional activation is partially mimicked by iodoacetamide but not by N-ethylmaleimide, two thiol-specific electrophiles, revealing a degree of specificity of cellular thiol targets in MeHg-induced transcriptional events.

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Methylmercury Activates Enhancer-of-Split and Bearded Complex Genes Independent of the Notch Receptor

Advance Access publication March Methylmercury Activates Enhancer-of-Split and Bearded Complex Genes Independent of the Notch Receptor Matthew D. Rand 1 2 Christin E. Bland 1 2 Jeffrey Bond 0 1 0 Department of Microbiology and Molecular Genetics , Bioinformatics Core , College of Medicine, University of Vermont , Vermont 05405 , USA 1 The Author 2008. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved 2 Department of Anatomy and Neurobiology, College of Medicine, University of Vermont , USA Methylmercury (MeHg) is a persistent environmental toxin that has targeted effects on fetal neural development. Although a number of cytotoxic mechanisms of MeHg have been characterized in cultured cells, its mode of action in the developing nervous system in vivo is less clear. Studies of MeHg-affected rodent and human brains show disrupted cortical and cerebellar architecture suggestive of mechanisms that augment cell signaling pathways potentially affecting cell migration and proliferation. We previously identified the Notch receptor pathway, a highly conserved signaling mechanism fundamental for neural development, as a target for MeHg-induced signaling in Drosophila neural cell lines. Here we have expanded our use of the Drosophila model to resolve a broader spectrum of transcriptional changes resulting from MeHg exposure in vivo and in vitro. Several Notch target genes within the Enhancer-of-split (E(spl)C) and Bearded (BrdC) complexes are upregulated with MeHg exposure in the embryo and in cultured neural cells. However, the profile of MeHg-induced E(spl)C and BrdC gene expression differs significantly from that seen with activation of the Notch receptor. Targeted knockdown of Notch and of the downstream coactivator Suppressor of Hairless (Su(H)), shows no effect on MeHg-induced transcription, indicating a novel Notch-independent mechanism of action for MeHg. MeHg transcriptional activation is partially mimicked by iodoacetamide but not by N-ethylmaleimide, two thiol-specific electrophiles, revealing a degree of specificity of cellular thiol targets in MeHginduced transcriptional events. - Methylmercury (MeHg) is a persistent environmental toxin that targets the developing nervous system of the fetus and of young children. Cytotoxicity of MeHg stems from its high affinity for cysteine thiol groups on proteins (Hughes, 1957) . A number of studies in cultured cells and in vivo have documented effects of MeHg on fundamental cellular processes including intracellular calcium ion homeostasis (Sirois and Atchison, 2000) , protein phosphorylation (Parran et al., 2003), generation of reactive oxygen species (ROS) (Sarafian, 1999) , cell cycle control (Ponce et al., 1994) , induction of apoptosis (Wilke et al., 2003) and disruption of protein synthesis and microtubule assembly (Cheung and Verity, 1985; Miura et al., 2000) . These pleiotropic effects of MeHg reflect its ability to interact with a number of essential proteins that are pivotal to the above-mentioned cellular processes. Less clear are mechanisms of MeHg toxicity that are specific to developing neural tissues in vivo. Fetal Minimata Disease, a condition resulting from high-level MeHg exposure akin to the catastrophic exposures that occurred in Minimata, Japan and Iraq, is characterized by atrophy and disorganization of cortical layers, loss of cells in the cerebellum and cerebrum, and ectopic cells in the white matter of the cortices in human brains, as well as in primate and rodent brains (Choi et al., 1978; Kakita et al., 2002; Peckham and Choi, 1988) . It is of note that cell death is not a hallmark of prenatal MeHg exposure and that aberrant cell migration is thought to contribute to the abnormal patterning of cellular layers (Choi et al., 1978) . Thus, the neuropathology is consistent with the notion that MeHg can redirect the program of normal neural development, presumably through altering cell–cell signaling events. In support of this hypothesis, studies in embryonic carcinoma cells demonstrate MeHg-induced changes in expression levels of Eph and Ephrin, a receptor–ligand signaling pair important for axonal guidance (Wilson et al., 2005) . In addition, MeHg alters neurotrophin signaling through the TrkA receptor in the neuronal PC12 cell line (Parran et al., 2004) and in developing rat brains, which may parallel disruption of cortical lamina (Barone et al., 1998) . Aside from these examples, our understanding of the signaling pathways targeted by MeHg during neural development is incomplete. In an effort to identify the fundamental developmental signaling pathways targeted by MeHg, we have begun to exploit Drosophila as a model. Using Drosophila-derived neural cell lines, we previously identified the Notch receptor pathway as a target for MeHg-induced signaling (Bland and Rand, 2006) . Notch signals are a means of cell–cell communication used to orchestrate cell fate decisions and early morphogenic activities of neural cells (Artavanis-Tsakonas et al., 1999; Skeath and Doe, 1998; Udolph et al., 2001) . Upon stimulation by its ligand, Delta, the Notch receptor is activated by sequential cleavages resulting in an intracellular domain product that signals in the nucleus (Kopan et al., 1996; Struhl and Adachi, 1998) . Nuclear Notch, in complex with the Suppressor of Hairless (Su(H)) transcription factor, upregulates transcription of genes in the Enhancer of Split complex (E(spl)C) and Bearded complex (BrdC) (Lai et al., 2000; Wurmbach et al., 1999) . The E(spl)C contains 13 genes encoding three types of proteins: m3, m5, m7, m8, mb, md, mc encode basic helix-loop-helix (bHLH) transcriptional repressor proteins; ma, m2, m4, and m6 encode Bearded family-related proteins; and m1 encodes a putative protease inhibitor (Delidakis and Artavanis-Tsakonas, 1992; Klambt et al., 1989; Wurmbach et al., 1999) . The Bearded complex encodes six proteins: Bearded (Brd), twin of m4 (Tom), Ocho, and Brother of Bearded (Bob) A, B and C genes (Lai et al., 2000; Leviten et al., 1997) . Brd proteins are small basic amphipathic helical proteins that are antagonistic toward Notch signaling (Apidianakis et al., 1999; Bardin and Schweisguth, 2006; Lai et al., 2000) . Notch activity induces