Target Genes In Response Research Articles

BTK Modulates p53 Activity to Enhance Apoptotic and Senescent Responses.

p53 is a tumor suppressor that prevents the emergence of transformed cells by inducing apoptosis or senescence, among other responses. Its functions are regulated tightly by posttranslational modifications. Here we show that Bruton's tyrosine kinase (BTK) is a novel modulator of p53. We found that BTK is induced in response to DNA damage and p53 activation. BTK induction leads to p53 phosphorylation, which constitutes a positive feedback loop that increases p53 protein levels and enhances the transactivation of its target genes in response to stress. Inhibiting BTK reduced both p53-dependent senescence and apoptosis. Further, BTK expression also upregulated DNA damage signals and apoptosis. We conclude that despite being involved in oncogenic signals in blood malignancies, BTK has antineoplastic properties in other contexts, such as the enhancement of p53's tumor suppressor responses. Along with evidence that BTK expression correlates with good prognosis in some epithelial tumors, our findings may encourage a reevaluation of the clinical uses of BTK inhibitors in cancer therapy. Cancer Res; 76(18); 5405-14. ©2016 AACR.

Time-Course Small RNA Profiling Reveals Rice miRNAs and Their Target Genes in Response to Rice Stripe Virus Infection.

It has been known that many microRNAs (miRNAs) are involved in the regulation for the plant development and defense mechanism by regulating the expression of the target gene. Several previous studies has demonstrated functional roles of miRNAs in antiviral defense mechanisms. In this study, we employed high-throughput sequencing technology to identify rice miRNAs upon rice stripe virus (RSV) infection at three different time points. Six libraries from mock and RSV-infected samples were subjected for small RNA sequencing. Bioinformatic analyses revealed 374 known miRNAs and 19 novel miRNAs. Expression of most identified miRNAs was not dramatically changed at 3 days post infection (dpi) and 7 dpi by RSV infection. However, many numbers of miRNAs were up-regulated in mock and RSV-infected samples at 15 dpi by RSV infection. Moreover, expression profiles of identified miRNAs revealed that only few numbers of miRNAs were strongly regulated by RSV infection. In addition, 15 resistance genes were targets of six miRNAs suggesting that those identified miRNAs and 15 NBS-LRR resistance genes might be involved in RSV infection. Taken together, our results provide novel insight into the dynamic expression profiles of rice miRNAs upon RSV infection and clues for the understanding of the regulatory roles of miRNAs via time-course.

Sphingosylphosphorylcholine regulates the Hippo signaling pathway in a dual manner

Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid which regulates many cancer-related processes, including cellular proliferation. The Hippo signaling pathway consists of a cascade of tumor suppressive kinases Mst1/2 and Lats1/2 and their downstream targets YAP and TAZ which are generally pro-proliferative transcriptional regulators. Direct phosphorylation by Lats1/2 causes inhibition or degradation of YAP/TAZ and down-regulation of their target genes. We found SPC treatment of MDA-MB-435S breast cancer cells to strongly inhibit their proliferation and to induce a sustained Lats2 protein expression (6–24h). Therefore, we hypothesized that Hippo signaling might mediate the anti-proliferative SPC response. We also saw a cell density-dependent increase in S127-phosphorylated YAP (pS127-YAP) and a decrease in mRNA levels of YAP target genes (CTGF, Cyr61) in response to long (9h) SPC treatment. Knockdown of S1P receptor 2 (S1P2) prevented the SPC-induced up-regulation of Lats2 and attenuated the anti-proliferative effect of SPC. However, while knockdown of Lats2 alone or in combination with Lats1 expectedly increased basal proliferation it did not attenuate the SPC-induced inhibition of proliferation. Exogenous expression of wild-type or kinase-dead Lats2 and knockdown of YAP/TAZ also had no effect on the anti-proliferative SPC response. It has been previously shown that activation of S1P2-G12/13 by sphingosine-1-phosphate (S1P) leads to rapid de-phosphorylation and up-regulation of YAP. Similarly, we saw a decrease in pS127-YAP and an increase in total YAP levels with short (1h) SPC treatment as well as a subsequent transient increase in YAP target gene expression. Inhibition of S1P2 prevented the SPC-induced YAP de-phosphorylation. The rapid YAP activation and subsequent up-regulation of Lats2 mRNA does not constitute a negative feedback loop as knockdown of YAP/TAZ did not inhibit SPC-induced Lats2 expression. In conclusion, in this study we show that SPC is able to regulate Hippo signaling in a dual and opposite manner, causing an initial activation of YAP followed by an inhibition. However, even the strong SPC-induced effects seen in Lats2 and YAP did not mediate the anti-proliferative SPC response.

