Surviving a Genome Collision: Genomic Signatures of Allopolyploidization in the Recent Crop Species

Polyploidization has played a major role in crop plant evolution, leading to advantageous traits that have been selected by humans. Here, we describe restructuring patterns in the genome of Brassica napus L., a recent allopolyploid species. Widespread segmental deletions, duplications, and homeologous chromosome exchanges were identified in diverse genome sequences from 32 natural and 20 synthetic accessions, indicating that homeologous exchanges are a major driver of postpolyploidization genome diversification. Breakpoints of genomic rearrangements are rich in microsatellite sequences that are known to interact with the meiotic recombination machinery. In both synthetic and natural B. napus, a subgenome bias was observed toward exchanges replacing larger chromosome segments from the C-subgenome by their smaller, homeologous A-subgenome segments, driving postpolyploidization genome size reduction. Selection in natural B. napus favored segmental deletions involving genes associated with immunity, reproduction, and adaptation. Deletions affecting mismatch repair system genes, which are assumed to control homeologous recombination, were also found to be under selection. Structural exchanges between homeologous subgenomes appear to be a major source of novel genetic diversity in de novo allopolyploids. Documenting the consequences of genomic collision by genomic resequencing gives insights into the adaptive processes accompanying allopolyploidization

Capturing sequence variation among flowering-time regulatory gene homologs in the allopolyploid crop species Brassica napus

Flowering, the transition from the vegetative to the generative phase, is a decisive time point in the lifecycle of a plant. Flowering is controlled by a complex network of transcription factors, photoreceptors, enzymes and miRNAs. In recent years, several studies gave rise to the hypothesis that this network is also strongly involved in the regulation of other important lifecycle processes ranging from germination and seed development through to fundamental developmental and yield-related traits. In the allopolyploid crop species Brassica napus, (genome AACC), homoeologous copies of flowering time regulatory genes are implicated in major phenological variation within the species, however the extent and control of intraspecific and intergenomic variation among flowering-time regulators is still unclear. To investigate differences among B. napus morphotypes in relation to flowering-time gene variation, we performed targeted deep sequencing of 29 regulatory flowering-time genes in four genetically and phenologically diverse B. napus accessions. The genotype panel included a winter-type oilseed rape, a winter fodder rape, a spring-type oilseed rape (all B. napus ssp. napus) and a swede (B. napus ssp. napobrassica), which show extreme differences in winter-hardiness, vernalization requirement and flowering behaviour. A broad range of genetic variation was detected in the targeted genes for the different morphotypes, including non-synonymous SNPs, copy number variation and presence-absence variation. The results suggest that this broad variation in vernalisation, clock and signaling genes could be a key driver of morphological differentiation for flowering-related traits in this recent allopolyploid crop species

Statistische Auswertung und Interpretation von hochdimensionalen molekularbiologischen Datensätzen

