Inflammation Impacts Androgen Receptor Signaling in Basal Prostate Stem Cells Through Interleukin 1 Receptor Antagonist.

The majority of patients with benign prostate hyperplasia (BPH) exhibit chronic prostate inflammation and the extent of inflammation correlates with the severity of symptoms. How inflammation contributes to prostate enlargement and/or BPH symptoms and the underlying mechanisms are not clearly understood. We established a unique mouse model Prostate Ovalbumin Expressing Transgenic 3 (POET3) that mimics chronic non-bacterial prostatitis in men to study the role of inflammation in prostate hyperplasia. After the injection of ovalbumin peptide-specific T cells, POET3 prostates exhibited an influx of inflammatory cells and an increase in pro-inflammatory cytokines that led to epithelial and stromal hyperplasia. We have previously demonstrated with the POET3 model that inflammation expands the basal prostate stem cell (bPSC) population and promotes bPSC differentiation in organoid cultures. In this study, we investigated the mechanisms underlying the impact of inflammation on bPSC. We found that AR activity was enhanced in inflamed bPSC and was essential for bPSC differentiation in organoid cultures. Most importantly, we identified, for the first time, interleukin 1 receptor antagonist (IL-1RA) as a key regulator of AR in basal stem cells. IL-1RA was one of the top genes upregulated by inflammation and inhibition of IL-1RA abrogated the enhanced AR nuclear accumulation and activity in organoids derived from inflamed bPSC. The mirroring effects of IL-1RA recombinant protein and IL-1α neutralizing antibody suggest that IL-1RA may function by antagonizing IL-1α inhibition of AR expression. Furthermore, we established a lineage tracing model to follow bPSC during inflammation and under castrate conditions. We found that inflammation induced bPSC proliferation and differentiation into luminal cells even under castrate conditions, indicating that AR activation driven by inflammation in bPSC is sufficient for their proliferation and differentiation under androgen-deprived conditions. However, proliferation of the differentiated bPSC in the luminal layer significantly diminished with castration, suggesting inflammation may not maintain AR activity in stromal cells, as stromal cells deprived of androgen after castration could no longer provide paracrine growth factors essential for luminal proliferation. Taken together, we have discovered novel mechanisms through which inflammation modulates AR signaling in bPSC and induces bPSC luminal differentiation that contributes to prostate hyperplasia.

Inbreeding Depression in Genotypically Matched Diploid and Tetraploid Maize.

The genetic and molecular basis of heterosis has long been studied but without a consensus about mechanism. The opposite effect, inbreeding depression, results from repeated self-pollination and leads to a reduction in vigor. A popular explanation for this reaction is the homozygosis of recessive, slightly deleterious alleles upon inbreeding. However, extensive studies in alfalfa indicated that inbreeding between diploids and autotetraploids was similar despite the fact that homozygosis of alleles would be dramatically different. The availability of tetraploid lines of maize generated directly from various inbred lines provided the opportunity to examine this issue in detail in perfectly matched diploid and tetraploid hybrids and their parallel inbreeding regimes. Identical hybrids at the diploid and tetraploid levels were inbred in triplicate for seven generations. At the conclusion of this regime, F1 hybrids and selected representative generations (S1, S3, S5, S7) were characterized phenotypically in randomized blocks during the same field conditions. Quantitative measures of the multiple generations of inbreeding provided little evidence for a distinction in the decline of vigor between the diploids and the tetraploids. The results suggest that the homozygosis of completely recessive, slightly deleterious alleles is an inadequate hypothesis to explain inbreeding depression in general.

Genome-Wide Association Study in Pseudo-F2 Populations of Switchgrass Identifies Genetic Loci Affecting Heading and Anthesis Dates.

Switchgrass (Panicum virgatum) is a native prairie grass and valuable bio-energy crop. The physiological change from juvenile to reproductive adult can draw important resources away from growth into producing reproductive structures, thereby limiting the growth potential of early flowering plants. Delaying the flowering of switchgrass is one approach by which to increase total biomass. The objective of this research was to identify genetic variants and candidate genes for controlling heading and anthesis in segregating switchgrass populations. Four pseudo-F2 populations (two pairs of reciprocal crosses) were developed from lowland (late flowering) and upland (early flowering) ecotypes, and heading and anthesis dates of these populations were collected in Lafayette, IN and DeKalb, IL in 2015 and 2016. Across 2 years, there was a 34- and 73-day difference in heading and a 52- and 75-day difference in anthesis at the Lafayette and DeKalb locations, respectively. A total of 37,901 single nucleotide polymorphisms obtained by exome capture sequencing of the populations were used in a genome-wide association study (GWAS) that identified five significant signals at three loci for heading and two loci for anthesis. Among them, a homolog of FLOWERING LOCUS T on chromosome 5b associated with heading date was identified at the Lafayette location across 2 years. A homolog of ARABIDOPSIS PSEUDO-RESPONSE REGULATOR 5, a light modulator in the circadian clock associated with heading date was detected on chromosome 8a across locations and years. These results demonstrate that genetic variants related to floral development could lend themselves to a long-term goal of developing late flowering varieties of switchgrass with high biomass yield.