NEAT: an efficient network enrichment analysis test.

BackgroundNetwork enrichment analysis is a powerful method, which allows to integrate gene enrichment analysis with the information on relationships between genes that is provided by gene networks. Existing tests for network enrichment analysis deal only with undirected networks, they can be computationally slow and are based on normality assumptions.ResultsWe propose NEAT, a test for network enrichment analysis. The test is based on the hypergeometric distribution, which naturally arises as the null distribution in this context. NEAT can be applied not only to undirected, but to directed and partially directed networks as well. Our simulations indicate that NEAT is considerably faster than alternative resampling-based methods, and that its capacity to detect enrichments is at least as good as the one of alternative tests. We discuss applications of NEAT to network analyses in yeast by testing for enrichment of the Environmental Stress Response target gene set with GO Slim and KEGG functional gene sets, and also by inspecting associations between functional sets themselves.ConclusionsNEAT is a flexible and efficient test for network enrichment analysis that aims to overcome some limitations of existing resampling-based tests. The method is implemented in the R package neat, which can be freely downloaded from CRAN (https://cran.r-project.org/package=neat).

Water-deficit stress-responsive microRNAs and their targets in four durum wheat genotypes.

MicroRNAs (miRNAs) guide regulation at the post-transcriptional level by inducing messenger RNA (mRNA) degradation or translational inhibition of their target protein-coding genes. Durum wheat miRNAs may contribute to the genotypic water-deficit stress response in different durum varieties. Further investigation of the interactive miRNA-target regulatory modules and experimental validation of their response to water stress will contribute to our understanding of the small RNA-mediated molecular networks underlying stress adaptation in durum wheat. In this study, a comprehensive genome-wide in silico analysis using the updated Triticum transcriptome assembly identified 2055 putative targets for 113 conserved durum miRNAs and 131 targets for four novel durum miRNAs that putatively contribute to genotypic stress tolerance. Predicted mRNA targets encode various transcription factors, binding proteins and functional enzymes, which play vital roles in multiple biological pathways such as hormone signalling and metabolic processes. Quantitative PCR profiling further characterised 43 targets and 5 miRNAs with stress-responsive and/or genotype-dependent differential expression in two stress-tolerant and two stress-sensitive durum genotypes subjected to pre-anthesis water-deficit stress. Furthermore, a 5' RLM-RACE approach validated nine mRNA targets cleaved by water-deficit stress-responsive miRNAs, which, to our knowledge, has not been previously reported in durum wheat. The present study provided experimental evidence of durum miRNAs and target genes in response to water-deficit stress in contrasting durum varieties, providing new insights into the regulatory roles of the miRNA-guided RNAi mechanism underlying stress adaptation in durum wheat.

Sensory systems and ionocytes are targets for silver nanoparticle effects in fish

Some nanoparticles (NPs) may induce adverse health effects in exposed organisms, but to date the evidence for this in wildlife is very limited. Silver nanoparticles (AgNPs) can be toxic to aquatic organisms, including fish, at concentrations relevant for some environmental exposures. We applied whole mount in-situ hybridisation (WISH) in zebrafish embryos and larvae for a suite of genes involved with detoxifying processes and oxidative stress, including metallothionein (mt2), glutathionine S-transferase pi (gstp), glutathionine S-transferase mu (gstm1), haem oxygenase (hmox1) and ferritin heavy chain 1 (fth1) to identify potential target tissues and effect mechanisms of AgNPs compared with a bulk counterpart and ionic silver (AgNO3). AgNPs caused upregulation in the expression of mt2, gstp and gstm1 and down regulation of expression of both hmox1 and fth1 and there were both life stage and tissue-specific responses. Responding tissues included olfactory bulbs, lateral line neuromasts and ionocytes in the skin with the potential for effects on olfaction, behaviour and maintenance of ion balance. Silver ions induced similar gene responses and affected the same target tissues as AgNPs. AgNPs invoked levels of target gene responses more similar to silver treatments compared to coated AgNPs indicating the responses seen were due to released silver ions. In the Nrf2 zebrafish mutant, expression of mt2 (24 hpf) and gstp (3 dpf) were either non-detectable or were at lower levels compared with wild type zebrafish for exposures to AgNPs, indicating that these gene responses are controlled through the Nrf2-Keap pathway.