Der Einsatz von Hochdurchsatz-Methoden ist in der molekularbiologischen Forschung zu einem elementaren Werkzeug zur Aufklärung der komplexen zellulären Vorgänge geworden. Ein wichtiger Aspekt bei der Durchführung von Hochdurchsatz-Methoden ist die Etablierung von geeigneten Algorithmen zur Auswertung der Daten. Für die DNA-Chiptechnologie sind in den letzen Jahren standardisierte Methoden zur Auswertung der Daten etabliert worden. Es hat sich jedoch gezeigt, dass die Interpretation der Daten im Kontext der biologischen Fragestellung sich trotz effizienter Datenanalyse als schwierig gestaltet. Deshalb wurde stärker darauf fokussiert, bioinformatische Methoden zur funktionellen Interpretation der Daten zu etablieren. In Kapitel 3 werden einige dieser Methoden dargestellt und anschließend auf einen Mikroarray-Datensatz angewendet, mit dem die c-Myc abhängige Genexpression in T-Lymphozyten von transgenen Mäusen untersucht wird. Es konnte gezeigt werden, dass die hier angewendeten Methoden für die funktionelle Interpretation von Genlisten geeignet sind. Da die Methoden zu unterschiedlichen Ergebnissen führen, bzw. auf unterschiedliche Schwerpunkte fokussieren, ist es sinnvoll, mehrere Methoden parallel anzuwenden, um eine möglichst effiziente Interpretation zu erzielen. Ein Aspekt der funktionellen Analyse ist die Untersuchung von ko-regulierten Genen eines Datensatzes auf eine mögliche Regulation durch einen gemeinsamen Transkriptionsfaktor. Eine Methode zur Detektion signifikant überrepräsentierter cis-regulativer Motive in den Promotersequenzen eines Sets von ko-regulierten Genen wurde im Rahmen der Arbeit etabliert. Diese Methode beinhaltet folgende Schritte: • Erstellung einer Datenbank mit orthologen Promotorsequenzen (Maus/Mensch) • Maskierung von repetitiven Sequenzelementen • Alignment der orthologen Sequenzen • Untersuchung von konservierten Sequenzbereichen auf Transkriptionsfaktor-Bindungsstellen (TFBS) • Korrektur der Anzahl der Bindungsstellen auf die Länge der konservierten Promotorsequenzen • Untersuchung einer Gruppe von ko-regulierten Genen auf die Anreicherung von TFBS im Vergleich zu einem Hintergrundset Die Methode wurde auf den in Kapitel 3 verwendeten Mikroarray-Datensatzes angewendet. Position weight matrices (PWM) mit E-Box-Motiven, die als Bindungsmotive für c-Myc bereits beschrieben wurden, konnten als signifikant überrepräsentiert gefunden werden. Zudem findet sich eine Anreicherung der PWM für den Transkriptionsfaktor YY1, dessen konstitutive Repression durch die Überexpression von c-Myc reduziert wird. Neben der Regulation der Genexpression z.B. durch Trankriptionsfaktoren gibt es weiteren Faktoren, die eine Rolle bei der Regulation der zellulären Prozesse spielen. Hierzu gehören u.a. die miRNAs, die als zelluläre Regulatoren in den letzten Jahren vermehrt in den Fokus der molekularbiologischen Forschung gerückt sind. Es treten z.B. zeit- und gewebespezifische miRNA-Expressions¬muster bei der Entwicklung von Pflanzen und Tieren oder bei der Regulation von physiolo¬gischen Prozessen wie der Apoptose, der Zellteilung und der Zelldifferenzierung auf. Da häufig eine Vielzahl von miRNAs an der Regulation eines Prozesses beteiligt sind, ist es notwendig, die Expression der verschiedenen miRNAs gleichzeitig zu messen, um so die entsprechenden Expressionmuster zu finden. Hierzu eignen sich miRNA-Mikroarrays, deren Auswertung im Rahmen dieser Arbeit etabliert wurde. Als Basis hierfür wurden die in Kapitel 2 beschriebenen Methoden verwendet. Die Algorithmen wurden dann in zwei Versuchen zur Untersuchung der N-Myc abhängigen miRNA Expression in vivo und in vitro eingesetzt. Die Ergebnisse beider Experimente zeigen eine deutliche Überlappung auf, somit konnte sowohl in den Vorversuchen, die im Rahmen der Etablierung durchgeführt wurden, als auch in den Experimenten gezeigt werden, dass sich die miRNA Mikroarray-Plattform für die experimentelle Anwendung eignet und die Auswertungsroutine zu Ergebnissen führt. Es wurde jedoch auch deutlich, dass durch eine Optimierung des derzeitigen Array-Designs, andere Normalisierungsmethoden angewendet werden könnten, die zu einer besseren Reduzierung von systematischen Fehlern führen könnten. Mit der RNAi-Screening Technologie steht eine weitere Hochdurchsatz-Technologie zur Verfügung, die eine Untersuchung der direkten Interaktion verschiedener Genprodukte ermöglicht. Die Screens generieren sehr große Datenmengen, die häufig eine sehr hohe Variabilität ausweisen, somit ebenfalls eine effiziente Datenanalyse erfordern. In dieser Arbeit wurde anhand der Auswertung eines shRNA-Screens, mit dem der Einfluss verschiedener Kinasen auf die Stabilität von c-Myc getestet werden sollte, die Etablierung einer Auswertungsroutine beschrieben

