c-Crk proto-oncogene contributes to transcriptional repression of p120-catenin in non-small cell lung cancer cells

As a member of adherens junction, p120-catenin (p120ctn) plays a major role in cell adhesions through stabilization of E-cadherin. p120ctn is transcriptionally down-regulated in non-small cell lung cancer (NSCLC), although the molecular mechanisms underlying p120ctn repression are incompletely defined. Here we further investigated transcriptional regulation of p120ctn in NSCLC. We prepared a promoter reporter plasmid construct that contained p120ctn promoter region from position −1082 to +320 relative to transcription start site. Through serial deletion mutation analysis of the p120ctn promoter, we pinpointed cis-acting elements involved in regulation of p120ctn. We identified transcription factor SP1 as a transcriptional repressor of p120ctn that directly binds to segment (−9 to +36) of the p120ctn promoter. SP1 can receive multiple signals from several intracellular signaling pathways. Through examination of SP1 binding partners, we identified proto-oncogene c-Crk to be involved in transcriptional down-regulation of p120ctn. RNAi mediated silencing of CRK in A549, H157 and H358 cells increased p120ctn protein levels. On the other hand, over-expression of CRK-I and CRK-II in NSCLC cells down-regulated p120ctn, an effect that was abrogated by simultaneous silencing of SP1. In summary, our data provide evidence for the role of c-Crk proto-oncogene in transcriptional repression of p120ctn that further clarifies the mechanism by which this biochemical signal promotes metastasis in NSCLC

Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems

<p>Abstract</p> <p>Background</p> <p>Alfalfa, [<it>Medicago sativa </it>(L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling.</p> <p>Results</p> <p>Cell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. In addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773 using the GS FLX Titanium platform. The first alfalfa (<it>Medicago sativa</it>) gene index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the GS FLX Titanium EST sequences, and the <it>de novo </it>assembled Illumina sequences. MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1,294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Out of 55 SNPs randomly selected for experimental validation, 47 (85%) were polymorphic between the two genotypes. We also identified numerous allelic variations within each genotype. Digital gene expression analysis identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes.</p> <p>Conclusions</p> <p>Our results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a forage crop and cellulosic feedstock.</p

Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells

available in PMC 2011 November 01.Cellular RNA levels are determined by the interplay of RNA production, processing and degradation. However, because most studies of RNA regulation do not distinguish the separate contributions of these processes, little is known about how they are temporally integrated. Here we combine metabolic labeling of RNA at high temporal resolution with advanced RNA quantification and computational modeling to estimate RNA transcription and degradation rates during the response of mouse dendritic cells to lipopolysaccharide. We find that changes in transcription rates determine the majority of temporal changes in RNA levels, but that changes in degradation rates are important for shaping sharp 'peaked' responses. We used sequencing of the newly transcribed RNA population to estimate temporally constant RNA processing and degradation rates genome wide. Degradation rates vary significantly between genes and contribute to the observed differences in the dynamic response. Certain transcripts, including those encoding cytokines and transcription factors, mature faster. Our study provides a quantitative approach to study the integrative process of RNA regulation.Human Frontier Science Program (Strasbourg, France)Howard Hughes Medical InstituteBurroughs Wellcome Fund (Career Award at the Scientific Interface

Deprescribing interventions and their impact on medication adherence in community-dwelling older adults with polypharmacy: a systematic review

Background: Polypharmacy, and the associated adverse drug events such as non-adherence to prescriptions, is a common problem for elderly people living with multiple comorbidities. Deprescribing, i.e. the gradual withdrawal from medications with supervision by a healthcare professional, is regarded as a means of reducing adverse effects of multiple medications including non-adherence. This systematic review examines the evidence of deprescribing as an effective strategy for improving medication adherence amongst older, community dwelling adults. Methods: A mixed methods review was undertaken. Eight bibliographic database and two clinical trials registers were searched between May and December 2017. Results were double screened in accordance with pre-defined inclusion/exclusion criteria related to polypharmacy, deprescribing and adherence in older, community dwelling populations. The Mixed Methods Appraisal Tool (MMAT) was used for quality appraisal and an a priori data collection instrument was used. For the quantitative studies, a narrative synthesis approach was taken. The qualitative data was analysed using framework analysis. Findings were integrated using a mixed methods technique. The review was performed in accordance with the PRISMA reporting statement. Results: A total of 22 original studies were included, of which 12 were RCTs. Deprescribing with adherence as an outcome measure was identified in randomised controlled trials (RCTs), observational and cohort studies from 13 countries between 1996 and 2017. There were 17 pharmacy-led interventions; others were led by General Practitioners (GP) and nurses. Four studies demonstrated an overall reduction in medications of which all studies corresponded with improved adherence. A total of thirteen studies reported improved adherence of which 5 were RCTs. Adherence was reported as a secondary outcome in all but one study. Conclusions: There is insufficient evidence to show that deprescribing improves medication adherence. Only 13 studies (of 22) reported adherence of which only 5 were randomised controlled trials. Older people are particularly susceptible to non-adherence due to multi-morbidity associated with polypharmacy. Bio-psycho-social factors including health literacy and multi-disciplinary team interventions influence adherence. The authors recommend further study into the efficacy and outcomes of medicines management interventions. A consensus on priority outcome measurements for prescribed medications is indicated

RNA-seq Analysis Reveals That an ECF σ Factor, AcsS, Regulates Achromobactin Biosynthesis in Pseudomonas syringae pv. syringae B728a