The role of retinoic acid receptors and their cognate ligands in reproduction in a context of triorganotin based endocrine disrupting chemicals.

Retinoic acid (RA), an active form of vitamin A, regulates the embryonic development, male and female reproduction and induces important effects on the cell development, proliferation, and differentiation. These effects are mediated by the retinoid (RAR) and rexinoid nuclear receptors (RXR), which are considered to be a ligand-activated, DNA-binding, trans-acting, and transcription-modulating proteins, involved in a general molecular mechanism responsible for the transcriptional responses in target genes. Organotin compounds are typical environmental contaminants and suspected endocrine disrupting substances. They may affect processes of reproductive system in mammals, predominantly via nuclear receptor signaling pathways. Triorganotins, such as tributyltin chloride (TBTCl) and triphenyltin chloride (TPTCl), are capable to bind to RXR molecules, and thus represent potent agonists of RXR subtypes of nuclear receptors not sharing any structural characteristics with endogenous ligands of nuclear receptors. Th is article summarizes selected effects of biologically active retinoids and rexinoids on both male and female reproduction and also deals with the effects of organotin compounds evoking endocrine disrupting actions in reproduction.

Genome-wide identification and characterization of novel lncRNAs in Populus under nitrogen deficiency.

Long non-coding RNAs (lncRNAs) have been identified as important regulatory factors of gene expression in eukaryotic species, such as Homo sapiens, Arabidopsis thaliana, and Oryza sativa. However, the systematic identification of potential lncRNAs in trees is comparatively rare. In particular, the characteristics, expression, and regulatory roles of lncRNAs in trees under nutrient stress remain largely unknown. A genome-wide strategy was used in this investigation to identify and characterize novel and low-nitrogen (N)-responsive lncRNAs in Populus tomentosa; 388 unique lncRNA candidates belonging to 380gene loci were detected and only seven lncRNAs were found to belong to seven conserved non-coding RNA families indicating the majority of P. tomentosa lncRNAs are species-specific. In total, 126 lncRNAs were significantly altered under low-N stress; 8 were repressed, and 118 were induced. Furthermore, 9 and 5 lncRNAs were detected as precursors of 11 known and 14 novel Populus miRNAs, respectively, whereas 4 lncRNAs were targeted by 29 miRNAs belonging to 5 families, including 22 conserved and 7 non-conserved miRNAs. In addition, 15 antisense lncRNAs were identified to be generated from opposite strands of 14 corresponding protein-coding genes. In total, 111 protein-coding genes with regions complementary to 38 lncRNAs were also predicted with some lncRNAs corresponding to multiple genes and vice versa, and their functions were annotated, which further demonstrated the complex regulatory relationship between lncRNAs and protein-coding genes in plants. Moreover, an interaction network among lncRNAs, miRNAs, and mRNAs was investigated. These findings enrich our understanding of lncRNAs in Populus, expand the methods of miRNA identification. Our results present the first global characterization of lncRNAs and their potential target genes in response to nitrogen stress in trees, which provides more information on low-nutrition adaptation mechanisms in woody plants.

Transcript degradation and noise of small RNA-controlled genes in a switch activated network in Escherichia coli.