Multiple testing with discrete data: Proportion of true null hypotheses and two adaptive FDR procedures.

We consider multiple testing with false discovery rate (FDR) control when p values have discrete and heterogeneous null distributions. We propose a new estimator of the proportion of true null hypotheses and demonstrate that it is less upwardly biased than Storey's estimator and two other estimators. The new estimator induces two adaptive procedures, that is, an adaptive Benjamini-Hochberg (BH) procedure and an adaptive Benjamini-Hochberg-Heyse (BHH) procedure. We prove that the adaptive BH (aBH) procedure is conservative nonasymptotically. Through simulation studies, we show that these procedures are usually more powerful than their nonadaptive counterparts and that the adaptive BHH procedure is usually more powerful than the aBH procedure and a procedure based on randomized p-value. The adaptive procedures are applied to a study of HIV vaccine efficacy, where they identify more differentially polymorphic positions than the BH procedure at the same FDR level.

Transcriptome profiling of aging Drosophila photoreceptors reveals gene expression trends that correlate with visual senescence.

BACKGROUND: Aging is associated with functional decline of neurons and increased incidence of both neurodegenerative and ocular disease. Photoreceptor neurons in Drosophila melanogaster provide a powerful model for studying the molecular changes involved in functional senescence of neurons since decreased visual behavior precedes retinal degeneration. Here, we sought to identify gene expression changes and the genomic features of differentially regulated genes in photoreceptors that contribute to visual senescence. RESULTS: To identify gene expression changes that could lead to visual senescence, we characterized the aging transcriptome of Drosophila sensory neurons highly enriched for photoreceptors. We profiled the nuclear transcriptome of genetically-labeled photoreceptors over a 40 day time course and identified increased expression of genes involved in stress and DNA damage response, and decreased expression of genes required for neuronal function. We further show that combinations of promoter motifs robustly identify age-regulated genes, suggesting that transcription factors are important in driving expression changes in aging photoreceptors. However, long, highly expressed and heavily spliced genes are also more likely to be downregulated with age, indicating that other mechanisms could contribute to expression changes at these genes. Lastly, we identify that circular RNAs (circRNAs) strongly increase during aging in photoreceptors. CONCLUSIONS: Overall, we identified changes in gene expression in aging Drosophila photoreceptors that could account for visual senescence. Further, we show that genomic features predict these age-related changes, suggesting potential mechanisms that could be targeted to slow the rate of age-associated visual decline.

Molecular response to the pathogen Phytophthora sojae among ten soybean near isogenic lines revealed by comparative transcriptomics.

BACKGROUND: Phytophthora root and stem rot (PRR) of soybean, caused by Phytophthora sojae, is controlled by Rps genes. However, little is known regarding the Rps-induced molecular responses to P. sojae and how they actually overlap. We thus sequenced, analyzed, and compared the transcriptomes of 10 near isogenic lines (NILs), each with a unique Rps gene/allele, and the susceptible parent Williams, pre- and post-inoculation with the pathogen. RESULTS: A total of 4,330 differentially expressed genes (DEGs) were identified in Williams versus 2,014 to 5,499 DEGs in individual NILs upon inoculation with the pathogen. Comparisons of the DEGs between the NILs and Williams identified incompatible interaction genes (IIGs) and compatible interaction genes (CIGs). Hierarchical cluster and heatmap analyses consistently grouped the NILs into three clusters: Cluster I (Rps1-a), Cluster II (Rps1-b, 1-c and 1-k) and Cluster III (Rps3-a, 3-b, 3-c, 4, 5, and 6), suggesting an overlap in Rps-induced defense signaling among certain NILs. Gene ontology (GO) analysis revealed associations between members of the WRKY family and incompatible reactions and between a number of phytohormone signaling pathways and incompatible/compatible interactions. These associations appear to be distinguished according to the NIL clusters. CONCLUSIONS: This study characterized genes and multiple branches of putative regulatory networks associated with resistance to P. sojae in ten soybean NILs, and depicted functional "fingerprints" of individual Rps-mediated resistance responses through comparative transcriptomic analysis. Of particular interest are dramatic variations of detected DEGs, putatively involved in ethylene (ET)-, jasmonic acid (JA)-, (reactive oxygen species) ROS-, and (MAP-kinase) MAPK- signaling, among these soybean NILs, implicating their important roles of these signaling in differentiating molecular defense responses. We hypothesize that different timing and robustness in defense signaling to the same pathogen may be largely responsible for such variations.

