Steady-state Abundance Research Articles

LMO2 Oncoprotein Stability in T-Cell Leukemia Requires Direct LDB1 Binding.

LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study, we systematically dissected the LMO2/LDB1-binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif, R(320)LITR, required for LMO2 binding. Most strikingly, coexpression of full-length, wild-type LDB1 increased LMO2 steady-state abundance, whereas coexpression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Mass spectrometric analysis of LDB1 binding partners in leukemic lines supports the notion that LMO2/LDB1 function in leukemia occurs in the context of multisubunit complexes, which also protect the LMO2 oncoprotein from degradation. Collectively, these data suggest that the assembly of LMO2 into complexes, via direct LDB1 interaction, is a potential molecular target that could be exploited in LMO2-driven leukemias resistant to existing chemotherapy regimens.

Galactose-1 phosphate uridylyltransferase (GalT) gene: A novel positive regulator of the PI3K/Akt signaling pathway in mouse fibroblasts

The vital importance of the Leloir pathway of galactose metabolism has been repeatedly demonstrated by various uni-/multicellular model organisms, as well human patients who have inherited deficiencies of the key GAL enzymes. Yet, other than the obvious links to the glycolytic pathway and glycan biosynthetic pathways, little is known about how this metabolic pathway interacts with the rest of the metabolic and signaling networks. In this study, we compared the growth and the expression levels of the key components of the PI3K/Akt growth signaling pathway in primary fibroblasts derived from normal and galactose-1 phosphate uridylyltransferase (GalT)-deficient mice, the latter exhibited a subfertility phenotype in adult females and growth restriction in both sexes. The growth potential and the protein levels of the pAkt(Thr308), pAkt(Ser473), pan-Akt, pPdk1, and Hsp90 proteins were significantly reduced by 62.5%, 60.3%, 66%, 66%, and 50%, respectively in the GalT-deficient cells. Reduced expression of phosphorylated Akt proteins in the mutant cells led to diminished phosphorylation of Gsk-3β (−74%). Protein expression of BiP and pPten were 276% and 176% higher respectively in cells with GalT-deficiency. Of the 24 genes interrogated using QIAGEN RT2 Profiler PCR Custom Arrays, the mRNA abundance of Akt1, Pdpk1, Hsp90aa1 and Pi3kca genes were significantly reduced at least 2.03-, 1.37-, 2.45-, and 1.78-fold respectively in mutant fibroblasts. Both serum-fasted normal and GalT-deficient cells responded to Igf-1-induced activation of Akt phosphorylation at +15 min, but the mutant cells have lower phosphorylation levels. The steady-state protein abundance of Igf-1 receptor was also significantly reduced in mutant cells. Our results thus demonstrated that GalT deficiency can effect down-regulation of the PI3K/Akt growth signaling pathway in mouse fibroblasts through distinct mechanisms targeting both gene and protein expression levels.

Eukaryotic translation initiation factor 6 is a novel regulator of reactive oxygen species‐dependent megakaryocyte maturation

Ribosomopathies constitute a class of inherited disorders characterized by defects in ribosome biogenesis and function. Classically, bone marrow (BM) failure is a clinical symptom shared between these syndromes, including Shwachman-Bodian-Diamond syndrome (SBDS). Eukaryotic translation initiation factor 6 (eIF6) is a critical translation factor that rescues the quasilethal effect of the loss of the SBDS protein. To determine whether eIF6 activity is necessary for BM development. We used eIF6(+/-) mice and primary BM megakaryocytes to investigate the involvement of eIF6 in the regulation of hematopoiesis. We provide evidence that reduced eIF6 expression negatively impacts on megakaryopoiesis. We show that inhibition of eIF6 leads to a reduction in cell size and mean ploidy level of megakaryocytes and a delay in megakaryocyte maturation by blocking the G1 /S transition. Consistent with this phenotype, only few megakaryocyte-forming proplatelets were found in eIF6(+/-) cells. We also discovered that, in eIF6(+/-) cells, the steady-state abundance of mitochondrial respiratory chain complex I-encoding mRNAs is decreased, resulting in decreased reactive oxygen species (ROS) production. Intriguingly, connectivity map analysis showed that eIF6-mediated changes overlap with specific translational inhibitors. eIF6 is a translation factor acting downstream of insulin/phorbol 12-myristate 13-acetate (PMA) stimulation. PMA treatment significantly restored eIF6(+/-) megakaryocyte maturation, indicating that activation of eIF6 is essential for the rescue of the phenotype. Taken together, our results show a role for eIF6-driven translation in megakaryocyte development, and unveil the novel connection between translational control and ROS production in this cell subset.