expression of these target genes in several developmental contexts to exert control of cell fate decisions, most notably in the embryonic neurectoderm (Bardin and Schweisguth, 2006; Zaffran and Frasch, 2000) . In a previous study we demonstrated that MeHg promotes upregulation of E(spl)mc and mb genes in several Drosophila cell lines, suggesting that MeHg activates Notch signaling (Bland and Rand, 2006) . We also identified a potential role for MeHg in promoting Notch receptor cleavage, consistent with the current model for activation of the receptor (Bland and Rand, 2006) . In this study we expand our use of Drosophila to further characterize MeHg influence on Notch signaling. Using microarray analysis of embryonic transcripts and quantitative real-time PCR (qPCR) analysis of neuronal cell line messenger RNA (mRNA), we show an increased expression of several E(spl)C and BrdC target genes in response to MeHg exposure. Unexpectedly, we find that activation of E(spl)C and BrdC target genes by MeHg does not require the Notch receptor or the downstream transcriptional coactivator Su(H). Furthermore, MeHg activation of E(spl)C and BrdC target genes is partially mimicked with the thiol-alkylating reagent iodoacetamide (IA), whereas a second thiol-specific reagent, N-ethylmaleimide (NEM), fails to induce Notch target genes. Our results support the hypothesis that MeHg acts through specific thiol-containing protein targets to regulate transcriptional activation of E(spl)C and BrdC genes. METHODS Fly stocks and MeHg treatments. All treatments and assays were performed with the standard laboratory wild type Canton S strain. MeHg was administered through additions to food preparations. Food consisted of a cornmeal, molasses, and agar mixture (Jazz Mix AS153, Fisher Scientific), prepared in batches and distributed in culture vials or in bottles. Methylmercury chloride (Aldrich 442534) was prepared as a 50mM stock solution in dimethyl sulfoxide (DMSO) and was added to the warm food mixture before it solidified. For embryo exposures a mating population of > 300 flies were starved overnight and then fed on control (DMSO) or 5–20lM MeHg food for 5 days. Embryos were collected in population cages on grape juice-agar plates with yeast paste and developmentally staged by aging for appropriate times to allow nervous system development (e.g., 18 h at 18 C for stage 14) prior to fixation for immunostaining or lysis for RNA isolation or total mercury analyses. Total mercury determination and development assays. Larval hatching rates were determined on embryos from control- and MeHg-treated flies. Embryos were transferred to a new grape juice plate in batches of 50. Hatching of first instar larvae was determined manually under a stereo dissecting microscope 24 h after transfer of embryos to the new plate. For mercury determinations, pooled embryos (> 50 embryos per sample) were solubilized with 50 ll of tetramethyl ammonium hydroxide (Alfa Aesar, Ward Hill, MA) and total mercury in each tube was determined by a combustion-trapping-atomic absorption technique (EPA, 1998) by the Trace Element Research Laboratory (Texas A&M, College Station, TX). Samples were analyzed with a Milestone DMA 80 Hg analyzer for quantification of Hg over the range 0.001-0.700 lg. Instrument calibration utilized dry certified reference materials as standards. Laboratory quality control samples included a method blank and certified reference materials (National Research Council Canada DOLT-2 and DORM-2). The method blank was below the detection limit of 0.00004 lg Hg, and the reference material recoveries were 107 and 99% of the certified values, respectively. Immunostaining. Embryos were dechorionated with 2% bleach for 3 min, rinsed thoroughly, and fixed in 4% paraformaldehyde 50% heptane by vigorous shaking for 18 min. Vitelline membranes were removed by manually shaking in methanol:heptane 50:50 and washed and stored at 4 C in methanol until staining. For immunostaining, embryos were permeablized in 0.5% triton X-100 in phosphate buffered saline (PBST). Subsequent blocking, primary and secondary antibody incubations were done in PBST with 5% normal donkey serum. Primary antibody was rabbit anti-horseradish peroxidase (Jackson Labs, West Grove, PA) and secondary antibody was horseradish peroxidase (HRP)– conjugated anti-rabbit (ECL, GE Healthcare, Piscataway, NJ) for diaminobenzidine substrate detection using a standard protocol. Embryos were visualized by brightfield microscopy methods and recorded with digital imaging using a Spot One camera and software (MVI, Avon, MA). RNA isolation and microarray analyses. Total RNA was isolated from dechorionated embryos pooled and homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA). RNA was DNase treated (Ambion, Austin, TX) and processed for probe generation and hybridization to ‘‘Affy’’ GeneChips, Drosophila Version 2, by the National Institutes of Health Neuroscience Microarray Consortium (NIH NMC). Data sets are posted at the NIH NMC data repository (http://arrayconsortium.tgen.org/np2/home.do). Affymetrix data set analyses were performed using tools made available through the Bioconductor Project (Balasubramanian et al., 2004) . Probe set statistics were calculated from probe intensities (.CEL files) using the Affymetrix package (RMA; Bolstad et al., 2003) background correction, qspline normalization (Workman et al., 2002) , and median polish summary statistic RMA; Bolstad et al., 2003). A linear model (Seber, 1977) of the RMA-like probe set expression statistic that included batch and MeHg exposure factors was used to calculate the response associated with the treatment, M, as well a p value, p, reflecting the probability of the response under the null hypothesis. It is generally recognized (Yang and Speed, 2003) that for small sample sizes, it is not advisable to partition genes based exclusively on either M (because different probe sets exhibit different variances) or p (because of the high false discovery rate with small sample sizes). Instead it has proven useful to use both of these differential expression statistics simultaneously; for example, via Boolean filters or Volcano plots. We identified the 5% most differentially expressed genes based on the joint distribution of M and log(p) under the null hypothesis, obtained by (1) permuting sample labels, (2) binning the results, and (3) contouring the resulting density (see Fig. 2D). Gene Ontology Annotation (GOA, Camon et al., 2004) , the DAVID 2.0 functional annotation tool (http://david.abcc.ncifcrf.gov/home.jsp) (Dennis et al., 