Post-transcriptional regulatory processes may change transcript levels and affect cell-to-cell variability or noise. We study small-RNA downregulation to elucidate its effects on noise in the iron homeostasis network of Escherichia coli. In this network, the small-RNA RyhB undergoes stoichiometric degradation with the transcripts of target genes in response to iron stress. Using single-molecule fluorescence in situ hybridization, we measured transcript numbers of the RyhB-regulated genes sodB and fumA in individual cells as a function of iron deprivation. We observed a monotonic increase of noise with iron stress but no evidence of theoretically predicted, enhanced stoichiometric fluctuations in transcript numbers, nor of bistable behavior in transcript distributions. Direct detection of RyhB in individual cells shows that its noise is much smaller than that of these two targets, when RyhB production is significant. A generalized two-state model of bursty transcription that neglects RyhB fluctuations describes quantitatively the dependence of noise and transcript distributions on iron deprivation, enabling extraction of in vivo RyhB-mediated transcript degradation rates. The transcripts’ threshold-linear behavior indicates that the effective in vivo interaction strength between RyhB and its two target transcripts is comparable. Strikingly, the bacterial cell response exhibits Fur-dependent, switch-like activation instead of a graded response to iron deprivation.

Asymmetric arginine dimethylation of RelA provides a repressive mark to modulate TNFα/NF-κB response

Nuclear factor kappa B (NF-κB) is an inducible transcription factor that plays critical roles in immune and stress responses and is often implicated in pathologies, including chronic inflammation and cancer. Although much has been learned about NF-κB-activating pathways, the specific repression of NF-κB is far less well understood. Here we identified the type I protein arginine methyltransferase 1 (PRMT1) as a restrictive factor controlling TNFα-induced activation of NF-κB. PRMT1 forms a cellular complex with NF-κB through direct interaction with the Rel homology domain of RelA. We demonstrate that PRMT1 methylates RelA at evolutionary conserved R30, located in the DNA-binding L1 loop, which is a critical residue required for DNA binding. Asymmetric R30 dimethylation inhibits the binding of RelA to DNA and represses NF-κB target genes in response to TNFα. Molecular dynamics simulations of the DNA-bound RelA:p50 predicted structural changes in RelA caused by R30 methylation or a mutation that interferes with the stability of the DNA-NF-κB complex. Our findings provide evidence for the asymmetric arginine dimethylation of RelA and unveil a unique mechanism controlling TNFα/NF-κB signaling.

New insights into cell non-autonomous mechanisms of theC. eleganshypoxic response

The hypoxic response is a well-studied and highly conserved biological response to low oxygen availability. First described more than 20 y ago, the traditional model for this response is that declining oxygen levels lead to stabilization of hypoxia-inducible transcription factors (HIFs), which then bind to hypoxia responsive elements (HREs) in target genes to mediate the transcriptional changes collectively known as the hypoxic response.(1,2) Recent work in C. elegans has forced a re-evaluation of this model by indicating that the worm HIF (HIF-1) can mediate effects in a cell non-autonomous fashion and, in at least one case, increase expression of an intestinal hypoxic response target gene in cells lacking HIF-1.

Gad8 Protein Is Found in the Nucleus Where It Interacts with the MluI Cell Cycle Box-binding Factor (MBF) Transcriptional Complex to Regulate the Response to DNA Replication Stress

The target of rapamycin (TOR) kinase is found at the core of two evolutionarily conserved complexes known as TOR complexes 1 and 2 (TORC1 and TORC2). In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. We have previously reported that loss of function of TORC2 renders cells highly sensitive to DNA replication stress; however, the mechanism underlying this sensitivity is unknown. TORC2 has one known direct substrate, the kinase Gad8, which is related to AKT in human cells. Here we show that both TORC2 and its substrate Gad8 are found in the nucleus and are bound to the chromatin. We also demonstrate that Gad8 physically interacts with the MluI cell cycle box-binding factor (MBF) transcription complex that regulates the G1/S progression and the response to DNA stress. In mutant cells lacking TORC2 or Gad8, the binding of the MBF complex to its cognate promoters is compromised, and the induction of MBF target genes in response to DNA replication stress is reduced. Consistently, the protein levels of Cdt2 and Cig2, two MBF target genes, are reduced in the absence of TORC2-Gad8 signaling. Taken together, our findings highlight critical functions of TORC2 in the nucleus and suggest a role in surviving DNA replication stress via transcriptional regulation of MBF target genes.