Genomic approaches to DNA repair and mutagenesis.

DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways.

AZT-induced mitochondrial toxicity: an epigenetic paradigm for dysregulation of gene expression through mitochondrial oxidative stress.

Mitochondrial dysfunction causes oxidative stress and cardiomyopathy. Oxidative stress also is a side effect of dideoxynucleoside antiretrovirals (NRTI) and is observed in NRTI-induced cardiomyopathy. We show here that treatment with the NRTI AZT {1-[(2R,4S,5S)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione} modulates cardiac gene expression epigenetically through production of mitochondrially derived reactive oxygen species. Transgenic mice with ubiquitous expression of mitochondrially targeted catalase (MCAT) and C57Bl/6 wild-type mice littermates (WT) were administered AZT (0.22 mg/day po, 35 days), and cardiac DNA and mRNA were isolated. In AZT-treated WT, 95 cardiac genes were differentially expressed compared with vehicle-treated WTs. When MCAT mice were treated with AZT, each of those 95 genes reverted toward the expression of vehicle-treated WTs. In AZT-treated WT hearts, Mthfr [5,10-methylenetetrahydrofolate reductase; a critical enzyme in synthesis of methionine cycle intermediates including S-adenosylmethionine (SAM)], was overexpressed. Steady-state abundance of SAM in cardiac extracts from AZT-treated MCAT mice increased 60% above that of vehicle-treated MCAT. No such change occurred in WT. AZT caused hypermethylation (47%) and hypomethylation (53%) of differentially methylated DNA regions in WT cardiac DNA. AZT-treated MCAT heart DNA exhibited greater hypermethylation (91%) and less hypomethylation (9%) compared with vehicle-treated MCAT controls. The gene encoding protein kinase C-α displayed multifocal epigenetic regulation caused by oxidative stress. Results show that mitochondrially derived oxidative stress in the heart hinders cardiac DNA methylation, alters steady-state abundance of SAM, alters cardiac gene expression, and promotes characteristic pathophysiological changes of cardiomyopathy. This mechanism for NRTI toxicity offers insight into long-term side effects from these commonly used antiviral agents.

Effect of ribonucleic acid (RNA) isolation methods on putative reference genes messenger RNA abundance in human spermatozoa.

Although the male gamete participates in a significant proportion of infertility cases, there are currently no proven molecular markers of sperm quality. The search for significant gene expression markers is partially hindered by the lack of a recognized set of reference genes (RGs) to normalize reverse transcription quantitative PCR (RT-qPCR) data across studies. The aim of this study is to define a set of RGs in assisted reproduction patients undergoing different sample collection and RNA isolation methods. Twenty-two normozoospermic men were included in the study. From each man, semen was either cryopreserved by slow freezing or analyzed fresh, and, for each, RNA was extracted with either phenol-free or phenol-based methods. In two cases, both methods were used to isolate RNA. Twenty putative RGs were analyzed and their mRNA abundance across samples was estimated by RT-qPCR. To determine the genes whose steady-state mRNA abundance remains unchanged, three different algorithms (geNorm, BestKeeper and NormFinder) were applied to the qPCR data. We found that RGs such as GAPDH or ACTB, useful in other biological contexts, cannot be used as reference for human spermatozoa. It is possible to compare gene expression from fresh and cryopreserved sperm samples using the same isolation method, while the mRNA abundance of expressed genes becomes different depending on the RNA isolation technique employed. In our conditions, the most appropriate RGs for RT-qPCR analysis were RPLP1, RPL13A, and RPLP2. Published discrepancies in gene expression studies in human spermatozoa may be due in part to inappropriate RGs selection, suggesting a possible different interpretation of PCR data in several reports, which were normalized using unstable RGs.

RNA around the clock - regulation at the RNA level in biological timing.

