TAZ AS A REGULATOR OF MESENCHYMAL TRANSFORMATION AND CLINICAL AGGRESSIVENESS IN GLIOMAS

Glioblastoma multiforme (GBM) is an aggressive, high grade brain tumor. Microarray studies have shown a subset of GBMs with a mesenchymal gene signature. This subset is associated with poor clinical outcome and resistance to treatment. To establish the molecular drivers of this mesenchymal transition, we correlated transcription factor expression to the mesenchymal signature and identified transcriptional co-activator with PDZ-binding motif (TAZ) to be highly associated with the mesenchymal shift. High TAZ expression correlated with worse clinical outcome and higher grade. These data led to the hypothesis that TAZ is critical to the mesenchymal transition and aggressive clinical behavior seen in GBM. We investigated the expression of TAZ, its binding partner TEAD, and the mesenchymal marker FN1 in human gliomas. Western analyses demonstrated increased expression of TAZ, TEAD4, and FN1 in GBM relative to lower grade gliomas. We also identified CpG islands in the TAZ promoter that are methylated in most lower grade gliomas, but not in GBMs. TAZ-methylated glioma stem cell (GSC) lines treated with a demethylation agent showed an increase in mRNA and protein TAZ expression; therefore, methylation may be another novel way TAZ is regulated since TAZ is epigenetically silenced in tumors with a better clinical outcome. To further characterize the role of TAZ in gliomagenesis, we stably silenced or over-expressed TAZ in GSCs. Silencing of TAZ decreased invasion, self-renewal, mesenchymal protein expression, and tumor-initiating capacity. Over-expression of TAZ led to an increase in invasion, mesenchymal protein expression, mesenchymal differentiation, and tumor-initiating ability. These actions are dependent on TAZ interacting with TEAD since all these effects were abrogated with TAZ could not bind to TEAD. We also show that TAZ and TEAD directly bind to mesenchymal gene promoters. Thus, TAZ-TEAD interaction is critically important in the mesenchymal shift and in the aggressive clinical behavior of GBM. We identified TAZ as a regulator of the mesenchymal transition in gliomas. TAZ could be used as a biomarker to both estimate prognosis and stratify patients into clinically relevant subgroups. Since mesenchymal transition is correlated to tumor aggressiveness, strategies to target and inhibit TAZ-TEAD and the downstream gene targets may be warranted in alternative treatment.

Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21.

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.

Identification of putative noncoding RNAs among the RIKEN mouse full-length cDNA collection

With the sequencing and annotation of genomes and transcriptomes of several eukaryotes, the importance of noncoding RNA (ncRNA)-RNA molecules that are not translated to protein products-has become more evident. A subclass of ncRNA transcripts are encoded by highly regulated, multi-exon, transcriptional units, are processed like typical protein-coding mRNAs and are increasingly implicated in regulation of many cellular functions in eukaryotes. This study describes the identification of candidate functional ncRNAs from among the RIKEN mouse full-length cDNA collection, which contains 60,770 sequences, by using a systematic computational filtering approach. We initially searched for previously reported ncRNAs and found nine murine ncRNAs and homologs of several previously described nonmouse ncRNAs. Through our computational approach to filter artifact-free clones that lack protein coding potential, we extracted 4280 transcripts as the largest-candidate set. Many clones in the set had EST hits, potential CpG islands surrounding the transcription start sites, and homologies with the human genome. This implies that many candidates are indeed transcribed in a regulated manner. Our results demonstrate that ncRNAs are a major functional subclass of processed transcripts in mammals.

Evolutionary turnover of mammalian transcription start sites

Alignments of homologous genomic sequences are widely used to identify functional genetic elements and study their evolution. Most studies tacitly equate homology of functional elements with sequence homology. This assumption is violated by the phenomenon of turnover, in which functionally equivalent elements reside at locations that are nonorthologous at the sequence level. Turnover has been demonstrated previously for transcription-factor-binding sites. Here, we show that transcription start sites of equivalent genes do not always reside at equivalent locations in the human and mouse genomes. We also identify two types of partial turnover, illustrating evolutionary pathways that could lead to complete turnover. These findings suggest that the signals encoding transcription start sites are highly flexible and evolvable, and have cautionary implications for the use of sequence-level conservation to detect gene regulatory elements.