BMI1 is a target gene of E2F-1 and is strongly expressed in primary neuroblastomas

The oncogene BMI1 encodes a polycomb group transcription factor that is required for embryonic development and self-renewal of stem cells. Despite these important functions little is known about the regulation of BMI1 expression. A cDNA microarray based search for target genes of E2F-1 in neuroblastoma cells expressing a 4-OHT-regulated E2F-1-ER fusion protein identified many hitherto unknown E2F-1 regulated genes. A total of 10% of these genes, including BMI1, encode proteins that function primarily in the regulation of gene expression. The BMI1 promoter contains a putative E2F binding site that was required for the activation of a BMI1 promoter-dependent reporter construct by E2F-1. Chromatin immunoprecipitation revealed 4-OHT-dependent binding of E2F-1-ER and binding of endogenous E2F-1 to the BMI1 promoter in tumor cells. We have previously shown activation of the oncogene MYCN by E2F. Thus, in neuroblastomas deregulated E2F-1 can activate two oncogenes, MYCN and BMI1 that are known to co-operate in tumor formation. Consistent with a role of Bmi1 in neuroblastoma tumorigenesis we found strong Bmi1 expression in primary neuroblastomas. Our results reveal a novel link between E2F and polycomb transcription factors and suggest a role of Bmi1 in neuroblastomas

Myc regulates keratinocyte adhesion and differentiation via complex formation with Miz1

Myc plays a key role in homeostasis of the skin. We show that Miz1, which mediates Myc repression of gene expression, is expressed in the epidermal basal layer. A large percentage of genes regulated by the Myc–Miz1 complex in keratinocytes encode proteins involved in cell adhesion, and some, including the α6 and β1 integrins, are directly bound by Myc and Miz1 in vivo. Using a Myc mutant deficient in Miz1 binding (MycV394D), we show that Miz1 is required for the effects of Myc on keratinocyte responsiveness to TGF-β. Myc, but not MycV394D, decreases keratinocyte adhesion and spreading. In reconstituted epidermis, Myc induces differentiation and loss of cell polarization in a Miz1-dependent manner. In vivo, overexpression of β1 integrins restores basal layer polarity and prevents Myc-induced premature differentiation. Our data show that regulation of cell adhesion is a major function of the Myc–Miz1 complex and suggest that it may contribute to Myc-induced exit from the epidermal stem cell compartment

Endophytic Life Strategies Decoded by Genome and Transcriptome Analyses of the Mutualistic Root Symbiont Piriformospora indica

Recent sequencing projects have provided deep insight into fungal lifestyle-associated genomic adaptations. Here we report on the 25 Mb genome of the mutualistic root symbiont Piriformospora indica (Sebacinales, Basidiomycota) and provide a global characterization of fungal transcriptional responses associated with the colonization of living and dead barley roots. Extensive comparative analysis of the P. indica genome with other Basidiomycota and Ascomycota fungi that have diverse lifestyle strategies identified features typically associated with both, biotrophism and saprotrophism. The tightly controlled expression of the lifestyle-associated gene sets during the onset of the symbiosis, revealed by microarray analysis, argues for a biphasic root colonization strategy of P. indica. This is supported by a cytological study that shows an early biotrophic growth followed by a cell death-associated phase. About 10% of the fungal genes induced during the biotrophic colonization encoded putative small secreted proteins (SSP), including several lectin-like proteins and members of a P. indica-specific gene family (DELD) with a conserved novel seven-amino acids motif at the C-terminus. Similar to effectors found in other filamentous organisms, the occurrence of the DELDs correlated with the presence of transposable elements in gene-poor repeat-rich regions of the genome. This is the first in depth genomic study describing a mutualistic symbiont with a biphasic lifestyle. Our findings provide a significant advance in understanding development of biotrophic plant symbionts and suggest a series of incremental shifts along the continuum from saprotrophy towards biotrophy in the evolution of mycorrhizal association from decomposer fungi