The circadian timing system in plants synchronizes their physiological functions with the environment. This is achieved by a global control of gene expression programs with a considerable part of the transcriptome undergoing 24-h oscillations in steady-state abundance. These circadian oscillations are driven by a set of core clock proteins that generate their own 24-h rhythm through periodic feedback on their own transcription. Additionally, post-transcriptional events are instrumental for oscillations of core clock genes and genes in clock output. Here we provide an update on molecular events at the RNA level that contribute to the 24-h rhythm of the core clock proteins and shape the circadian transcriptome. We focus on the circadian system of the model plant Arabidopsis thaliana but also discuss selected regulatory principles in other organisms.

The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression.

The Artemisia annua L. β-pinene synthase QH6 was previously determined to be circadian-regulated at the transcriptional level, showing a rhythmic fluctuation of steady-state transcript abundances. Here we isolated both the genomic sequence and upstream promoter region of QH6. Different regulatory elements, such as G-box (TGACACGTGGCA, −421 bp from the translation initiation site) which might have effects on rhythmic gene expression, were found. Using the yeast one-hybrid and electrophoretic mobility shift assay (EMSA), we confirmed that the bZIP transcription factor HY5 binds to this motif of QH6. Studies with promoter truncations before and after this motif suggested that this G-box was important for the diurnal fluctuation of the transgenic β-glucuronidase gene (GUS) transcript abundance in Arabidopsis thaliana. GUS gene driven by the promoter region immediately after G-box showed an arrhythmic expression in both light/dark (LD) and constant dark (DD) conditions, whereas the control with G-box retained its fluctuation in both LD and DD. We further transformed A. thaliana with the luciferase gene (LUC) driven by an 1400 bp fragment upstream QH6 with its G-box intact or mutated, respectively. The luciferase activity assay showed that a peak in the early morning disappeared in the mutant. Gene expression analysis also demonstrated that the rhythmic expression of LUC was abolished in the hy5-1 mutant.

Quantitative changes in Gimap3 and Gimap5 expression modify mitochondrial DNA segregation in mice.

Mammalian mitochondrial DNA (mtDNA) is a high-copy maternally inherited genome essential for aerobic energy metabolism. Mutations in mtDNA can lead to heteroplasmy, the co-occurence of two different mtDNA variants in the same cell, which can segregate in a tissue-specific manner affecting the onset and severity of mitochondrial dysfunction. To investigate mechanisms regulating mtDNA segregation we use a heteroplasmic mouse model with two polymorphic neutral mtDNA haplotypes (NZB and BALB) that displays tissue-specific and age-dependent selection for mtDNA haplotypes. In the hematopoietic compartment there is selection for the BALB mtDNA haplotype, a phenotype that can be modified by allelic variants of Gimap3. Gimap3 is a tail-anchored member of the GTPase of the immunity-associated protein (Gimap) family of protein scaffolds important for leukocyte development and survival. Here we show how the expression of two murine Gimap3 alleles from Mus musculus domesticus and M. m. castaneus differentially affect mtDNA segregation. The castaneus allele has incorporated a uORF (upstream open reading frame) in-frame with the Gimap3 mRNA that impairs translation and imparts a negative effect on the steady-state protein abundance. We found that quantitative changes in the expression of Gimap3 and the paralogue Gimap5, which encodes a lysosomal protein, affect mtDNA segregation in the mouse hematopoietic tissues. We also show that Gimap3 localizes to the endoplasmic reticulum and not mitochondria as previously reported. Collectively these data show that the abundance of protein scaffolds on the endoplasmic reticulum and lysosomes are important to the segregation of the mitochondrial genome in the mouse hematopoietic compartment.

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased Kd value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R49 that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.

