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Purpose This study aims to examine the drivers of sustainable entrepreneurship intention (SEI) among university students in Pakistan. Design/methodology/approach A survey-based methodology was used to collect the data from the participants. Further, this study used covariance-based structural equation modeling to test the proposed hypotheses. Findings The findings of this study confirm that the model of sustainable entrepreneurship is effective as it explains 83% variance to predict SEI among the students. Originality/value This research has contributed to the literature of sustainable entrepreneurship and proposed valuable insights in understanding the influence of sustainability education, sustainability attitude, self-efficacy and self-identity on SEI.

In order to investigate the purchase intention of the customers, and its relationship with perceived humor, this research analysesdifferent types of humor that havebeen used in advertising campaigns. In this regard, multi-mediation analysis has been applied keeping in mind the perspective of humor in advertising; moreover, attitudes towards the advertisement, and attitudes towards the brand are used as sequential mediators. In addition, self-monitoring and message arguments have also been included as moderators in order to estimate the interaction effect in theproposedmodel. The data has been gathered from 617 respondentsafter showing them relevant advertisements. After checking the normality of the data, the Confirmatory Factor Analysis was applied using AMOS. Also, Simple Linear Regression was used to test simple relationships; however, complex models have also been tested by usingthe hierarchal regression (to test moderation), and the bootstrapping (to test mediation) methods. A good fitbetween the data and the tested model was observed. As predicted, the purchase intentions were positively related to perceived humor, and evidence of full mediation effect was found. However, for the moderation of self-monitoring and message arguments, the findings were distinctive. In reality, this research can be used by mature brands which mostly require the use of persuasive advertising. As the results of different types of humor, collectively, are significant, it can be inferred that companies must use a mix method approach in terms of types of humor they might resort to in their advertisementcampaigns. Findings are particularly noteworthy for national and multinational media agencies in Pakistan, as well in the other parts of the world.

The main objective of this study is to analyze the brand image, price and service quality dimensions in creating customer satisfaction in apparel industry in Karachi. Brand image, price image, service quality and customer satisfaction are the variables used in this research. The purpose of this study is to define, what elements of brand image, price image and service quality are affecting the customer satisfaction when taking a purchase decision. Furthermore, after investigating on the mentioned variables, new opportunities are opening their doors for the marketers to create a sustainable market in the future, and understand the buyer?s black box which will broaden the marketer?s scope and create awareness to enhance the customer satisfaction. Primary data is collected throughsurvey questionnaire from 270 respondents. Regression analysis is used to infer the data. The results show that there is a significant positive relationship between brand, price image, service quality and customer satisfaction.

The purpose of this study is to investigate the ways of achieving Science, Technology and Innovation (STI) in UAE. Previously, sustainability within an entrepreneurship context has been related to economic viability as opposed to sustainability in its broadest sense. Through a survey research method, we have highlighted the effects of three independent variables and two intervening variables on three important outcomes, innovation, need for achievement and motivation, which ultimately contributes towards STI. These data have been collected from the students of a well-known university in Al-Ain, UAE. The responses of 251 students have been utilized for analysis. For hypotheses testing, we used AMOS 18 (Structural Equation Modeling) and SPSS 20. The study revealed that all of the predictors have a strong effect on the outcome variables, which leads to STI in UAE. There is a strong need to revise the curriculum of higher education institutions of UAE to develop self-confidence, locus of control and risk taking propensity among students. The study provides novel insight into entrepreneurship education and serves as an initial benchmark in the field.

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function as posttranscriptional regulators of gene expression. Many miRNAs are expressed in the developing brain and regulate multiple aspects of neural development, including neurogenesis, dendritogenesis, and synapse formation. Rett syndrome (RTT) is a progressive neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG–binding protein 2 ( MECP2 ). Although Mecp2 is known to act as a global transcriptional regulator, miRNAs that are directly regulated by Mecp2 in the brain are not known. Using massively parallel sequencing methods, we have identified miRNAs whose expression is altered in cerebella of Mecp2 -null mice before and after the onset of severe neurological symptoms. In vivo genome-wide analyses indicate that promoter regions of a significant fraction of dysregulated miRNA transcripts, including a large polycistronic cluster of brain-specific miRNAs, are DNA-methylated and are bound directly by Mecp2. Functional analysis demonstrates that the 3′ UTR of messenger RNA encoding Brain-derived neurotrophic factor ( Bdnf ) can be targeted by multiple miRNAs aberrantly up-regulated in the absence of Mecp2. Taken together, these results suggest that dysregulation of miRNAs may contribute to RTT pathoetiology and also may provide a valuable resource for further investigations of the role of miRNAs in RTT.

