Distinct amino acid compositional requirements for formation and maintenance of the [PSI+] prion in yeast

Multiple yeast prions have been identified that result from the structural conversion of proteins into a self-propagating amyloid form. Amyloid-based prion activity in yeast requires a series of discrete steps. First, the prion protein must form an amyloid nucleus that can recruit and structurally convert additional soluble proteins. Subsequently, maintenance of the prion during cell division requires fragmentation of these aggregates to create new heritable propagons. For the Saccharomyces cerevisiae prion protein Sup35, these different activities are encoded by different regions of the Sup35 prion domain. An N-terminal glutamine/asparagine-rich nucleation domain is required for nucleation and fiber growth, while an adjacent oligopeptide repeat domain is largely dispensable for prion nucleation and fiber growth but is required for chaperone-dependent prion maintenance. Although prion activity of glutamine/asparagine-rich proteins is predominantly determined by amino acid composition, the nucleation and oligopeptide repeat domains of Sup35 have distinct compositional requirements. Here, we quantitatively define these compositional requirements in vivo. We show that aromatic residues strongly promote both prion formation and chaperone-dependent prion maintenance. In contrast, nonaromatic hydrophobic residues strongly promote prion formation but inhibit prion propagation. These results provide insight into why some aggregation-prone proteins are unable to propagate as prions

Multiple Roles for the Non-Coding RNA SRA in Regulation of Adipogenesis and Insulin Sensitivity

Peroxisome proliferator-activated receptor-γ (PPARγ) is a master transcriptional regulator of adipogenesis. Hence, the identification of PPARγ coactivators should help reveal mechanisms controlling gene expression in adipose tissue development and physiology. We show that the non-coding RNA, Steroid receptor RNA Activator (SRA), associates with PPARγ and coactivates PPARγ-dependent reporter gene expression. Overexpression of SRA in ST2 mesenchymal precursor cells promotes their differentiation into adipocytes. Conversely, knockdown of endogenous SRA inhibits 3T3-L1 preadipocyte differentiation. Microarray analysis reveals hundreds of SRA-responsive genes in adipocytes, including genes involved in the cell cycle, and insulin and TNFα signaling pathways. Some functions of SRA may involve mechanisms other than coactivation of PPARγ. SRA in adipocytes increases both glucose uptake and phosphorylation of Akt and FOXO1 in response to insulin. SRA promotes S-phase entry during mitotic clonal expansion, decreases expression of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, and increases phosphorylation of Cdk1/Cdc2. SRA also inhibits the expression of adipocyte-related inflammatory genes and TNFα-induced phosphorylation of c-Jun NH2-terminal kinase. In conclusion, SRA enhances adipogenesis and adipocyte function through multiple pathways

Somatic, germline and sex hierarchy regulated gene expression during Drosophila metamorphosis

<p>Abstract</p> <p>Background</p> <p><it>Drosophila melanogaster </it>undergoes a complete metamorphosis, during which time the larval male and female forms transition into sexually dimorphic, reproductive adult forms. To understand this complex morphogenetic process at a molecular-genetic level, whole genome microarray analyses were performed.</p> <p>Results</p> <p>The temporal gene expression patterns during metamorphosis were determined for all predicted genes, in both somatic and germline tissues of males and females separately. Temporal changes in transcript abundance for genes of known functions were found to correlate with known developmental processes that occur during metamorphosis. We find that large numbers of genes are sex-differentially expressed in both male and female germline tissues, and relatively few are sex-differentially expressed in somatic tissues. The majority of genes with somatic, sex-differential expression were found to be expressed in a stage-specific manner, suggesting that they mediate discrete developmental events. The <it>Sex-lethal </it>paralog, <it>CG3056</it>, displays somatic, male-biased expression at several time points in metamorphosis. Gene expression downstream of the somatic, sex determination genes <it>transformer </it>and <it>doublesex (dsx) </it>was examined in two-day old pupae, which allowed for the identification of genes regulated as a consequence of the sex determination hierarchy. These include the homeotic gene <it>abdominal A</it>, which is more highly expressed in females as compared to males, as a consequence of <it>dsx</it>. For most genes regulated downstream of <it>dsx </it>during pupal development, the mode of regulation is distinct from that observed for the well-studied direct targets of DSX, <it>Yolk protein 1 </it>and <it>2</it>.</p> <p>Conclusion</p> <p>The data and analyses presented here provide a comprehensive assessment of gene expression during metamorphosis in each sex, in both somatic and germline tissues. Many of the genes that underlie critical developmental processes during metamorphosis, including sex-specific processes, have been identified. These results provide a framework for further functional studies on the regulation of sex-specific development.</p