A stochastic biodiversity model with overlapping niche structure

The niche is a fundamental ecological concept that underpins many explanations of patterns of biodiversity. The complexity of niche processes in ecological systems, however, means that it is difficult to capture them accurately in theoretical models of community assembly. In this study, we build upon simple neutral biodiversity models by adding the important ingredient of overlapping niche structure. Our model is spatially implicit and contains a fixed number of equal-sized habitats. Each species in the metacommunity arises through a speciation event; at which time, it is randomly assigned a fundamental niche or set of environments/habitats in which it can persist. Within each habitat, species compete with other species that have different but overlapping fundamental niches. Species abundances then change through ecological drift; each, however, is constrained by its maximum niche breadth and by the presence of other species in its habitats. Using our model, we derive analytical expressions for steady-state species abundance distributions, steady-state distributions of niche breadth across individuals and across species, and dynamic distributions of niche breadth across species. With this framework, we identify the conditions that produce the log-series species abundance distribution familiar from neutral models. We then identify how overlapping niche structure can lead to other species abundance distributions and, in particular, ask whether these new distributions differ significantly from species abundance distributions predicted by non-overlapping niche models. Finally, we extend our analysis to consider additional distributions associated with realized niche breadths. Overall, our results show that models with overlapping niches can exhibit behavior similar to neutral models, with the caveat that species with narrow fundamental niche breadths will be very rare. If narrow-niche species are common, it must be because they are in a non-overlapping niche or have countervailing advantages over broad-niche species. This result highlights the role that niches can play in establishing demographic neutrality.

Loss of Epidermal p38α Signaling Prevents UVR-Induced Inflammation via Acute and Chronic Mechanisms

Ultraviolet B radiation (UVB) is a component of solar radiation primarily responsible for causing damage and cancer in irradiated skin, and disrupting immune homeostasis. The immediate harm and long-term health risks of excessive sunlight exposure are impacting the lives of nearly all people worldwide. Inflammation is a key mechanism underlying UVB’s various detrimental effects. Here we show that activation of the protein kinase p38α is restricted to the epidermis in UVB-exposed skin, and p38α ablation targeted to the epithelial compartment is sufficient to suppress UVB-induced inflammation. Mechanistically, loss of epithelial p38α signaling attenuates the expression of genes required to induce vascular leakage and edema, and also increases the steady-state abundance of epidermal γδ T cells, which are known to promote the repair of damaged epidermis. These effects of p38α deficiency delineate a molecular network operating at the organism-environment interface, and reveal conditions crucial to preventing the pathology resulting from sun-damaged skin.

Evolution of the Galaxy and the Birth of the Solar System: The Short-Lived Nuclides Connection

An attempt is made, probably for the first time, to understand the origin of the solar system in context with the evolution of the galaxy as a natural consequence of the birth of several generations of stellar clusters. The galaxy is numerically simulated to deduce the inventories of the short-lived nuclides, 26Al, 36Cl, 41Ca, 53Mn and 60Fe, from the stellar nucleosynthetic contributions of the various stellar clusters using an N-body simulation with updated prescriptions of the astrophysical processes. The galaxy is evolved by considering the discreteness associated with the stellar clusters and individual stars. We estimate the steady state abundance of the radionuclides around 4.56 billion years ago at the time of formation of the solar system. Further, we also estimate the present 26Al/27Al and 60Fe/56Fe of the interstellar medium that match within a factor of two with the observed estimates. On contrary to the conventional galactic chemical evolution (GCE) model, the present adopted numerical approach provides a natural framework to understand the astrophysical environment related with the origin of the solar system. We deduce the nature of the two stellar clusters; the one that formed and evolved prior to the solar system formation, and the other within which the solar system probably formed. The former could have contributed the short-lived nuclides 129I and 53Mn, whereas, the supernova associated with the most massive star in the latter contributed 26Al and 60Fe to the solar system. The analysis was performed with the revised solar metallicity of 0.014.