2003) , and Gentleman’s GOHyperG (Falcon and Gentleman, 2007) procedure were used to identify probe sets associated with a biological process, molecular function, or cellular component. Variability was observed in the signal intensity for individual probe sets among replicate microarray data sets. Of the 10 Notch-related genes that were upregulated with MeHg only four (m4, ma, mc, and m8) gave p values < 0.05 among the three replicate Genechip data sets. For example, where E(spl)mc gave replicates of 2.05-, 2.03-, and 1.31-fold increase and p ¼ 0.021, BobA showed high variability with replicates of 6.98-, 1.62-, and 1.23-fold increase with p ¼ 0.94. Statistical significance was therefore obtained through combined analyses of M and p values and annotation based analyses using GO terms as explained and justified above. It is likely the variability in this system comes from the developmental complexity and dynamics of mRNA expression in the tissue source (i.e. the whole embryo). In addition, the point at which MeHg accumulates to a toxic threshold during embryo development is likely to vary from treatment to treatment and among embryos of a single treatment. This notion is supported by the observation that a variety of neural phenotypes result within a single treatment population of MeHg-exposed embryos (see Fig. 1C). Cell culture and treatments. The Drosophila central nervous system (CNS) cell line ML-DmBG2-C6 (C6) (Ui et al., 1994; Ui-Tei et al., 1995) (available at the Drosophila Genome Resource Center) were routinely maintained at 23–25 C in Sang’s M3 medium (JRH Biosciences, Lenexa, KS) containing 12.5% fetal calf serum (Atlanta Biologicals, Lawrenceville, GA), 10 lg/ml insulin, bactopeptone (2.5 g/l) and yeastolate (1 g/l) (13 BPYE) supplement (Difco, Sparks, MD), 100 units/ml penicillin, and 100 lg/ml streptomycin (Invitrogen, Carlsbad, CA). Cells were grown to 60–90% confluence in M3 medium with serum. Media was removed, and cells were washed with serum-free M3 medium. Cells were then cultured in serum-free M3 media with various concentrations of MeHg or DMSO-solvent control, for 0–18 h as indicated. Dose selection was based on our earlier observations of the effectiveness of 10lM MeHg in the C6 cell line (Bland and Rand, 2006) . A number of in vitro studies using insect or mammalian cells have investigated MeHg effects at 0.1–50lM concentration, which show a wide variety of effects with respect to the toxic endpoints in question and durations of exposure (Braeckman et al., 1997; Morken et al., 2005; Sanfeliu et al., 2001; Shanker et al., 2004) , Although difficult to extrapolate our dose levels in Drosophila to a relevant dosage in affected human brains, concentrations of 1–10 ppm (~5– 50lM on a wet weight basis) MeHg have been documented in human brains showing definitive morphological abnormalities (Choi et al., 1978). Cells were harvested by direct lysis in TRIzol reagent and subsequently processed for RNA isolation. Notch activation in C6 cells was stimulated directly by exposure to ethylenediaminetetraacetic acid (EDTA) (Krejci and Bray, 2007; Rand et al., 2000) . Cells were treated with a 5-min pulse of 5mM EDTA in PBS buffer followed 1-h incubation in complete M3 medium. Cell were harvested by direct lysis in TRIzol reagent and subsequently processed for RNA isolation and qPCR analyses. Stock solutions (100mM) of IA (Biorad #163-2109, Hercules, CA) and NEM (Calbiochem #34115, Gibbstown, NJ) were freshly prepared in water just prior to use. Cell treatments were carried out in Robb’s Drosophila PBS (DPBS) (2mM Na2HPO4, 0.35mM KH2PO4, 50mM NaCl, 40mM KCl, 0.5mM CaCl2, 1.25mM MgCl2, 1.25mM MgSO4, 50mM sucrose, 5mM glucose), at pH8.3 for IA and pH6.8 for NEM. Cells were washed twice with Robb’s DPBS and incubated for 1 h with various concentrations of IA or NEM, followed by 2-h incubation in complete M3 medium. Cell were harvested in TRIzol reagent and subsequently processed for RNA isolation and qPCR analyses. RNA interference (RNAi) used to knockdown expression of Notch and Su(H) was done essentially as described previously (Bland and Rand, 2006; Clemens et al., 2000) . For Su(H) RNAi double stranded RNA encompassing the 5# coding region (bp 1067–1579) was synthesized from a PCR DNA FIG. 1. Mercury accumulation, larval hatching and nervous system defects in maternally dosed embryos. (A) Total mercury in embryos from female flies fed the indicated concentration of MeHg-containing food. (B) Percent of embryos hatching to larva subsequent to feeding of female flies on indicated concentration of MeHg. Total number of embryos counted for each determination is indicated above each bar. (C) Immunostaining with anti-HRP reveals all neural cells in the ventral nerve cord (VNC) of embryos. (I–III) Stage 16 (18 h) embryos from wild type Canton S females fed on MeHgcontaining food (20lM) for 5 days prior to embryo collection. Arrows in III denote regions devoid of neural cells. Control embryos (Cont.), show a regular segmental pattern of condensed neurons in the VNC. The ‘‘ladder-like’’ formation of the longitudinal tracts and anterior and posterior commissural bundles (‘‘rungs’’) is clearly developed. See text for discussion. template with terminal T7 sequence amended primers. Primer design was assisted by reference to the GenomeRNAi web site (http://www.dkfz.de/ signaling2/rnai/) and utilized probe ID HFA03445. Quantitative PCR. qPCR was done using SYBR-Green JumpStart Taq ready mix (Sigma, St Louis, MO) on an ABI PRISM 7500 Fast Sequence Detection System. Primer sequences for E(spl) m1, m2, m3, m7, mb, mc, md, BobA, Brd, Notch, Su(H), and the RP49 control gene can be found in supplemental data. Primers for E(spl)m1, m2, m3, m7, and md are derived from Krejci and Bray (2007) . The linear response of amplification with each primer set was validated using template dilutions, and single product amplification was determined by gel analysis and melt curve determination. Gene expression levels were determined by the comparative CT method (Livak and Schmittgen, 2001) and compared between control- and MeHg-treated samples. Western blotting. Cell lysates were prepared and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene flouride PVDF membranes for Western blot analyses by standard protocols and as previously described (Bland and Rand, 2006) . Antibodies used included: monoclonal C17-9C6 anti-dNotch intracellular domain (gift from Spyros Artavanis-Tsakonas, Harvard Medical School, also available at the Developmental studies hybridoma-bank, http://dsub. biology.uiowa.edu/, and polyclonal