GONOME: measuring correlations between GO terms and genomic positions

Background: Current methods to find significantly under- and over-represented gene ontology (GO) terms in a set of genes consider the genes as equally probable balls in a bag, as may be appropriate for transcripts in micro-array data. However, due to the varying length of genes and intergenic regions, that approach is inappropriate for deciding if any GO terms are correlated with a set of genomic positions. Results: We present an algorithm - GONOME - that can determine which GO terms are significantly associated with a set of genomic positions given a genome annotated with (at least) the starts and ends of genes. We show that certain GO terms may appear to be significantly associated with a set of randomly chosen positions in the human genome if gene lengths are not considered, and that these same terms have been reported as significantly over-represented in a number of recent papers. This apparent over-representation disappears when gene lengths are considered, as GONOME does. For example, we show that, when gene length is taken into account, the term development is not significantly enriched in genes associated with human CpG islands, in contradiction to a previous report. We further demonstrate the efficacy of GONOME by showing that occurrences of the proteosome-associated control element (PACE) upstream activating sequence in the S. cerevisiae genome associate significantly to appropriate GO terms. An extension of this approach yields a whole-genome motif discovery algorithm that allows identification of many other promoter sequences linked to different types of genes, including a large group of previously unknown motifs significantly associated with the terms 'translation' and 'translational elongation'. Conclusion: GONOME is an algorithm that correctly extracts over-represented GO terms from a set of genomic positions. By explicitly considering gene size, GONOME avoids a systematic bias toward GO terms linked to large genes. Inappropriate use of existing algorithms that do not take gene size into account has led to erroneous or suspect conclusions. Reciprocally GONOME may be used to identify new features in genomes that are significantly associated with particular categories of genes.

Mice and men: Their promoter properties

Using the two largest collections of Mus musculus and Homo sapiens transcription start sites ( TSSs) determined based on CAGE tags, ditags, full- length cDNAs, and other transcript data, we describe the compositional landscape surrounding TSSs with the aim of gaining better insight into the properties of mammalian promoters. We classified TSSs into four types based on compositional properties of regions immediately surrounding them. These properties highlighted distinctive features in the extended core promoters that helped us delineate boundaries of the transcription initiation domain space for both species. The TSS types were analyzed for associations with initiating dinucleotides, CpG islands, TATA boxes, and an extensive collection of statistically significant cis- elements in mouse and human. We found that different TSS types show preferences for different sets of initiating dinucleotides and ciselements. Through Gene Ontology and eVOC categories and tissue expression libraries we linked TSS characteristics to expression. Moreover, we show a link of TSS characteristics to very specific genomic organization in an example of immune- response- related genes ( GO: 0006955). Our results shed light on the global properties of the two transcriptomes not revealed before and therefore provide the framework for better understanding of the transcriptional mechanisms in the two species, as well as a framework for development of new and more efficient promoter- and gene- finding tools.

Increased DNA Methylation at the AXIN1 Gene in a Monozygotic Twin from a Pair Discordant for a Caudal Duplication Anomaly

The AXIN1 gene has been implicated in caudal duplication anomalies. Its coding region was sequenced in both members of a monozygotic ( MZ) twin pair discordant for a caudal duplication anomaly, but no mutation was found. Using bisulfite sequencing, we examined methylation at the promoter region of the AXIN1 gene in these twins and in twin and age-matched singleton controls. Methylation of the promoter region in peripheral blood mononucleated cells was variable among individuals, including MZ pairs. In the MZ pair discordant for the caudal duplication, this region of the affected twin was significantly more methylated than that of the unaffected twin (), which was significantly more P < .0001 methylated than those of the controls (). We have confirmed that this CpG island does function as a promoter P = .02 in vitro and that its activity is inversely proportional to the extent of methylation. This finding raises the possibility that hypermethylation of the AXIN1 promoter, by mechanisms as yet undetermined, is associated with the malformation. This case may be paradigmatic for some cases of MZ discordance.

Computational analysis of base composition pattern and promoter elements in the putative promoter regions in relation to expression profiles of 682 human genes on chromosome 22

The base composition pattern (BCP) in the putative promoter region (PPRs) up to 5 Kb lengths of 682 human genes on Chromosome 22 (Chr22) was examined. Two-dimensional (2D) and three-dimensional (3D) functions were designed to delineate the DNA base composition, with four major patterns identified. It is found that 17.6% genes include TATA box, 28.0% GC box, 18.9% CAAT box and 38.4% CpG islands, and approximately 10% genes have one of four putative initiator (Inr) motifs. The occurrence of the promoter elements is tightly associated with the base composition features in the promoter regions, and the associations of the base composition features with occurrence of the promoter elements in the promoter regions mediate tissue-wide expression of the genes in human. The occurrence of two or more promoter elements in the promoter regions is required for the medium- and wide-range expression profiles of the human genes on Chr22. Thus, the reported data shed light on the characteristics of the PPRs of the human genes on Chr22, which may improve our understanding of regulatory roles of the PPRs with occurrence of the promoter elements in gene expression.