MicroRNA and target gene responses to salt stress in grafted cucumber seedlings

Grafting is widely applied to enhance cucumber growth and tolerance to various biotic and abiotic adversities. Except for regulating plant growth and plant development, microRNAs (miRNAs) respond to various adversities through negative mediation of target gene expression at the post-transcriptional level. In the present study, cucumber (Cucumis sativus) scions were grafted onto pumpkin (Cucurbita moschata) rootstocks to characterize the molecular mechanisms of miRNAs and target regulatory networks that confer increased salt stress tolerance. The expression levels of 17 miRNAs and their predicted target genes were determined by quantitative real-time PCR using auto-grafted (self-grafted cucumber seedlings) and hetero-grafted (cucumber scion grafted onto pumpkin rootstock) cucumber seedlings after 24 h of 100 mmol l−1 NaCl treatment. Compared with that of auto-grafted cucumber seedlings, the expression level of most miRNAs in the leaves of hetero-grafted cucumber seedlings increased under normal conditions, decreased considerably after 2 h of salt stress, and increased after 6 and 24 h of salt stress. The expression level of most miRNAs in the roots of hetero-grafted cucumber seedlings increased under normal conditions and salt-stressed conditions. Compared with that of auto-grafted cucumber seedlings, the expression level of most target genes in the leaves of hetero-grafted cucumber seedlings decreased under normal and salt-stressed conditions. However, the expression level of most target genes in the roots of hetero-grafted cucumber seedlings increased significantly. These results provide useful information relevant to the functional analysis of miRNAs mediating graft-dependent salt tolerance.

Cooperation of Secondary Transporters and Sensor Kinases in Transmembrane Signalling: The DctA/DcuS and DcuB/DcuS Sensor Complexes of Escherichia coli.

Many membrane-bound sensor kinases require accessory proteins for function. The review describes functional control of membrane-bound sensors by transporters. The C4-dicarboxylate sensor kinase DcuS requires the aerobic or anaerobic C4-dicarboxylate transporters DctA or DcuB, respectively, for function and forms DctA/DcuS or DcuB/DcuS sensor complexes. Free DcuS is in the permanent (ligand independent) ON state. The DctA/DcuS and DcuB/DcuS complexes, on the other hand, control expression in response to C4-dicarboxylates. In DctA/DcuS, helix 8b of DctA and the PASC domain of DcuS are involved in interaction. The stimulus is perceived by the extracytoplasmic sensor domain (PASP) of DcuS. The signal is transmitted across the membrane by a piston-type movement of TM2 of DcuS which appears to be pulled (by analogy to the homologous citrate sensor CitA) by compaction of PASP after C4-dicarboxylate binding. In the cytoplasm, the signal is perceived by the PASC domain of DcuS. PASC inhibits together with DctA the kinase domain of DcuS which is released after C4-dicarboxylate binding. DcuS exhibits two modes for regulating expression of target genes. At higher C4-dicarboxylate levels, DcuS is part of the DctA/DcuS complex and in the C4-dicarboxylate-responsive form which stimulates expression of target genes in response to the concentration of the C4-dicarboxylates (catabolic use of C4-dicarboxylates, mode I regulation). At limiting C4-dicarboxylate concentrations (≤0.05mM), expression of DctA drops and free DcuS appears. Free DcuS is in the permanent ON state (mode II regulation) and stimulates low level (C4-dicarboxylate independent) DctA synthesis for DctA/DcuS complex formation and anabolic C4-dicarboxylate uptake.

Autism-Associated Chromatin Regulator Brg1/SmarcA4 Is Required for Synapse Development and Myocyte Enhancer Factor 2-Mediated Synapse Remodeling.