Robust adjustment of sequence tag abundance.

MOTIVATION: The majority of next-generation sequencing technologies effectively sample small amounts of DNA or RNA that are amplified (i.e. copied) before sequencing. The amplification process is not perfect, leading to extreme bias in sequenced read counts. We present a novel procedure to account for amplification bias and demonstrate its effectiveness in mitigating gene length dependence when estimating true gene expression. RESULTS: We tested the proposed method on simulated and real data. Simulations indicated that our method captures true gene expression more effectively than classic censoring-based approaches and leads to power gains in differential expression testing, particularly for shorter genes with high transcription rates. We applied our method to an unreplicated Arabidopsis RNA-seq dataset resulting in disparate gene ontologies arising from gene set enrichment analyses. AVAILABILITY AND IMPLEMENTATION: R code to perform the RASTA procedures is freely available on the web at www.stat.purdue.edu/∼doerge/.

The fate of duplicated genes in a polyploid plant genome.

Polyploidy is generally not tolerated in animals, but is widespread in plant genomes and may result in extensive genetic redundancy. The fate of duplicated genes is poorly understood, both functionally and evolutionarily. Soybean (Glycine max L.) has undergone two separate polyploidy events (13 and 59 million years ago) that have resulted in 75% of its genes being present in multiple copies. It therefore constitutes a good model to study the impact of whole-genome duplication on gene expression. Using RNA-seq, we tested the functional fate of a set of approximately 18 000 duplicated genes. Across seven tissues tested, approximately 50% of paralogs were differentially expressed and thus had undergone expression sub-functionalization. Based on gene ontology and expression data, our analysis also revealed that only a small proportion of the duplicated genes have been neo-functionalized or non-functionalized. In addition, duplicated genes were often found in collinear blocks, and several blocks of duplicated genes were co-regulated, suggesting some type of epigenetic or positional regulation. We also found that transcription factors and ribosomal protein genes were differentially expressed in many tissues, suggesting that the main consequence of polyploidy in soybean may be at the regulatory level.

The nonsense-mediated decay pathway maintains synapse architecture and synaptic vesicle cycle efficacy.

A systematic Drosophila forward genetic screen for photoreceptor synaptic transmission mutants identified no-on-and-no-off transient C (nonC) based on loss of retinal synaptic responses to light stimulation. The cloned gene encodes phosphatidylinositol-3-kinase-like kinase (PIKK) Smg1, a regulatory kinase of the nonsense-mediated decay (NMD) pathway. The Smg proteins act in an mRNA quality control surveillance mechanism to selectively degrade transcripts containing premature stop codons, thereby preventing the translation of truncated proteins with dominant-negative or deleterious gain-of-function activities. At the neuromuscular junction (NMJ) synapse, an extended allelic series of Smg1 mutants show impaired structural architecture, with decreased terminal arbor size, branching and synaptic bouton number. Functionally, loss of Smg1 results in a ~50% reduction in basal neurotransmission strength, as well as progressive transmission fatigue and greatly impaired synaptic vesicle recycling during high-frequency stimulation. Mutation of other NMD pathways genes (Upf2 and Smg6) similarly impairs neurotransmission and synaptic vesicle cycling. These findings suggest that the NMD pathway acts to regulate proper mRNA translation to safeguard synapse morphology and maintain the efficacy of synaptic function.

Differential sensitivity of the Arabidopsis thaliana transcriptome and enhancers to the effects of genome doubling.

Two fundamental types of polyploids are known: allopolyploids, in which different parental chromosome sets were combined by ancestral hybridization and duplication; and autopolyploids, which derive from multiplication of the same chromosome set. In autopolyploids, changes to the nuclear environment are not as profound as in allopolyploids, and therefore the effects of genome doubling on gene regulation remain unclear. To investigate the consequences of autopolyploidization per se, we performed a microarray analysis in three equivalent lineages of matched diploids and autotetraploids of Arabidopsis thaliana. Additionally, we compared the expression levels of GFP transgenes driven by endogenous enhancer elements (enhancer traps) in diploids and autotetraploid of 16 transgenic lines. We expected that true ploidy-dependent changes should occur in independently derived autopolyploid lineages. By this criterion, our microarray analysis detected few changes associated with polyploidization, while the enhancer-trap analysis revealed altered GFP expression at multiple plant life stages for 25% of the lines tested. Genes on individual traps were coordinately regulated while endogenous gene expression was not affected except for one line. The unique sensitivity of enhancer traps to ploidy, in contrast to the observed stability of genes, could derive from lower complexity of regulatory pathways acting on traps versus endogenous genes.