Molecular ions in the protostellar shock L1157-B1

We perform a complete census of molecular ions with an abundance larger than 1e-10 in the protostellar shock L1157-B1 by means of an unbiased high-sensitivity survey obtained with the IRAM-30m and Herschel/HIFI. By means of an LVG radiative transfer code the gas physical conditions and fractional abundances of molecular ions are derived. The latter are compared with estimates of steady-state abundances in the cloud and their evolution in the shock calculated with the chemical model Astrochem. We detect emission from HCO+, H13CO+, N2H+, HCS+, and, for the first time in a shock, from HOCO+, and SO+. The bulk of the emission peaks at blueshifted velocity, ~ 0.5-3 km/s with respect to systemic, has a width of ~ 4-8 km/s, and is associated with the outflow cavities (T_kin ~ 20-70 K, n(H2) ~ 1e5 cm-3). Observed HCO+ and N2H+ abundances are in agreement with steady-state abundances in the cloud and with their evolution in the compressed and heated gas in the shock for cosmic rays ionization rate Z = 3e-16 s-1. HOCO+, SO+, and HCS+ observed abundances, instead, are 1-2 orders of magnitude larger than predicted in the cloud; on the other hand they are strongly enhanced on timescales shorter than the shock age (~2000 years) if CO2, S or H2S, and OCS are sputtered off the dust grains in the shock. The performed analysis indicates that HCO+ and N2H+ are a fossil record of pre-shock gas in the outflow cavity, while HOCO+, SO+, and HCS+ are effective shock tracers and can be used to infer the amount of CO2 and sulphur-bearing species released from dust mantles in the shock. The observed HCS+ (and CS) abundance indicates that OCS should be one of the main sulphur carrier on grain mantles. However, the OCS abundance required to fit the observations is 1-2 orders of magnitude larger than observed. Further studies are required to fully understand the chemistry of sulphur-bearing species.

Kinetic Analyses Reveal Potent and Early Blockade of Hepatitis C Virus Assembly by NS5A Inhibitors

All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear. To study their mechanisms, we compared their kinetics of antiviral suppression with those of other classes of DAA, using the hepatitis C virus genotype 1a cell culture-infectious virus H77S.3. We performed detailed kinetic analyses of specific steps in the hepatitis C virus life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors. Assays were designed to measure active viral RNA synthesis and steady-state RNA abundance, polyprotein synthesis, virion assembly, and infectious virus production. Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA synthesis compared with protease or polymerase inhibitors. By 24 hours after addition of an NS5A inhibitor, polyprotein synthesis was reduced <50%, even at micromolar concentrations. In contrast, inhibition of virus release by NS5A inhibitors was potent and rapid, with onset of inhibition as early as 2 hours. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing hepatitis C virus particles, indicating a block in virus assembly. DAAs that target NS5A rapidly inhibit intracellular assembly of genotype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against preformed replicase, thereby resulting in slow shut-off of viral RNA synthesis.

Protein-to-mRNA ratios are conserved between Pseudomonas aeruginosa strains.

Recent studies have shown that the concentrations of proteins expressed from orthologous genes are often conserved across organisms and to a greater extent than the abundances of the corresponding mRNAs. However, such studies have not distinguished between evolutionary (e.g., sequence divergence) and environmental (e.g., growth condition) effects on the regulation of steady-state protein and mRNA abundances. Here, we systematically investigated the transcriptome and proteome of two closely related Pseudomonas aeruginosa strains, PAO1 and PA14, under identical experimental conditions, thus controlling for environmental effects. For 703 genes observed by both shotgun proteomics and microarray experiments, we found that the protein-to-mRNA ratios are highly correlated between orthologous genes in the two strains to an extent comparable to protein and mRNA abundances. In spite of this high molecular similarity between PAO1 and PA14, we found that several metabolic, virulence, and antibiotic resistance genes are differentially expressed between the two strains, mostly at the protein but not at the mRNA level. Our data demonstrate that the magnitude and direction of the effect of protein abundance regulation occurring after the setting of mRNA levels is conserved between bacterial strains and is important for explaining the discordance between mRNA and protein abundances.

Mapping in an apple (Malus x domestica) F1segregating population based on physical clustering of differentially expressed genes