anti-Su(H) (Santa Cruz Biotechnology, #sc15813). Secondary IRDye700 and 800 secondary antibodies (Rockland, Gilbertsville, PA) were used for development on a LiCor Odyssey (Li-Cor, Lincoln, NE) infrared scanner. RESULTS Delivery and Developmental Effects of MeHg in the Drosophila Embryo We previously demonstrated the ability of MeHg to activate expression of two Notch target genes, E(spl) mc and mb, in cultured Drosophila cells (Bland and Rand, 2006) consistent with the hypothesis that MeHg can stimulate Notch receptors. We sought to further characterize this activity, first by examining whether a similar effect of MeHg occurs in vivo. We approached this by examining changes in transcripts in whole embryos exposed to MeHg (see below). We first set out to validate whether MeHg exposure can be achieved in Drosophila embryos. Dosages were introduced by culturing female flies on MeHg-containing food for 5 days prior to embryo collection (see ‘‘Methods’’). Total mercury content in embryos from MeHgfed female flies was measured to determine the possibility that maternally ingested MeHg accumulates in the embryo. Total mercury amounts were determined in lysates of a pool of > 50 embryos using combustion-trapping atomic absorption (see ‘‘Methods’’). We observed a linear increase in total mercury accumulation in embryos with respect to MeHg concentration in the food (Fig. 1A). To estimate the concentration of MeHg in the embryo we assume that the total Hg is essentially all MeHg due to the stability of MeHg. By approximating the embryo volume from its average dimensions (500 lm 3 180 lm) and its nearellipsoid geometry, we calculate an estimated volume of 8.4 nl per embryo. With 0.12 ng Hg/embryo achieved at the 20lM food concentration, an approximate 3.5-fold increase (71.4lM) in MeHg is seen in the embryo relative to the food. Embryos exposed to a maternal feeding of MeHg showed developmental defects as seen by a decrease in larval hatching rates and malformation of the nervous system. From controltreated females, 98.5% of embryos successfully hatched to larvae (Fig. 1B). A significant decrease in hatching was observed with embryos from MeHg-fed females, with 82.5% hatching (16% decrease) on 10lM food and 52.7% hatching (42% decrease) on 20lM food (Fig. 1B). As an additional line of evidence that MeHg was reaching the embryo, the gross morphology of the embryonic central nervous system was assayed using the pan-neural specific antibody anti-HRP. This antibody recognizes a surface antigen expressed on all neurons in Drosophila (Sun and Salvaterra, 1995) . Compared with control embryos, treatment of embryos with MeHg results in distinct developmental defects as seen by abnormal morphology of the nervous system (Fig. 1C). MeHg-exposed embryos show a range of phenotypes including expanded regions of HRPpositive cells (Fig. 1CI) or alternatively, regions where neurons fail to form (Fig. 1CIII, arrows). These two phenotypes suggest that neurogenic pathways (such as Notch in the latter case) are targets for MeHg. As well, deficits in formation of axon tracts (not shown) and an overall ‘‘twisted’’ formation of the CNS (Fig. 1CII) was observed indicative of additional developmental signaling pathways being disrupted. The percent of embryos demonstrating an abnormal neural phenotype was found to correlate with the concentration of MeHg in the food: 5lM MeHg food produces 3.4% (39/1099) of embryos with a neural phenotype, whereas 20lM MeHg food gives a neural phenotype in 45% (370/798) of embryos. Overall, these data confirm that embryonic MeHg exposure, which results in developmental abnormalities, can be achieved through maternal dosage of the fly. The diversity of neural phenotypes we have observed so far fall into discrete classes that implicate several distinct pathways, including Notch, as targets for MeHg and will be the subject of a separate investigation. Transcript Profiling Identifies the Notch Pathway as a MeHg Target in the Embryo A whole-genome transcriptional profiling approach was taken to determine genes that are up- and downregulated in expression subsequent to MeHg exposure in the embryo. RNA from embryos of female flies fed either control or 20lM MeHg food was analyzed. This concentration of MeHg was chosen because it gives a significant fraction of neural phenotypes by immunostaining (see above). Three replicates of control and MeHg treatments were analyzed for a total of six Affymetrix GeneChip data sets. The results show that the experimental system of transcript profiling provides power for detecting differentially expressed genes. The median standard deviation of the RMA gene expression statistic is 0.13 (Fig. 2A). A threshold of |DRMA| ¼ 1 (customarily taken to represent twofold change in expression) is therefore ~8 times the median standard deviation. For the data set the median standard deviation corresponds to a coefficient of variation of < 9% in the expression signal intensity. A histogram shows that for most genes, the p values are consistent with the null hypothesis that MeHg treatment does not ubiquitously alter transcription (Fig. 2B, 97% of probe sets are not differentially expressed). We identified 287 ‘‘hits’’ that change at least 1.5-fold up or down (more precisely, |DRMA| > 0.59). Of these, the signal for 74 probe sets is upregulated and 213 probe sets are downregulated (see Fig. 3 and Supplementary Data). We find a nonrandom association of Notch signaling pathway genes with the pool of differentially expressed genes with MeHg exposure. Of the 286 differentially expressed hits, 146 are associated with a biological process. GOA (Camon et al., 2004) associates 42 genes with the Notch Signaling Pathway GO term represented among the > 18,000 probe sets on the Affymetrix GeneChips. Ten of these 42 Notch genes are differentially regulated with MeHg exposure (Fig. 2C), with a significant (p < 4 3 10 9) association between differential expression and the Notch Signaling Pathway as determined using Gentleman’s Bioconductor GOstats package (Balasubramanian et al., 2004) . A Volcano plot for the entire data set is illustrated in Figure 2D. The contour illustrated demarks the 5% quantile under the null hypothesis. Superimposed on the plot are the points for the 42 Notch pathway genes identified by GO. The skewed distribution of the Notch-related genes toward the ‘‘right’’ of the plot reflects the significant increase of several Notch pathway probe sets with MeHg treatment. The 10 Notch family genes showing upregulation and lying outside the 5% quantile contour are illustrated (Fig. 