An empirical Bayesian method for estimating biological networks from temporal microarray data.

Gene regulatory networks refer to the interactions that occur among genes and other cellular products. The topology of these networks can be inferred from measurements of changes in gene expression over time. However, because the measurement device (i.e., microarrays) typically yields information on thousands of genes over few biological replicates, these systems are quite difficult to elucidate. An approach with proven effectiveness for inferring networks is the Dynamic Bayesian Network. We have developed an iterative empirical Bayesian procedure with a Kalman filter that estimates the posterior distributions of network parameters. We compare our method to similar existing methods on simulated data and real microarray time series data. We find that the proposed method performs comparably on both model-based and data-based simulations in considerably less computational time. The R and C code used to implement the proposed method are publicly available in the R package ebdbNet.

DAG lipase activity is necessary for TRP channel regulation in Drosophila photoreceptors.

In Drosophila, a phospholipase C-mediated signaling cascade links photoexcitation of rhodopsin to the opening of the TRP/TRPL channels. A lipid product of the cascade, diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs), have both been proposed as potential excitatory messengers. A crucial enzyme in the understanding of this process is likely to be DAG lipase (DAGL). However, DAGLs that might fulfill this role have not been previously identified in any organism. In this work, the Drosophila DAGL gene, inaE, has been identified from mutants that are defective in photoreceptor responses to light. The inaE-encoded protein isoforms show high sequence similarity to known mammalian DAG lipases, exhibit DAG lipase activity in vitro, and are highly expressed in photoreceptors. Analyses of norpA inaE double mutants and severe inaE mutants show that normal DAGL activity is required for the generation of physiologically meaningful photoreceptor responses.

Identification of novel high-frequency DNA methylation changes in breast cancer.

Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.

Natural variation among Arabidopsis thaliana accessions for transcriptome response to exogenous salicylic acid.

Little is known about how gene expression variation within a given species controls phenotypic variation under different treatments or environments. Here, we surveyed the transcriptome response of seven diverse Arabidopsis thaliana accessions in response to two treatments: the presence and absence of exogenously applied salicylic acid (SA), an important signaling molecule in plant defense. A factorial experiment was conducted with three biological replicates per accession with and without applications of SA and sampled at three time points posttreatment. Transcript level data from Affymetrix ATH1 microarrays were analyzed on both per-gene and gene-network levels to detect expression level polymorphisms associated with SA response. Significant variation in transcript levels for response to SA was detected among the accessions, with relatively few genes responding similarly across all accessions and time points. Twenty-five of 54 defined gene networks identified from other microarray studies (pathogen-challenged Columbia [Col-0]) showed a significant response to SA in one or more accessions. A comparison of gene-network relationships in our data to the pathogen-challenged Col-0 data demonstrated a higher-order conservation of linkages between defense response gene networks. Cvi-1 and Mt-0 appeared to have globally different SA responsiveness in comparison to the other five accessions. Expression level polymorphisms for SA response were abundant at both individual gene and gene-network levels in the seven accessions, suggesting that natural variation for SA response is prevalent in Arabidopsis.

A two-step multiple comparison procedure for a large number of tests and multiple treatments.

For situations where the number of tested hypotheses is increasingly large, the power to detect statistically significant multiple treatment effects decreases. As is the case with microarray technology, often researchers are interested in identifying differentially expressed genes for more than two types of cells or treatments. A two-step procedure is proposed for the purpose of increasing power to detect significant effects (i.e., to identify differentially expressed genes). Specifically, in the first step, the null hypothesis of equality across the mean expression levels for all treatments is tested for each gene. In the second step, only pairwise comparisons corresponding to the genes for which the treatment means are statistically different in the first step are tested. We propose an approach to estimate the overall FDR for both fixed rejection regions and fixed FDR significance levels. Also proposed is a procedure to find the FDR significance levels used in the first step and the second step such that the overall FDR can be controlled below a pre-specified FDR significance level. When compared via simulation the two-step approach has increased power over a one-step procedure, and controls the FDR at a desire significance level.

Genomic changes in synthetic Arabidopsis polyploids.