Ecological investigations in the wadden area off Borkum, East Frisian Islands, Germany

Die Bodentiergemeinschaft des Wattenmeeres ist von Frühjahr bis Herbst eines jeden Jahres durch extrem hohe Dichten von Jungtieren charakterisiert. Die Kenntnisse über die Ansiedlung von fplanktischen Larven im Wattenmeer, sowie die Dynamik postlarvaler Stadien sind aufgrund der üblicherweise verwendeten, großen Siebmaschenweiten gering. Gerade aber diesen Altersstadien kommt möglicherweise eine besondere Stellung im Energiefluß des Wattenmeeres zu. An 5 Stationen (von NWL bis HWL, B1-B5) im Rückseitenwatt der ostfriesischen Insel Borkum wurden 1986 Ansiedlung, räumliche Verteilung, Wachstum, Mortalität und Produktion der Altersklasse 0 von Macoma balthica, Mya arenaria und Cerastoderma edule untersucht. Um die Ansiedlung der planktotrophen Larven dieser Arten zu beschreiben, wurden ihre Dichten in Plankton und Bodenproben miteinander verglichen. Die Untersuchungen zur Dynamik der benthischen Stadien wurden mit zwei in der Probenfläche und der Siebmaschenweite unterschiedlichen Probenserien durchgeführt. Die Drift postlarvaler Stadien wurde durch bodennahe Planktonfänge innerhalb des Eulitorals nachgewiesen. Parallel zu den Untersuchungen an der Endofauna wurden das Vorkommen und die Größe epibenthischer Räuber im Untersuchungsgebiet erfaßt. Die Hauptansiedlung von M. balthica- und M. arenaria-Larven erfolgte nahezu gleichzeitig Ende Mai/Anfang Juni. Die meisten Larven beider Arten gingen an der prielnächsten (tiefsten) Station (B1) zum Bodenleben über, gefolgt von der nächst höher gelegenen Station B2. Während frühe Bodenstadien von M. arenaria nicht im oberen Bereich des Watts (B3,B4) gefunden wurden, ist eine geringfügige Erstansiedlung von M. balthica in diesem Gebiet nicht auszuschließen. Ein die Ansiedlung limitierender Einfluß der relativ dichten Mya arenaria-Siedlung an den Stationen B1 und B2 sowie der Alttiere von M. balthica konnte nicht festgestellt werden. Die Ähnlichkeit des Ansiedlungsprozesses bei beiden Arten, die sich im Zahlenverhältnis Larvenangebot zu Anzahl der ersten Bodenstadien widerspiegelt, kann ein Hinweis auf eine überwiegend passive Ansiedlung der Larven am Boden sein. Der Ort der Hauptansiedlung von C. edule wurde durch den Transekt nicht erfaßt. Die Station B2 war zwar durch ein Herzmuschelfeld charakterisiert, dieses war aber nach zwei Eiswintern nahezu vollständig eliminiert. Der Abundanz der planktischen Larven zufolge war der Hauptansiedlungszeitraum ebenfalls Ende Mai/Anfang Juni. Zu dieser Zeit wurden nur vereinzelt frühe Bodenstadien an den Stationen B1 und B2 gefunden, keine an den Stationen B3 und B4. Während die frühen postlarvalen Stadien von M. arenaria überwiegend am Ort der Ansiedlung blieben, verbreiteten sich die von M. balthica bis in den oberen Bereich des Untersuchungsgebietes (B3-B5). Analog zu der Besiedlung dieser Gebiete durch postlarvale M. balthica wurde die im Verlauf des Untersuchungsjahres stattfindende Kolonisierung der Station B1 durch C. edule ebenfalls postlarvalem Transport zugeschrieben. Demzufolge spielt bei beiden Muschelarten postlarvaler Transport eine wichtige Rolle bei der Besiedlung von Habitaten. Planktonfänge innerhalb der bodennahen Wasserschicht bestätigten, daß im Untersuchungsgebiet M. balthica die am stärksten verdriftende Muschelart war, gefolgt von C. edule. Mortalität, Wachstum, mittlere Biomasse, Produktion und P/B-Verhältnis wurden für M. balthica an den Stationen B1, B3 und B4 sowie für M. arenaria an der Station B1 bestimmt. Wachstum und damit auch Produktion beider Arten erwiesen sich hier - wie an den höher gelegen Stationen (nur M. balthica) - als durch größenselektiven Feinddruck beeinflußt. Der Effekt postlarvalen Transports auf Wachstum wird diskutiert. Übergreifend über die auf Artebene diskutierten Ergebnisse wird die Bedeutung der Dispersion postlarvaler Stadien und die Wirkung epibenthischen Feinddrucks im Wattenmeer erörtert. Der Vergleich postlarvalen Transportes mit der Dispersion planktischer Larvenstadien, der Dispersion von Meiofauna und der Mobilität adulter Stadien der Makrofauna verdeutlicht, daß es sich hierbei um eine Strategie handeln kann, innerhalb eines unvorhersagbaren Biotops freiwerdende Ressourcen zu nutzen und dadurch Konkurrenz zu vermeiden. Es wird die Hypothese aufgestellt, daß Initialansiedlung und Immigration einerseits sowie Feinddruck und Emigration andererseits einen Regelkreis darstellen, der in verschiedenen Teilbereichen des Watts mit unterschiedlicher Geschwindigkeit abläuft.

Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility ( het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer ( HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated ""gene dumps'' and, perhaps, simultaneously, as "" gene factories''.