2D). The Notch-related genes upregulated with MeHg are E(spl)ma, mc, md, m4, m5, m8, Bearded (Brd), Brother of Bearded (BobA), Ocho, and Bigbrain (Bib) (Fig. 3A). Remarkably, all but one of these Notch pathway genes share the common property of being a downstream target for Notch receptor activation and comprise nearly half of the 19 Notch target genes contained in the E(spl)C and BrdC complexes. It is of note that the E(spl)C and BrdC loci are separated by a large distance, being located on different arms of chromosome 3 (Fig. 3B). The Bib gene, which encodes an aquaporin-related protein, has genetic interactions with the Notch pathway, however, its regulation by Notch signaling is not well understood (Doherty et al., 1997) . Identification of these targets is consistent with our previous studies in cell-based assays, which similarly shows upregulated transcription of the E(spl)mc target gene with MeHg exposure in Drosophila neural cells (Bland and Rand, 2006) . Additional Responses in Transcript Profiles with MeHg Exposure Aside from the Notch pathway, annotation clusters with enrichments greater than 1.5 as identified by the DAVID functional annotation tool include: genes with lipid transporter activity (p < 5.5 3 10 6), thiol-specific antioxidants (p < 8.8 3 10 4), and defense response (p < 1.2 3 10 3). The lipid transporter-related genes identified correspond with transcripts for CG6300, CG4830, and CG11407, that are identified by sequence similarity and have not been functionally characterized. The antioxidant gene with the greatest differential expression is the peroxiredoxin (Prx) 2540 (CG11765), for which an increase was seen in two independent probe sets. The increase in Prx probe sets also contributes to the annotation cluster of defense response. Other defense response probe sets showing upregulation are the heat shock proteins Hsp70A and B (CG18743 and CG6489, respectively). Notable annotation clusters identified by probe sets with downregulated intensity include insect cuticle genes (p < 1.0 3 10 11), proteases of the C1 (cysteine)-type (p < 2.1 3 10 4), and EF-hand containing calcium binding proteins (p < 1.9 3 10 3). Concentration and Time-Dependent Activation of E(spl)mc Transcription Results from microarray analyses of MeHg-treated embryos highlight the potential for a broader range of Notch targets (BrdC genes in addition to E(spl)C genes) to respond to MeHg than previously observed. We therefore sought to determine more precisely the spectrum of Notch targets that respond to MeHg stimulation. Although effective in producing a global perspective on the transcriptional response in vivo, the embryo presents a complexity of cell types and developmental dynamics that could confound interpretation of transcriptional sensitivity in specific tissues, e.g. developing neural tissues. We therefore turned to the C6 neural-derived cell system with which we have considerable experience in the more quantitative method of qPCR (Bland and Rand, 2006) . We refined our experimental conditions by first analyzing the time- and concentration-dependent MeHg response of a representative target, the E(spl)mc mRNA (mc). Treatments of 10lM MeHg induce a time-dependent increase in mc with a 14-fold rise in expression seen at 18 h (Fig. 4A). Closer examination of early time points shows a significant increase of mc at 3-h treatment. We next examined various concentrations of MeHg at a 3-h exposure time and found a concentration-dependent increase with a nearly 15-fold increase in expression of mc with 50lM MeHg (Fig. 4B). To account for potential changes in receptor expression we examined the level of Notch mRNA. We find that Notch transcript levels remain essentially unchanged over the time and concentration ranges examined (Figs. 4A and 4B), indicating changes in E(spl)mc mRNA were not merely resulting from upregulation of Notch expression. It remained possible that the increase in E(spl)mc mRNA with MeHg was occurring by one of two mechanisms: an increase in mRNA synthesis or, alternatively, inhibition of constitutive degradation of the mRNA transcript. To distinguish between these two possibilities we examined the mc transcriptional response to MeHg in the presence versus absence of actinomycin D (AMD), an inhibitor of RNA transcriptional complexes (Sobell, 1985) . We see that AMD completely abolishes the MeHg-induced increase of mc transcript in C6 cells (Fig. 5). These data confirm that MeHg functions by inducing transcription of the mc gene and is therefore likely to exert a similar effect upon the other E(spl)C and BrdC genes. In addition, these data define an appropriate treatment regimen to analyze increases in other E(spl)C and BrdC transcript levels in response to MeHg in these cells. MeHg Exposure and Notch Activation Differentially Activate Transcription of E(spl)C and BrdC Genes Our previous model proposed that MeHg activates Notch by stimulating cleavage of the receptor (Bland and Rand, 2006) . This model predicts that stimulation of the Notch receptor and MeHg exposure would result in an identical pattern of Notch target gene activation. We therefore examined the profile of expression of several representatives of the E(spl) and Brd family genes in C6 cells where endogenous Notch is stimulated directly as compared with MeHg exposure. Notch receptor activation is achieved with a brief exposure to the calciumchelating agent EDTA, which causes dissociation of the Notch heterodimer and acute activation of the receptor (Rand et al., 2000). This approach has proven a useful tool in analysis of Notch activation of E(spl) genes in cultured Drosophila and mammalian cells (Krejci and Bray, 2007; Rand et al., 2000) . A comprehensive analysis of E(spl) and Brd genes using qPCR was restricted by the ability to generate valid primer sets for this quantitative method, preventing us from analyzing all E(spl) and Brd genes (e.g., m4, Ocho were excluded). Nonetheless, we found a robust upregulation of several E(spl)C genes in response to EDTA activation, notably a 95-fold increase in the m3 gene (Fig. 6A). As well, the m7 and mc transcripts show a marked increase of 55-fold and 38-fold respectively (Fig. 6A). In contrast, the md gene is minimally responsive (twofold increase, Fig. 6A). Also, there are insignificant changes in the BrdC genes, BobA and Brd, with Notch activation (1.3- and 1.2-fold, respectively, Fig. 6A). To confirm that the EDTA response relies exclusively on the Notch receptor, we examined cells where Notch expression is knocked down with RNAi. We see that EDTA response of m3 and