Common Motifs in the Response of Cereal Primary Metabolism to Fungal Pathogens are not Based on Similar Transcriptional Reprogramming

During compatible interactions with their host plants, biotrophic plant–pathogens subvert host metabolism to ensure the sustained provision of nutrient assimilates by the colonized host cells. To investigate, whether common motifs can be revealed in the response of primary carbon and nitrogen metabolism toward colonization with biotrophic fungi in cereal leaves, we have conducted a combined metabolome and transcriptome study of three quite divergent pathosystems, the barley powdery mildew fungus (Blumeria graminis f.sp. hordei), the corn smut fungus Ustilago maydis, and the maize anthracnose fungus Colletotrichum graminicola, the latter being a hemibiotroph that only exhibits an initial biotrophic phase during its establishment. Based on the analysis of 42 water-soluble metabolites, we were able to separate early biotrophic from late biotrophic interactions by hierarchical cluster analysis and principal component analysis, irrespective of the plant host. Interestingly, the corresponding transcriptome dataset could not discriminate between these stages of biotrophy, irrespective, of whether transcript data for genes of central metabolism or the entire transcriptome dataset was used. Strong differences in the transcriptional regulation of photosynthesis, glycolysis, the TCA cycle, lipid biosynthesis, and cell wall metabolism were observed between the pathosystems. However, increased contents of Gln, Asn, and glucose as well as diminished contents of PEP and 3-PGA were common to early post-penetration stages of all interactions. On the transcriptional level, genes of the TCA cycle, nucleotide energy metabolism and amino acid biosynthesis exhibited consistent trends among the compared biotrophic interactions, identifying the requirement for metabolic energy and the rearrangement of amino acid pools as common transcriptional motifs during early biotrophy. Both metabolome and transcript data were employed to generate models of leaf primary metabolism during early biotrophy for the three investigated interactions

Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

<p>Abstract</p> <p>Background</p> <p>Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the <it>Xiphophorus </it>melanoma model system, a mutated version of the EGF receptor Xmrk (<it>Xiphophorus </it>melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation.</p> <p>Methods</p> <p>Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene <it>FOSL1 </it>was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated.</p> <p>Results</p> <p>Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (<it>Fosl1</it>), early growth response 1 (<it>Egr1</it>), osteopontin (<it>Opn</it>), insulin-like growth factor binding protein 3 (<it>Igfbp3</it>), dual-specificity phosphatase 4 (<it>Dusp4</it>), and tumor-associated antigen L6 (<it>Taal6</it>). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that <it>FOSL1</it>, <it>OPN</it>, <it>IGFBP3</it>, <it>DUSP4</it>, and <it>TAAL6 </it>also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of <it>FOSL1 </it>in human melanoma cell lines reduced their proliferation and migration.</p> <p>Conclusion</p> <p>Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development. Specifically, a role of FOSL1 in melanomagenic processes is demonstrated. These data are the basis for future detailed analyses of the investigated target genes.</p

gsrc: an R package for genome structure rearrangement calling

Abstract Summary Genome structure rearrangements are a common phenomenon in allopolyploid species. Deletions, duplications and homeologous non-reciprocal translocations (HNRT) between the highly similar subgenomes can be observed, which are known to have a large impact on phenotypic traits. Current research is limited because these rearrangements can be located genome wide only by cost intensive sequencing approaches and not reliably in high-density array genotyping data. We developed gsrc, an R-package to detect genome structure rearrangements from genotyping data in allopolyploid species including exchanges between subgenomes. We exemplarily apply gsrc to a publicly available Brassica napus dataset. Availability and Implementation The compiled R-package and source code are available at http://cran.r-project.org/web/packages/gsrc/. Supplementary information Supplementary data are available at Bioinformatics online. </jats:sec