Iron is an essential micronutrient for Pseudomonas syringae pv. syringae strain B728a and many other microorganisms; therefore, B728a has evolved methods of iron acquirement including the use of iron-chelating siderophores. In this study an extracytoplasmic function (ECF) sigma factor, AcsS, encoded within the achromobactin gene cluster is shown to be a major regulator of genes involved in the biosynthesis and secretion of this siderophore. However, production of achromobactin was not completely abrogated in the deletion mutant, implying that other regulators may be involved such as PvdS, the sigma factor that regulates pyoverdine biosynthesis. RNA-seq analysis identified 287 genes that are differentially expressed between the AcsS deletion mutant and the wild type strain. These genes are involved in iron response, secretion, extracellular polysaccharide production, and cell motility. Thus, the transcriptome analysis supports a role for AcsS in the regulation of achromobactin production and the potential activity of both AcsS and achromobactin in the plant-associated lifestyle of strain B728a

Effects of genome-wide copy number variation on expression in mammalian cells

<p>Abstract</p> <p>Background</p> <p>There is only a limited understanding of the relation between copy number and expression for mammalian genes. We fine mapped <it>cis </it>and <it>trans </it>regulatory loci due to copy number change for essentially all genes using a human-hamster radiation hybrid (RH) panel. These loci are called copy number expression quantitative trait loci (ceQTLs).</p> <p>Results</p> <p>Unexpected findings from a previous study of a mouse-hamster RH panel were replicated. These findings included decreased expression as a result of increased copy number for 30% of genes and an attenuated relationship between expression and copy number on the X chromosome suggesting an <it>Xist </it>independent form of dosage compensation. In a separate glioblastoma dataset, we found conservation of genes in which dosage was negatively correlated with gene expression. These genes were enriched in signaling and receptor activities. The observation of attenuated X-linked gene expression in response to increased gene number was also replicated in the glioblastoma dataset. Of 523 gene deserts of size > 600 kb in the human RH panel, 325 contained <it>trans </it>ceQTLs with -log₁₀<it>P </it>> 4.1. Recently discovered genes, ultra conserved regions, noncoding RNAs and microRNAs explained only a small fraction of the results, suggesting a substantial portion of gene deserts harbor as yet unidentified functional elements.</p> <p>Conclusion</p> <p>Radiation hybrids are a useful tool for high resolution mapping of <it>cis </it>and <it>trans </it>loci capable of affecting gene expression due to copy number change. Analysis of two independent radiation hybrid panels show agreement in their findings and may serve as a discovery source for novel regulatory loci in noncoding regions of the genome.</p

A global view of porcine transcriptome in three tissues from a full-sib pair with extreme phenotypes in growth and fat deposition by paired-end RNA sequencing

<p>Abstract</p> <p>Background</p> <p>Elucidation of the pig transcriptome is essential for interpreting functional elements of the genome and understanding the genetic architecture of complex traits such as fat deposition, metabolism and growth.</p> <p>Results</p> <p>Here we used massive parallel high-throughput RNA sequencing to generate a high-resolution map of the porcine mRNA and miRNA transcriptome in liver, longissimus dorsi and abdominal fat from two full-sib F₂hybrid pigs with segregated phenotypes on growth, blood physiological and biochemical parameters, and fat deposition. We obtained 8,508,418-10,219,332 uniquely mapped reads that covered 78.0% of the current annotated transcripts and identified 48,045-122,931 novel transcript fragments, which constituted 17,085-29,499 novel transcriptional active regions in six tested samples. We found that about 18.8% of the annotated genes showed alternative splicing patterns, and alternative 3' splicing is the most common type of alternative splicing events in pigs. Cross-tissue comparison revealed that many transcriptional events are tissue-differential and related to important biological functions in their corresponding tissues. We also detected a total of 164 potential novel miRNAs, most of which were tissue-specifically identified. Integrated analysis of genome-wide association study and differential gene expression revealed interesting candidate genes for complex traits, such as <it>IGF2, CYP1A1, CKM </it>and <it>CES1 </it>for heart weight, hemoglobin, pork pH value and serum cholesterol, respectively.</p> <p>Conclusions</p> <p>This study provides a global view of the complexity of the pig transcriptome, and gives an extensive new knowledge about alternative splicing, gene boundaries and miRNAs in pigs. Integrated analysis of genome wide association study and differential gene expression allows us to find important candidate genes for porcine complex traits.</p

Chromatin Organization in Sperm May Be the Major Functional Consequence of Base Composition Variation in the Human Genome

Chromatin in sperm is different from that in other cells, with most of the genome packaged by protamines not nucleosomes. Nucleosomes are, however, retained at some genomic sites, where they have the potential to transmit paternal epigenetic information. It is not understood how this retention is specified. Here we show that base composition is the major determinant of nucleosome retention in human sperm, predicting retention very well in both genic and non-genic regions of the genome. The retention of nucleosomes at GC-rich sequences with high intrinsic nucleosome affinity accounts for the previously reported retention at transcription start sites and at genes that regulate development. It also means that nucleosomes are retained at the start sites of most housekeeping genes. We also report a striking link between the retention of nucleosomes in sperm and the establishment of DNA methylation-free regions in the early embryo. Taken together, this suggests that paternal nucleosome transmission may facilitate robust gene regulation in the early embryo. We propose that chromatin organization in the male germline, rather than in somatic cells, is the major functional consequence of fine-scale base composition variation in the human genome. The selective pressure driving base composition evolution in mammals could, therefore, be the need to transmit paternal epigenetic information to the zygote