mc expression is effectively abolished with Notch RNAi (inset Fig. 6A), confirming that the profile of EDTA stimulation of E(spl)C and BrdC expression directly reflects the activity of the Notch receptor. In contrast to Notch activation, MeHg shows a markedly different profile of E(spl) and Brd gene activation with a relatively small induction of m3 (sixfold) and the greatest stimulation of md (35-fold) (Fig. 6B). In addition, a significant increase in the BrdC genes (BobA and Brd) is seen with MeHg (six to eightfold increase, Fig. 6A), again differing from Notch activation where BobA and Brd fail to be activated. Altogether, these data demonstrate MeHg-induced transcription of conventional Notch target genes is distinct from activation of the Notch receptor itself. MeHg Activation of Notch Target Genes does not Require Notch The contrasting profile of E(spl)C and BrdC gene expression with MeHg and Notch activation prompted us to reinvestigate the role of the Notch receptor, as well as core components that propagate Notch signaling in the MeHg response. We examined the requirement for the Notch receptor directly by knocking down Notch expression with RNAi. First, we observe in control cells that 3 h of 50lM MeHg treatment shows no change in Notch mRNA transcript levels (Figs. 4A and 4B, 7B). With RNAi treatment, Notch transcript levels can effectively be reduced to < 30% of control and even greater reduction at the protein level (Figs. 7A and 7B). We find that knockdown of Notch shows no effect on MeHg stimulation of mc expression (Fig. 7C). This is in contrast to the robust effect Notch RNAi has on suppressing EDTA activation of E(spl) targets (see Fig. 6A inset). To further examine a possible role for the Notch pathway, we performed targeted RNAi knockdown of Su(H), the essential transcriptional coactivator that is required for Notchdependent E(spl) expression. First, we observed that 3 h of 50lM MeHg alone caused a significant reduction in the Su(H) transcript level (Fig. 8A). RNAi treatment similarly achieved ~50% reduction in Su(H) mRNA, which was further knocked down (~90% reduction) in combination with MeHg (Fig. 8A). Reduction in transcript level correlated with significant reduction in Su(H) protein expression (Fig. 8B). Despite substantial knockdown of Su(H), an insignificant reduction of MeHg-induced mc expression was observed (Fig. 8C). Altogether, these data demonstrate that activation of mc by MeHg does not require the Notch receptor or Su(H), two essential core components of the Notch signaling pathway. MeHg Activation of Notch Target Genes is Likely to Operate via a Thiol-Selective Mechanism The observation that MeHg acts independently of the Notch pathway to induce Notch target genes raised questions as to the FIG. 9. Effects of IA and NEM on E(spl) and Brd gene activation. C6 cells were treated with indicated concentration of IA or NEM as described in the methods and E(spl) or Brd gene expression was determined by qPCR (mean values, n ¼ 2). Expression levels of E(spl) mc (A) and E(spl) m3 (B) in response to various concentrations of IA or NEM. (C) Expression of E(spl) or Brd genes in response to 80lM IA relative to no IA control (see ‘‘Methods’’). specificity with which MeHg acts in this potentially novel mechanism. Because MeHg has a high affinity for protein thiols (Hughes, 1957) , we asked whether a similar activation of E(spl)C and BrdC genes could be achieved with two common thiol-specific alkylating reagents, IA and NEM. Although these compounds are both considered thiol-specific, they react via distinct mechanisms and have previously been shown to selectively modify different thiol-containing proteins (Dennehy et al., 2006) . Exposure of C6 cells to IA results in a concentrationdependent increase in both mc and m3 expression (Figs. 9A and 9B). In contrast, NEM at the same or higher concentrations fails to significantly induce mc or m3 expression (Fig. 9 and data not shown). Further analysis shows upregulated expression of all the E(spl) and Brd genes in a pattern that shows both similarity and differences with that of MeHg induction (Fig. 9C). As with MeHg, IA upregulated mc and m3 to similar levels relative to each other; however, unlike MeHg, these two targets were the most highly induced (Fig. 9C). Again, similar to MeHg but distinct from Notch activation, the Brd and BobA genes were upregulated with IA (Fig. 9C). As well, downregulation of Notch with RNAi showed no effect on subsequent induction of E(spl) and BrdC gene expression with IA exposure (data not shown), indicating that IA similarly works through a Notch-independent mechanism. In contrast to MeHg, md is not as highly responsive to IA exposure. Overall, IA demonstrates a specific activity in the induction of E(spl) and Brd genes confirming the sensitivity of expression of these Notch targets to cellular thiol modification. DISCUSSION MeHg Induces E(spl)C and BrdC Genes Independent of Notch We show that MeHg is a direct activator of several E(spl)C and BrdC genes, the well documented targets of the Notch signaling pathway. In the developing Drosophila embryo MeHg exposure upregulates expression of six transcripts of the E(spl)C (md, m4, ma, m5, mc, and m8) and three transcripts of the BrdC complex (BobA, Brd and Ocho). Similarly, we find MeHg induces expression of seven E(spl) genes (m1, m2, m3, m7, mb, mc, and md) and two Brd genes (BobA and Brd) in cultured Drosophila neural cells. All of these genes are characterized as having Su(H) binding sites in their upstream regulatory regions, a hallmark of Notch responsive genes (Nellesen et al., 1999) . Yet we demonstrate that MeHg induces expression of E(spl)C and BrdC genes independent of the Notch receptor and the Su(H) transcription factor, pointing to a novel mechanism for transcriptional activation by MeHg. The fact that distinct profiles of activation of E(spl)C and BrdC gene are seen with MeHg versus Notch stimulation further supports the hypothesis that MeHg induces transcription through a novel mechanism. It is of note that the lack of effect of Notch RNAi seen here differs significantly from our previously reported effect on Notch RNAi on MeHg activation of mc (Bland and Rand, 2006) . However, our former study examined effects of a longer exposure regimen (16-h treatment with 10lM MeHg), which may engage Notch in secondary regulatory responses controlling mc expression by mechanisms distinct from the acute effects seen in this study. Of the E(spl)C and BrdC genes, the activity of the bHLH encoding genes is best understood. In Drosophila, loss of