Interpretation of Mutations, Expression, Copy Number in Somatic Breast Cancer: Implications for Metastasis and Chemotherapy

Breast cancer (BC) patient management has been transformed over the last two decades due to the development and application of genome-wide technologies. The vast amounts of data generated by these assays, however, create new challenges for accurate and comprehensive analysis and interpretation. This thesis describes novel methods for fluorescence in-situ hybridization (FISH), array comparative genomic hybridization (aCGH), and next generation DNA- and RNA-sequencing, to improve upon current approaches used for these technologies. An ab initio algorithm was implemented to identify genomic intervals of single copy and highly divergent repetitive sequences that were applied to FISH and aCGH probe design. FISH probes with higher resolution than commercially available reagents were developed and validated on metaphase chromosomes. An aCGH microarray was developed that had improved reproducibility compared to the standard Agilent 44K array, which was achieved by placing oligonucleotide probes distant from conserved repetitive sequences. Splicing mutations are currently underrepresented in genome-wide sequencing analyses, and there are limited methods to validate genome-wide mutation predictions. This thesis describes Veridical, a program developed to statistically validate aberrant splicing caused by a predicted mutation. Splicing mutation analysis was performed on a large subset of BC patients previously analyzed by the Cancer Genome Atlas. This analysis revealed an elevated number of splicing mutations in genes involved in NCAM pathways in basal-like and HER2-enriched lymph node positive tumours. Genome-wide technologies were leveraged further to develop chemosensitivity models that predict BC response to paclitaxel and gemcitabine. A type of machine learning, called support vector machines (SVM), was used to create predictive models from small sets of biologically-relevant genes to drug disposition or resistance. SVM models generated were able to predict sensitivity in two groups of independent patient data. High variability between individuals requires more accurate and higher resolution genomic data. However the data themselves are insufficient; also needed are more insightful analytical methods to fully exploit these data. This dissertation presents both improvements in data quality and accuracy as well as analytical procedures, with the aim of detecting and interpreting critical genomic abnormalities that are hallmarks of BC subtypes, metastasis and therapy response

The Light Responsive Transcriptome of the Zebrafish: Function and Regulation

Most organisms possess circadian clocks that are able to anticipate the day/night cycle and are reset or “entrained” by the ambient light. In the zebrafish, many organs and even cultured cell lines are directly light responsive, allowing for direct entrainment of the clock by light. Here, we have characterized light induced gene transcription in the zebrafish at several organizational levels. Larvae, heart organ cultures and cell cultures were exposed to 1- or 3-hour light pulses, and changes in gene expression were compared with controls kept in the dark. We identified 117 light regulated genes, with the majority being induced and some repressed by light. Cluster analysis groups the genes into five major classes that show regulation at all levels of organization or in different subset combinations. The regulated genes cover a variety of functions, and the analysis of gene ontology categories reveals an enrichment of genes involved in circadian rhythms, stress response and DNA repair, consistent with the exposure to visible wavelengths of light priming cells for UV-induced damage repair. Promoter analysis of the induced genes shows an enrichment of various short sequence motifs, including E- and D-box enhancers that have previously been implicated in light regulation of the zebrafish period2 gene. Heterologous reporter constructs with sequences matching these motifs reveal light regulation of D-box elements in both cells and larvae. Morpholino-mediated knock-down studies of two homologues of the D-box binding factor Tef indicate that these are differentially involved in the cell autonomous light induction in a gene-specific manner. These findings suggest that the mechanisms involved in period2 regulation might represent a more general pathway leading to light induced gene expression