Purpose: In this study, the impact of Services cape on Loyalty has been studied in the presence of Social Class as a moderating variable. Methodology: The probability based systematic random sampling technique is used to collect the data through a self-administered survey to test the gained hypothesis from 308 respondents living in upper, middle and lower classes. A structured questionnaire was approved of actual information from the respondents to support the collection gained. The conceptual development of costumer’s loyalty measurement scale proposed by McMullan (2005) was adopted for analyzing the loyalty and for computing the services cape of the restaurant the instrument proposed by Hightower (2010) was acquired. SPSS and AMOS were being used to check the questionnaire. The instruments reliability was checked by using confirmatory factor analysis (CFA) and composite reliability test. SEM (structural equation modelling) through AMOS was used to examine the data. Moderation and moderation steps were analyzed by using orthogonalization and 5000 bootstrapping samples, SPSS MACRO developed by preacher & Hays to check the moderation. Findings: Ambient = 0.71, social = 0.75, design = 0.72 and customer loyalty 0.70 is the outcome of CFA. The positive relation of the loyalty of restaurant with ambiance, social dimension and design dimension of services cape was tested as the first three hypothesis using linear regression. Moreover, the analyzed result of hypothesis 4, 5 and 6 indicated the outcome that social class effects while picking the restaurant. Originality/ Value: By this research, the buying and spending pattern of consumers in Pakistan and the factors that influence the repeat selection of the restaurant in presence of their social class of the target market has been brought into attention. KeyWords: Services cape, Loyalty, Services Marketing, Social Dimension, Design Dimension, Ambiance, Physical Environment..

NF-kappaB is a key mediator of inflammation. Here, we mapped the genome-wide loci bound by the RELA subunit of NF-kappaB in lipopolysaccharide (LPS)-stimulated human monocytic cells, and together with global gene expression profiling, found an overrepresentation of the E2F1-binding motif among RELA-bound loci associated with NF-kappaB target genes. Knockdown of endogenous E2F1 impaired the LPS inducibility of the proinflammatory cytokines CCL3(MIP-1alpha), IL23A(p19), TNF-alpha, and IL1-beta. Upon LPS stimulation, E2F1 is rapidly recruited to the promoters of these genes along with p50/RELA heterodimer via a mechanism that is dependent on NF-kappaB activation. Together with the observation that E2F1 physically interacts with p50/RELA in LPS-stimulated cells, our findings suggest that NF-kappaB recruits E2F1 to fully activate the transcription of NF-kappaB target genes. Global gene expression profiling subsequently revealed a spectrum of NF-kappaB target genes that are positively regulated by E2F1, further demonstrating the critical role of E2F1 in the Toll-like receptor 4 pathway.

Identification of unconventional functional features such as fusion transcripts is a challenging task in the effort to annotate all functional DNA elements in the human genome. Paired-End diTag (PET) analysis possesses a unique capability to accurately and efficiently characterize the two ends of DNA fragments, which may have either normal or unusual compositions. This unique nature of PET analysis makes it an ideal tool for uncovering unconventional features residing in the human genome. Using the PET approach for comprehensive transcriptome analysis, we were able to identify fusion transcripts derived from genome rearrangements and actively expressed retrotransposed pseudogenes, which would be difficult to capture by other means. Here, we demonstrate this unique capability through the analysis of 865,000 individual transcripts in two types of cancer cells. In addition to the characterization of a large number of differentially expressed alternative 5′ and 3′ transcript variants and novel transcriptional units, we identified 70 fusion transcript candidates in this study. One was validated as the product of a fusion gene between BCAS4 and BCAS3 resulting from an amplification followed by a translocation event between the two loci, chr20q13 and chr17q23. Through an examination of PETs that mapped to multiple genomic locations, we identified 4055 retrotransposed loci in the human genome, of which at least three were found to be transcriptionally active. The PET mapping strategy presented here promises to be a useful tool in annotating the human genome, especially aberrations in human cancer genomes.

The protooncogene MYC encodes the c-Myc transcription factor that regulates cell growth, cell proliferation, cell cycle, and apoptosis. Although deregulation of MYC contributes to tumorigenesis, it is still unclear what direct Myc-induced transcriptomes promote cell transformation. Here we provide a snapshot of genome-wide, unbiased characterization of direct Myc binding targets in a model of human B lymphoid tumor using ChIP coupled with pair-end ditag sequencing analysis (ChIP-PET). Myc potentially occupies >4,000 genomic loci with the majority near proximal promoter regions associated frequently with CpG islands. Using gene expression profiles with ChIP-PET, we identified 668 direct Myc-regulated gene targets, including 48 transcription factors, indicating that Myc is a central transcriptional hub in growth and proliferation control. This first global genomic view of Myc binding sites yields insights of transcriptional circuitries and cis regulatory modules involving Myc and provides a substantial framework for our understanding of mechanisms of Myc-induced tumorigenesis.

We have developed a DNA tag sequencing and mapping strategy called gene identification signature (GIS) analysis, in which 5' and 3' signatures of full-length cDNAs are accurately extracted into paired-end ditags (PETs) that are concatenated for efficient sequencing and mapped to genome sequences to demarcate the transcription boundaries of every gene. GIS analysis is potentially 30-fold more efficient than standard cDNA sequencing approaches for transcriptome characterization. We demonstrated this approach with 116,252 PET sequences derived from mouse embryonic stem cells. Initial analysis of this dataset identified hundreds of previously uncharacterized transcripts, including alternative transcripts of known genes. We also uncovered several intergenically spliced and unusual fusion transcripts, one of which was confirmed as a trans-splicing event and was differentially expressed. The concept of paired-end ditagging described here for transcriptome analysis can also be applied to whole-genome analysis of cis-regulatory and other DNA elements and represents an important technological advance for genome annotation.