function of the bHLH E(spl) genes, most commonly achieved through disabling Notch signaling, disrupts neural development resulting in hypertrophy of the embryonic CNS (Jennings et al., 1994; Lieber et al., 1993) . This activity of E(spl)s predicts that MeHg could elicit direct effects on neural development via altering E(spl) levels. Although several vertebrate E(spl) bHLH homologs (the HES and HERP genes reviewed in Iso et al., 2003) have been identified, a direct functional correlation between individual Drosophila and vertebrate E(spl) homologs has not been well characterized. However, the HES genes display similar control over neuronal differentiation in mice (Ohtsuka et al., 1999) . A similar transcriptional activity of MeHg on HES genes in a mammalian system is currently under investigation. The ability of MeHg to act as a transcriptional activator remains an important point of interest. Two studies suggest MeHg can support transcription by acting at the level of RNAPolII function or infuencing nucleosome stability. MeHg can stimulate RNAPolII activity in isolated nuclei of HeLa cells in a selective manner that is not achieved with other organic and inorganic mercury compounds (Chao and Frenkel, 1983) . Furthermore, NEM is only weakly effective at inducing RNAPolII activity (Chao and Frenkel, 1983) , consistent with our results. Alternatively, MeHg may serve to stabilize transcriptionally active conformations of nucleosomes, as demonstrated by the use of orgonomercurial supports to isolate transcriptionally active fragments of chromosomes ( ChenCleland et al., 1993 ). Both of these mechanisms predict MeHg would globally upregulate transcriptional activity in the cell. However, two observations support the notion that MeHg is acting with a higher degree of specificity toward individual regulatory regions of genes: first, our whole-genome transcript analyses show that a very limited number of transcripts change levels in response to MeHg in embryos (Fig. 2B); next, we see that some E(spl) genes respond to MeHg to a greater extent than others (e.g., md vs. mb in C6 cells, Fig. 6B), suggesting that upstream regulatory regions of certain genes harbor selective ‘‘MeHg-responsive’’ elements. This latter observation focuses attention on the upstream regions of individual E(spl) and Brd genes, which have been the topic of a number of studies. A recent analysis of conservation of E(spl) regulatory regions among nine species of Drosophila (Maeder et al., 2007) identifies consensus binding sites for as many as 27 distinct transcription factors distributed across the various E(spl) genes, with notable heterogeneity between individual E(spl) genes. Our data suggests that MeHg induces the association of one or more transcription factors with their cognate binding sites in the E(spl) and Brd genes, thereby stimulating transcription. Alternatively, MeHg may cause dissociation of repressors in these regions, similarly upregulating expression. Distinguishing the factors involved in mediating MeHg-induced transcriptional activation warrants a more in-depth analysis of the E(spl) and Brd regulatory region, which will be addressed in future experiments. Overall, these data implicate a potentially direct mechanism for MeHg to influence transcription at specific loci that are critical in neurogenesis. Thiol-Selective Mechanisms of Transcriptional Activation Additional MeHg-Sensitive Pathways in the Embryo By comparing MeHg, IA, and NEM exposure to C6 cells, Maternal dosage of flies results in a 3.5-fold increase in a profile of thiol-specific activation of E(spl)C and BrdC genes MeHg in embryos (20lM MeHg food concentrations give is revealed. Similar to MeHg, IA induces mc expression in ~70lM MeHg in the embryo). At these levels of MeHg we a concentration-dependent manner that is not seen with NEM. observe a significant drop in embryonic hatching rate, which We infer from these results that MeHg and IA have over- correlates with the appearance of a number of neural lapping protein targets. Previous studies show that IA and phenotypes. These concentrations are within the same range NEM can be differentiated by their thiol-selective reactivity where pronounced effects of MeHg on transcription in C6 toward cellular protein substrates (Dennehy et al., 2006) . This neuronal cells are observed. It is reasonable to predict that difference likely reflects the fact that IA is a smaller aliphatic a similar transcriptional response should occur in the embryo electrophile that reacts via a different chemistry than the with this exposure level. Yet the level of MeHg-induced bulkier heterocyclic dicarbonyl NEM (Dennehy et al., 2006) . It E(spl)C and BrdC gene expression in embryos, as determined follows that the relatively small size of IA and MeHg may by probe hybridization methods in microarrays, differs contribute to an ability to modify the same proteins. However, significantly from that seen in C6 cells using the qPCR a broader examination of the E(spl)C and BrdC genes induced methodology. Some of this discrepancy is likely due to the by IA demonstrates similarities and differences in transcrip- fundamentally different sensitivities of the respective technitional activation compared with MeHg. IA stimulates Brd and ques. However, differences in expression between embryos and BobA expression in a similar manner as MeHg. In contrast to cells are more likely to stem from the diversity of cell types in MeHg, IA only modestly stimulates md expression. These the embryo in contrast to the relatively homogeneous C6 cell results indicate that there is a level of specificity in MeHg line. This observation reinforces the importance of investigatreactivity that is not shared with IA. This difference may reflect ing MeHg effects in the context of the developing organism. a selective reactivity toward cysteine thiols based on the Nonetheless, the fact that E(spl) and Brd genes are targeted in composition of the neighboring amino acids in the protein, both contexts strongly supports the overall hypothesis that a factor previously reported to influence the specificity of IA a fundamental underlying MeHg-sensitive mechanism reguversus NEM reactivity (Dennehy et al., 2006) . lates their expression. With respect to cellular thiol modification, a particularly We observe changes in several transcripts in MeHg-exposed relevant thiol-responsive element is the Nrf2/Keap1system, embryos indicating a number of other pathways are potentially which regulates transcription in response to electrophiles and affected, consistent with the observation