Polyploids are common and arise frequently by genome duplication (autopolyploids) or interspecific hybridization (allopolyploids). Neoallopolyploids display sterility, lethality, phenotypic instability, gene silencing and epigenetic changes. Little is known about the molecular basis of these phenomena, and how much genomic remodeling happens upon allopolyploidization. Extensive genomic remodeling has been documented in wheat, but little remodeling occurs in cotton. Newly synthesized Arabidopsis allopolyploids, which display phenotypic instability and low fertility, displayed several, possibly related mechanisms that can remodel genomes. We detected transcriptional activity of several transposons although their transposition was limited. One represents a new family of conditionally active En-Spm-like transposons of Arabidopsis thaliana, which underwent remodeling of CG methylation upon allopolyploidization. A random amplified fragment length polymorphism survey suggested remodeling at few, specific loci. Meiotic analyses revealed the appearance of chromosomal fragments in a substantial fraction of anther meiocytes. In several individuals produced by hybrids between the synthetic and a natural allopolyploid pollen viability inversely correlated with meiotic instability. Activity of selected DNA transposons and the possibly related chromosomal breaks could cause changes by inducing translocations and rearrangements.

Role of transposable elements in heterochromatin and epigenetic control.

Heterochromatin has been defined as deeply staining chromosomal material that remains condensed in interphase, whereas euchromatin undergoes de-condensation. Heterochromatin is found near centromeres and telomeres, but interstitial sites of heterochromatin (knobs) are common in plant genomes and were first described in maize. These regions are repetitive and late-replicating. In Drosophila, heterochromatin influences gene expression, a heterochromatin phenomenon called position effect variegation. Similarities between position effect variegation in Drosophila and gene silencing in maize mediated by "controlling elements" (that is, transposable elements) led in part to the proposal that heterochromatin is composed of transposable elements, and that such elements scattered throughout the genome might regulate development. Using microarray analysis, we show that heterochromatin in Arabidopsis is determined by transposable elements and related tandem repeats, under the control of the chromatin remodelling ATPase DDM1 (Decrease in DNA Methylation 1). Small interfering RNAs (siRNAs) correspond to these sequences, suggesting a role in guiding DDM1. We also show that transposable elements can regulate genes epigenetically, but only when inserted within or very close to them. This probably accounts for the regulation by DDM1 and the DNA methyltransferase MET1 of the euchromatic, imprinted gene FWA, as its promoter is provided by transposable-element-derived tandem repeats that are associated with siRNAs.

Correct estimation of preferential chromosome pairing in autotetraploids.

In recent work, a statistical model was proposed for the purpose of estimating parameters associated with quantitative trait locus (QTL) mapping and preferential pairing within a polyploidy framework. The statistical model contained several parameters that, when estimated from experimental data, supplied information about QTL, including a preferential pairing factor. Among the results reported were estimates of preferential pairing, many of which indicated high levels of preferential pairing (p = 0.60) that were inconsistent with biological expectations. By using the biological inconsistencies as our motivation, we present a reformulated statistical method for estimating preferential pairing, and use this method to reanalyze the same autotetraploid alfalfa data and to conduct a simulation study. Our results directly contradict the current findings of significant preferential pairing and affirm the traditional view of random chromosome segregation in alfalfa.

Interacting quantitative trait loci control loss of peripheral tolerance and susceptibility to autoimmune ovarian dysgenesis after day 3 thymectomy in mice.

Day 3 thymectomy (D3Tx) results in a loss of peripheral tolerance mediated by CD4(+)CD25(+) T cells and the development of autoimmune ovarian dysgenesis (AOD) in A/J and (C57BL/6J x A/J)F(1) (B6AF(1)) hybrids but not in C57BL/6J mice. Quantitative trait loci (QTL) linkage analysis using a B6AF(1) x C57BL/6J backcross population verified Aod1 and Aod2 that were previously mapped as qualitative traits. Additionally, three new QTL intervals, Aod3, Aod4, and Aod5, on chromosomes 1, 2, and 7, respectively, influencing specific subphenotypes of AOD were identified. QTL linkage analysis using the A x B and B x A recombinant inbred lines verified Aod3 and confirmed linkage to H2. Aod5 colocalized with Mater, an ovarian-specific autoantigen recognized by anti-ovarian autoantibodies in the sera of D3Tx mice. Sequence analysis of Mater identified allelic, strain-specific splice variants between A/J and C57BL/6J mice making it an attractive candidate gene for Aod5. Interaction analysis revealed significant epistatic effects between Aod1-5 and Gasa2, a locus associated with susceptibility to D3Tx-induced autoimmune gastritis, as well as with H2. These results indicate that the QTL controlling D3Tx-induced autoimmune phenomenon are both organ specific and more generalized in their effects with respect to the genesis and activity of the immunoregulatory mechanisms maintaining peripheral tolerance.