Synapse development requires normal neuronal activities and the precise expression of synapse-related genes. Dysregulation of synaptic genes results in neurological diseases such as autism spectrum disorders (ASD). Mutations in genes encoding chromatin-remodeling factor Brg1/SmarcA4 and its associated proteins are the genetic causes of several developmental diseases with neurological defects and autistic symptoms. Recent large-scale genomic studies predicted Brg1/SmarcA4 as one of the key nodes of the ASD gene network. We report that Brg1 deletion in early postnatal hippocampal neurons led to reduced dendritic spine density and maturation and impaired synapse activities. In developing mice, neuronal Brg1 deletion caused severe neurological defects. Gene expression analyses indicated that Brg1 regulates a significant number of genes known to be involved in synapse function and implicated in ASD. We found that Brg1 is required for dendritic spine/synapse elimination mediated by the ASD-associated transcription factor myocyte enhancer factor 2 (MEF2) and that Brg1 regulates the activity-induced expression of a specific subset of genes that overlap significantly with the targets of MEF2. Our analyses showed that Brg1 interacts with MEF2 and that MEF2 is required for Brg1 recruitment to target genes in response to neuron activation. Thus, Brg1 plays important roles in both synapse development/maturation and MEF2-mediated synapse remodeling. Our study reveals specific functions of the epigenetic regulator Brg1 in synapse development and provides insights into its role in neurological diseases such as ASD.

Complex analysis of the p53 tumor suppressor in lung carcinoma.

Lung cancer is the leading cause of cancer-related deaths worldwide. The p53 tumor suppressor is a transcription factor controlling expression of its target genes in response to various stress stimuli. Mutations of the TP53 gene occur very frequently in lung carcinomas and they play an important role in both oncogenic transformation of lung epithelial cells and lung carcinoma progression. We determined the TP53 status in 42 samples of squamous cell lung carcinoma (SQCC) and 56 samples of lung adenocarcinoma (AC) by the functional analysis FASAY and its variant called split assay. Altogether, we detected 64 TP53 mutations in 63 patients and analyzed them by cDNA and gDNA sequencing. The TP53 mutations were found in 76.2% (32/42) of SQCC cases, and 55.4% (31/56) of ACs. Immunoblotting revealed the p53 protein accumulation in 18 samples (42.9%) among SQCC cases and 19 samples (33.9%) among AC cases. Using fluorescence in situ hybridization we detected loss of the TP53-specific 17p13.3 locus in 23 from 41 analyzed SQCC samples (56.1%) and in 20 from 54 analyzed AC samples (37.0%). We did not find any statistically significant differences in overall and disease-free survival in relation to TP53 status.

The Effect of IV Iron Therapy on HIF Pathway and HIF Regulated Genes in Elderly Patients with Unexplained Anemia