that several phenooxidative stress (Copple et al., 2008) . MeHg has been shown to types arise in MeHg-exposed embryonic nervous system. For activate this pathway in mammalian cells (Toyama et al., example, elevation of heat shock proteins (HSP70) in embryos 2007). Interestingly, a biotinylated form of iodacetamide shows with MeHg indicates a response shared with other stress a preferential activation of this pathway as compared with paradigms examined in the Drosophila model, including a bitotinylated maleimide (Hong et al., 2005) , in agreement temperature and starvation. (Landis et al., 2004; Sorensen with the differential effects of IA and NEM we observe here. It et al., 2005) . As well, upregulation of HSPs is seen with metal is of great interest that a homologous Nrf2/Keap1 signaling ion exposure in Drosophila cells (Bournias-Vardiabasis et al., pathway has recently been described in Drosophila (Sykiotis 1990). Heat shock proteins act as chaperones, aiding in protein and Bohmann, 2008), presenting the opportunity to investigate folding in under conditions of stress. Interestingly HSP70 has MeHg effects on this important antioxidant pathway in a fly been shown to suppress neurodegeneration in polyglutamine model. disease and preserve locomotor function under stress con An important implication stemming from our observations ditions (Feder et al., 1996; Klose et al., 2005) . Thus, one of thiol-selective transcriptional activity is the potential for possibility is that HSPs counter the effects of MeHg in other environmental oxidants or electrophiles to stimulate disrupting thiol disulfide bond formation in newly synthesized E(spl)C, BrdC, or other genes via modification of the same proteins. Although induction of HSPs is a potential marker for thiol targets. IA shares a thiol-reactive chemistry with aliphatic MeHg toxicity (Sacco et al., 1997), a functional role for HSPs epoxides and alkyl halides (Dennehy et al., 2006) , suggesting in attenuating MeHg toxicity has yet to be resolved, but of that the latter may function similarly to invoke transcription. remains a testable hypothesis in the Drosophila system. Our current observations and our system provide the rationale An additional family of genes upregulated in MeHg-exposed and an experimental model by which to compare, for example, embryos are the perioxiredoxins (Prx), a highly conserved inorganic mercury (Hg2þ) and other organomercurials such as family of proteins that exhibit thiol-dependent peroxidase ethylmercury (Thimerosal) with MeHg in influencing tran- activity and that are critical to maintaining intracellular redox scription. Overall, these data support the hypothesis that status (Rhee et al., 2005) . Drosophila carry five paralogs of Prx a discrete set of thiol-presenting proteins are involved in (Radyuk et al., 2001) . Two of these members are of the 1a MeHg-sensitive mechanism that regulates gene expression. Cysteine class (Prx2540, Prx6005) and three of these members (Prx 4156, 4783, 5037) are of the 2-Cysteine class (Radyuk et al., 2001) . Curiously, we see that with MeHg exposure, only the 1-Cys class is affected with upregulation of Prx2540 (Fig. 3A) and downregulation of Prx6005 (see Supplementary Data). Prx genes are characterized as playing a role in defense response to ROS (Rhee et al., 2005) . It is less clear why Prx6005 is downregulated; however, this result highlights a potential mechanism of differential expression in this gene family in response to MeHg. A role for Prx in attenuating MeHg toxicity has not been demonstrated directly. A number of microarray probe sets show downregulation of transcripts with MeHg and represent genes annotated as insect cuticle proteins by sequence similarity (Supplemental Data). This observation is consistent with the notion that cuticle formation is compromised in late embryos and leads to our observed decrease in larval hatching of MeHg-exposed embryos. Another possibility is that decreased cuticle gene expression is a downstream effect of altered Notch signaling because epidermal cell fate is compromised with altered Notch function (Hoppe and Greenspan, 1990) . The observed decrease in cysteine protease gene expression with MeHg exposure is difficult to interpret. The fact that mercury compounds can bind and inactivate cysteine proteases (Muller and Saenger, 1993) would predict a compensatory increase in expression of these gene transcripts, contracy to what we observe. In contrast, the observed downregulation of calcium binding proteins, particularly EF-hand proteins, may stem from the ability of mercury to substitute for Caþþ ions in activation of EF-hand proteins (e.g., calmodulin; Chao et al., 1984) . Overall, our microarray data are indicative of the complexity of events downstream of MeHg exposure in the developing embryo and highlight the necessity to evaluate transcriptional changes with MeHg in additional contexts and at various developmental stages to deduce the most universally responsive genes. To conclude, our results demonstrate that MeHg can regulate transcriptional activation of E(spl)C and BrdC genes independent of the Notch pathway in Drosophila. Our study also presents evidence of a thiol-selective mechanism in these events. Finally, our results demonstrate the utility of the Drosophila model in elucidating potentially fundamental mechanisms of MeHg toxicity. SUPPLEMENTARY DATA Supplementary data are available online at http://toxsci. oxfordjournals.org/. FUNDING National Institute of Health, National Institute of Environmental Health Science (R21ES013754 and R01ES015550) awarded to M.D.R. ACKNOWLEDGMENTS We thank the NIH Neuroscience Microarray consortium for assistance in sample handling, processing and probe set data analysis. This work was carried out with the excellent technical assistance of Julie Dao. We are grateful to Alena Krejci and Sarah Bray (University of Cambridge, UK) for sharing primer sequences. We thank Lucy Cherbas for cell culture consults and materials received through the Drosophila Genomics Resource Center. We also thank Felix Eckenstein (University of Vermont) for critical review of the manuscript. We acknowledge the help and services of the University of Vermont COBRE molecular core facility for use of qPCR instrumentation and assistance with data analysis. 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Rand, Matthew D., Bland, Christin E., Bond, Jeffrey. Methylmercury Activates Enhancer-of-Split and Bearded Complex Genes Independent of the Notch Receptor, Toxicological Sciences, 2008, 163-176, DOI: 10.1093/toxsci/kfn060