BackgroundApple tree breeding is slow and difficult due to long generation times, self-incompatibility, and complex genetics. The identification of molecular markers linked to traits of interest is a way to expedite the breeding process. In the present study, we aimed to identify genes whose steady-state transcript abundance was associated with inheritance of specific traits segregating in an apple (Malus × domestica) rootstock F1 breeding population, including resistance to powdery mildew (Podosphaera leucotricha) disease and woolly apple aphid (Eriosoma lanigerum).ResultsTranscription profiling was performed for 48 individual F1 apple trees from a cross of two highly heterozygous parents, using RNA isolated from healthy, actively-growing shoot tips and a custom apple DNA oligonucleotide microarray representing 26,000 unique transcripts. Genome-wide expression profiles were not clear indicators of powdery mildew or woolly apple aphid resistance phenotype. However, standard differential gene expression analysis between phenotypic groups of trees revealed relatively small sets of genes with trait-associated expression levels. For example, thirty genes were identified that were differentially expressed between trees resistant and susceptible to powdery mildew. Interestingly, the genes encoding twenty-four of these transcripts were physically clustered on chromosome 12. Similarly, seven genes were identified that were differentially expressed between trees resistant and susceptible to woolly apple aphid, and the genes encoding five of these transcripts were also clustered, this time on chromosome 17. In each case, the gene clusters were in the vicinity of previously identified major quantitative trait loci for the corresponding trait. Similar results were obtained for a series of molecular traits. Several of the differentially expressed genes were used to develop DNA polymorphism markers linked to powdery mildew disease and woolly apple aphid resistance.ConclusionsGene expression profiling and trait-associated transcript analysis using an apple F1 population readily identified genes physically linked to powdery mildew disease resistance and woolly apple aphid resistance loci. This result was especially useful in apple, where extreme levels of heterozygosity make the development of reliable DNA markers quite difficult. The results suggest that this approach could prove effective in crops with complicated genetics, or for which few genomic information resources are available.

Protein Quantitative Trait Loci Identify Novel Candidates Modulating Cellular Response to Chemotherapy

Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p≤0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms.

Massively parallel functional annotation of 3′ untranslated regions

Functional characterization of noncoding sequences is crucial for understanding the human genome and learning how genetic variation contributes to disease. 3' untranslated regions (UTRs) are an important class of noncoding sequences, but their functions remain largely uncharacterized. We developed a method for massively parallel functional annotation of sequences from 3' UTRs (fast-UTR) and used this approach to measure the effects of a total of >450 kilobases of 3' UTR sequences from >2,000 human genes on steady-state mRNA abundance, mRNA stability and protein production. We found widespread regulatory effects on mRNA that were coupled to effects on mRNA stability and protein production. Furthermore, we discovered 87 novel cis-regulatory elements and measured the effects of genetic variation within known and novel 3' UTR motifs. This work shows how massively parallel approaches can improve the functional annotation of noncoding sequences, advance our understanding of cis-regulatory mechanisms and quantify the effects of human genetic variation.

Molecular signatures in rainbow darter (Etheostoma caeruleum) inhabiting an urbanized river reach receiving wastewater effluents

Rainbow darter (Etheostoma caeruleum) is a small benthic fish species found in North America that are abundant and distributed throughout the Grand River watershed, ON, Canada. Rainbow darter exhibit intersex in males at sites adjacent to municipal wastewater effluents (MWWE). In October 2010, female and male rainbow darter were collected at 3 sites (1 upstream reference and 2 downstream exposed sites) in the Grand River near the cities of Kitchener and Waterloo. The primary objectives of this research were (1) to characterize the responses of whole organism endpoints (i.e. condition factor (K), liversomatic (LSI) and gonadosomatic index (GSI), histopathology) to MWWEs and (2) to identify transcripts showing altered steady state abundance with exposure to MWWE in fish inhabiting municipal wastewater effluent-exposed areas. Genes measured in this study included vitellogenin, Sry-box containing protein 9 (sox9), forkhead box L2 (foxl2), doublesex and mab-3 related transcription factor 1 (dmrt1), cytochrome P450, family 11, subfamily A, polypeptide 1 (cyp11a) as well as estrogen (esr1, esrb) and androgen (ar) receptors. There were no changes in condition factor; however, there was a significant increase in LSI and a decrease in GSI in fish inhabiting downstream environments when compared with fish collected from the reference site. Males had a high incidence (∼70%) of intersex in downstream sites; characterized by the presence of oocytes within the testis. In the gonad, there were sex specific differences for genes related to sexual differentiation; dmrt1 was only expressed in males whereas foxl2 and sox9 were highly expressed in females compared to males. Expression levels of ar and esr1 were higher in females than males. Conversely, esrb was not differentially expressed between sexes or among sites. There were no differences detected for the genes investigated within sex among sites. This study is the first to report on gene expression changes in the rainbow darter, with emphasis on the differences in transcript abundance between sexes and how these changes relate to exposures to MWWEs. Molecular approaches are being investigated for their potential application to field ecotoxicology, and molecular bioassays for relevant, sentinel species in environmental monitoring programs are required to better understand the impact of anthropogenic impacts on species at risk in river systems.