LK, SS Equal contributionsVG, JTP Equal credit as senior authorsProlyl hydroxylase 2 (encoded by EGLN1), the principle negative regulator of HIF-1 and HIF-2 (encoded by EPAS1), is an iron dependent enzyme (Kaelin WG et al. Mol Cell, 2008); thus iron deficiency can augment hypoxic responses (Zhang X et al Blood Cells Mol Dis, 2014). Because EPAS1 mRNA has a 5' iron regulatory element, iron deficiency may inhibit the translation of HIF-2α and downregulate its target genes (Sanchez M et al Nat Struct Mol Biol, 2007). The Partnership for Anemia: Clinical and Translational Trials in the Elderly conducted a trial to determine the efficacy of IV iron in patients with borderline iron deficiency (Price E et al. Blood Cells Mol Dis, 2014). We measured expression levels of HIF1A, EPAS1, EGLN1 and HIF target genes in platelets and granulocytes. Nineteen patients were treated with 200 mg IV iron sucrose weekly for 5 weeks. Blood was obtained at screening, one week after the second dose of IV iron and 8 weeks after completion of iron therapy. Granulocytes and platelets were isolated and their RNA reverse transcribed. Transcripts were quantified by real-time qT-PCR, and normalized to HPRT gene. The threshold cycle for the expression of each gene was calculated and compared to 5 healthy controls.We found a positive correlation between granulocyte and platelet mRNA of HIF1A, EPAS1 and EGLN1, and these relationships achieved a one-sided significance level of 0.1 or stronger for most data points for HIF1A and EPAS1 (Table 1). There was also a positive correlation for granulocyte and platelet mRNA of 5 of 7 hypoxia response genes (P of 0.1 or stronger in many comparisons; the strongest being for VEGF and PDK1).Table 1Spearman correlation rho between granulocyte and platelet mRNAs (N = 17).GeneScreenTV2FU2Regulators of the hypoxic responseHIF1A.52**.21.40*EPAS1.40*.54**.67**EGLN1.28.30.09Responders to the hypoxic responseGLUT1.44**.00.07HK1.25.20.36*PDK1.24.55**.41*VEGF.68**.33*.62**FOXO3-.10.22.45**BNIP3.03-.19-.08BNIP3L.15-.05.20*one-sided P <0.10; **P <0.05There were positive relationships in the expression of the three hypoxic response regulating genes with lower iron stores, as reflected in three measures, and these tended to be strongest for the TfR to log ferritin index (Table 2). These relationships also tended to hold for HK1, PDK1, VEGF and BNIP3L in both types of cells, but the relationships were stronger in platelets.Table 2Spearman correlation for relationship between iron status and gene expression at screening. A negative correlation with ferritin and positive correlation with TfR and TfR/log ftn indicates increased gene expression with lower iron stores.GranulocytesPlateletsFerritinTfRTfR/log ftnFerritinTfRTfR/log ftnRegulators of the hypoxic responseHIF1A-.04.05.15-.26.41*.52**EPAS1-.31.090.23-.016.15.09EGLN1-.18.26.31-.33*.57**.66**Responders to the hypoxic responseGLUT1.39*-.33-.38*.27.35*.23HK1-.20.016.07-.22.23.23PDK1-.22.31.39*-.38*.16.24VEGF-.22.12.30-.22.48**.52**FOXO3.10-.097-.061]-.19-.15-.14BNIP3-.04-.10-.007-.18-.30-.25BNIP3L-.08.13.21-.36*.43**.50**Trends for change in gene expression with iron therapy were also observed. Iron therapy led to decreased expression of HIF1A and EGLN1 in both cell types but increased expression of EPAS1; these relationships were stronger in granulocytes, where they achieved a one-sided significance of P <0.2. IV iron tended to be associated with reduced expression of a panel of 7 HIF target genes, more so in granulocytes than in platelets. Expression of FOXO3 did not decrease with IV iron, perhaps consistent with its being a target of HIF-2 as well as HIF-1.Expression of hypoxic response regulating genes tended to correlate between platelets and granulocytes, as did expression of one-half of hypoxic response genes tested. Furthermore, expected relationships with iron status were observed for the expression of HIF1A, EPAS1 and EGLIN1. However, variable responses were found in the expression of certain HIF target genes in response to iron therapy that may be related to a balance of decreased HIF-a protein with enhanced translation of HIF-2a. Transcripts of these HIF regulated and HIF pathway genes are being compared to normals and to non-anemic elderly subjects to determine if anemia in the elderly is associated with a blunted hypoxic response. DisclosuresNo relevant conflicts of interest to declare.

Boron-deficiency-responsive microRNAs and their targets in Citrus sinensis leaves.

BackgroundMicroRNAs play important roles in the adaptive responses of plants to nutrient deficiencies. Most research, however, has focused on nitrogen (N), phosphorus (P), sulfur (S), copper (Cu) and iron (Fe) deficiencies, limited data are available on the differential expression of miRNAs and their target genes in response to deficiencies of other nutrient elements. In this study, we identified the known and novel miRNAs as well as the boron (B)-deficiency-responsive miRNAs from citrus leaves in order to obtain the potential miRNAs related to the tolerance of citrus to B-deficiency.MethodsSeedlings of ‘Xuegan’ [Citrus sinensis (L.) Osbeck] were supplied every other day with B-deficient (0 μM H3BO3) or -sufficient (10 μM H3BO3) nutrient solution for 15 weeks. Thereafter, we sequenced two small RNA libraries from B-deficient and -sufficient (control) citrus leaves, respectively, using Illumina sequencing.ResultsNinety one (83 known and 8 novel) up- and 81 (75 known and 6 novel) down-regulated miRNAs were isolated from B-deficient leaves. The great alteration of miRNA expression might contribute to the tolerance of citrus to B-deficiency. The adaptive responses of miRNAs to B-deficiency might related to several aspects: (a) attenuation of plant growth and development by repressing auxin signaling due to decreased TIR1 level and ARF-mediated gene expression by altering the expression of miR393, miR160 and miR3946; (b) maintaining leaf phenotype and enhancing the stress tolerance by up-regulating NACs targeted by miR159, miR782, miR3946 and miR7539; (c) activation of the stress responses and antioxidant system through down-regulating the expression of miR164, miR6260, miR5929, miR6214, miR3946 and miR3446; (d) decreasing the expression of major facilitator superfamily protein genes targeted by miR5037, thus lowering B export from plants. Also, B-deficiency-induced down-regulation of miR408 might play a role in plant tolerance to B-deficiency by regulating Cu homeostasis and enhancing superoxide dismutase activity.ConclusionsOur study reveals some novel responses of citrus to B-deficiency, which increase our understanding of the adaptive mechanisms of citrus to B-deficiency at the miRNA (post-transcriptional) level.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0642-y) contains supplementary material, which is available to authorized users.