Context dependent substitution biases vary within the human genome

Background: Models of sequence evolution typically assume that different nucleotide positions evolve independently. This assumption is widely appreciated to be an over-simplification. The best known violations involve biases due to adjacent nucleotides. There have also been suggestions that biases exist at larger scales, however this possibility has not been systematically explored. Results: To address this we have developed a method which identifies over- and under-represented substitution patterns and assesses their overall impact on the evolution of genome composition. Our method is designed to account for biases at smaller pattern sizes, removing their effects. We used this method to investigate context bias in the human lineage after the divergence from chimpanzee. We examined bias effects in substitution patterns between 2 and 5 bp long and found significant effects at all sizes. This included some individual three and four base pair patterns with relatively large biases. We also found that bias effects vary across the genome, differing between transposons and non-transposons, between different classes of transposons, and also near and far from genes. Conclusions: We found that nucleotides beyond the immediately adjacent one are responsible for substantial context effects, and that these biases vary across the genome

Restless Legs Syndrome-associated intronic common variant in Meis1 alters enhancer function in the developing telencephalon

This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported).-- et al.Genome-wide association studies (GWAS) identified the MEIS1 locus for Restless Legs Syndrome (RLS), but causal single nucleotide polymorphisms (SNPs) and their functional relevance remain unknown. This locus contains a large number of highly conserved noncoding regions (HCNRs) potentially functioning as cis-regulatory modules. We analyzed these HCNRs for allele-dependent enhancer activity in zebrafish and mice and found that the risk allele of the lead SNP rs12469063 reduces enhancer activity in the Meis1 expression domain of the murine embryonic ganglionic eminences (GE). CREB1 binds this enhancer and rs12469063 affects its binding in vitro. In addition, MEIS1 target genes suggest a role in the specification of neuronal progenitors in the GE, and heterozygous Meis1-deficient mice exhibit hyperactivity, resembling the RLS phenotype. Thus, in vivo and in vitro analysis of a common SNP with small effect size showed allele-dependent function in the prospective basal ganglia representing the first neurodevelopmental region implicated in RLS.The project was supported by Fritz-Thyssen-Stiftung, Cologne, Germany (10.09.2.146; 10.12.2.183), KKF-TUM (8766156), DAAD (0811963), and COST (“HOX and TALE homeoproteins in Development and Disease”). B.S. was partially supported by DFG grants (WI 1820/4-1; WI 1820/5-1) and a TUM-Excellence stipend. The KORA study was financed by the Helmholtz Zentrum München, which is funded by the German Federal Ministry of Education and Research (BMBF) and by the State of Bavaria. KORA research was supported within the Munich Center of Health Sciences (MC Health), Ludwig-Maximilians-Universität, as part of LMUinnovativ. J.L.G.-S. and F.C. acknowledge funding of the Spanish and the Andalusian Governments and the Feder program for grants (BFU2010-14839, BFU2009-07044, CSD2007-00008, and Proyectos de Excelencia CVI-3488 and CVI 2658). This work was funded in part by a grant from the German Federal Ministry of Education and Research (BMBF) to the German Center for Diabetes Research (DZD), to the German Mouse Clinic (Infrafrontier: 01KX1012), to the German Center for Neurodegenerative Diseases (DZNE), Germany; by the Initiative and Networking Fund of the Helmholtz Association in the framework of the Helmholtz Alliance for Mental Research in an Ageing Society (HA-215); and the Munich Cluster for Systems Neurology (EXC 1010 SyNergy) and its Collaborative Research Center (CRC) 870/2 “Assembly and Function of Neuronal Circuits.”Peer Reviewe