Genome-Wide Mapping of the Binding Sites and Structural Analysis of Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 2 Reveal that It Is a DNA-Binding Transcription Factor.

The oncogenic herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is known to encode four viral interferon regulatory factors (vIRF1 to -4) to subvert the host antiviral immune response, but their detailed DNA-binding profiles as transcription factors in the host remain uncharacterized. Here, we first performed genome-wide vIRF2-binding site mapping in the human genome using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq). vIRF2 was capable of binding to the promoter regions of 100 putative target genes. Importantly, we confirmed that vIRF2 can specifically interact with the promoters of the genes encoding PIK3C3, HMGCR, and HMGCL, which are associated with autophagosome formation or tumor progression and metastasis, and regulate their transcription in vivo. The crystal structure of the vIRF2 DNA-binding domain (DBD) (referred to here as vIRF2DBD) showed variable loop conformations and positive-charge distributions different from those of vIRF1 and cellular IRFs that are associated with DNA-binding specificities. Structure-based mutagenesis revealed that Arg82 and Arg85 are required for the in vitro DNA-binding activity of vIRF2DBD and can abolish the transcription regulation function of vIRF2 on the promoter reporter activity of PIK3C3, HMGCR, and HMGCL. Collectively, our study provided unique insights into the DNA-binding potency of vIRF2 and suggested that vIRF2 could act as a transcription factor of its target genes in the host antiviral immune response. The oncogenic herpesvirus KSHV is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV has developed a unique mechanism to subvert the host antiviral immune responses by encoding four homologues of cellular interferon regulatory factors (vIRF1 to -4). However, none of their DNA-binding profiles in the human genome have been characterized until now, and the structural basis for their diverse DNA-binding properties remain poorly understood. In this study, we performed the first genome-wide vIRF2-binding site mapping in the human genome and found vIRF2 can bind to the promoter regions of 100 target cellular genes. X-ray structure analysis and functional studies provided unique insights into its DNA-binding potency and regulation of target gene expression. Our study suggested that vIRF2 could act as a transcription factor of its target genes and contribute to KSHV infection and pathogenesis through versatile functions.

Hormone-regulated defense and stress response networks contribute to heterosis in Arabidopsis F1 hybrids

Plant hybrids are extensively used in agriculture to deliver increases in yields, yet the molecular basis of their superior performance (heterosis) is not well understood. Our transcriptome analysis of a number of Arabidopsis F1 hybrids identified changes to defense and stress response gene expression consistent with a reduction in basal defense levels. Given the reported antagonism between plant immunity and growth, we suggest that these altered patterns of expression contribute to the greater growth of the hybrids. The altered patterns of expression in the hybrids indicate decreases to the salicylic acid (SA) biosynthesis pathway and increases in the auxin [indole-3-acetic acid (IAA)] biosynthesis pathway. SA and IAA are hormones known to control stress and defense responses as well as plant growth. We found that IAA-targeted gene activity is frequently increased in hybrids, correlating with a common heterotic phenotype of greater leaf cell numbers. Reduced SA concentration and target gene responses occur in the larger hybrids and promote increased leaf cell size. We demonstrated the importance of SA action to the hybrid phenotype by manipulating endogenous SA concentrations. Increasing SA diminished heterosis in SA-reduced hybrids, whereas decreasing SA promoted growth in some hybrids and phenocopied aspects of hybrid vigor in parental lines. Pseudomonas syringae infection of hybrids demonstrated that the reductions in basal defense gene activity in these hybrids does not necessarily compromise their ability to mount a defense response comparable to the parents.