Structural variations (SVs) contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. While genomic DNA paired-end-tag (DNA-PET) sequencing is an attractive approach to identify genomic SVs, the current application of PET sequencing with short insert size DNA can be insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We employed a recently developed procedure to generate PET sequencing data using large DNA inserts of 10–20 kb and compared their characteristics with short insert (1 kb) libraries for their ability to identify SVs. Our results suggest that although short insert libraries bear an advantage in identifying small deletions, they do not provide significantly better breakpoint resolution. In contrast, large inserts are superior to short inserts in providing higher physical genome coverage for the same sequencing cost and achieve greater sensitivity, in practice, for the identification of several classes of SVs, such as copy number neutral and complex events. Furthermore, our results confirm that large insert libraries allow for the identification of SVs within repetitive sequences, which cannot be spanned by short inserts. This provides a key advantage in studying rearrangements in cancer, and we show how it can be used in a fusion-point-guided-concatenation algorithm to study focally amplified regions in cancer.

Eukaryotic cells make many types of primary and processed RNAs that are found either in specific subcellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic subcellular localizations are also poorly understood. Because RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell's regulatory capabilities are focused on its synthesis, processing, transport, modification and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three-quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations, taken together, prompt a redefinition of the concept of a gene. This work was supported by the National Human Genome Research Institute (NHGRI) production grants U54HG004557, U54HG004555, U54HG004576 and U54HG004558, and by the NHGRI pilot grant R01HG003700. It was also supported by the NHGRI ARRA stimulus grant 1RC2HG005591, the National Science Foundation (SNF) grant 127375, the European Research Council (ERC) grant/n249968, a research grant for the RIKEN Omics Science Center from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and grants BIO2011-26205, CSD2007-00050 and INB GNV-1 from the Spanish Ministry of Science

Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor-mutated non-small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism-associated TKI resistance.

Somatic genome rearrangements are thought to play important roles in cancer development. We optimized a long-span paired-end-tag (PET) sequencing approach using 10-Kb genomic DNA inserts to study human genome structural variations (SVs). The use of a 10-Kb insert size allows the identification of breakpoints within repetitive or homology-containing regions of a few kilobases in size and results in a higher physical coverage compared with small insert libraries with the same sequencing effort. We have applied this approach to comprehensively characterize the SVs of 15 cancer and two noncancer genomes and used a filtering approach to strongly enrich for somatic SVs in the cancer genomes. Our analyses revealed that most inversions, deletions, and insertions are germ-line SVs, whereas tandem duplications, unpaired inversions, interchromosomal translocations, and complex rearrangements are over-represented among somatic rearrangements in cancer genomes. We demonstrate that the quantitative and connective nature of DNA–PET data is precise in delineating the genealogy of complex rearrangement events, we observe signatures that are compatible with breakagefusion-bridge cycles, and we discover that large duplications are among the initial rearrangements that trigger genome instability for extensive amplification in epithelial cancers. [Supplemental material is available for this article. The sequencing data from this study have been submitted to NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE26954.]

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome., National Human Genome Research Institute (U.S.), National Institutes of Health (U.S.)

Mammalian genomes are viewed as functional organizations that orchestrate spatial and temporal gene regulation. CTCF, the most characterized insulator-binding protein, has been implicated as a key genome organizer. However, little is known about CTCF-associated higher-order chromatin structures at a global scale. Here we applied chromatin interaction analysis by paired-end tag (ChIA-PET) sequencing to elucidate the CTCF-chromatin interactome in pluripotent cells. From this analysis, we identified 1,480 cis- and 336 trans-interacting loci with high reproducibility and precision. Associating these chromatin interaction loci with their underlying epigenetic states, promoter activities, enhancer binding and nuclear lamina occupancy, we uncovered five distinct chromatin domains that suggest potential new models of CTCF function in chromatin organization and transcriptional control. Specifically, CTCF interactions demarcate chromatin-nuclear membrane attachments and influence proper gene expression through extensive cross-talk between promoters and regulatory elements. This highly complex nuclear organization offers insights toward the unifying principles that govern genome plasticity and function.

MBF (or DSC1) is known to regulate transcription of a set of G 1 /S-phase genes encoding proteins involved in regulation of DNA replication. Previous studies have shown that MBF binds not only the promoter of G 1 /S-phase genes, but also the constitutive genes; however, it was unclear if the MBF bindings at the G 1 /S-phase and constitutive genes were mechanistically distinguishable. Here, we report a chromatin immunoprecipitation-microarray (ChIP-chip) analysis of MBF binding in the Schizosaccharomyces pombe genome using high-resolution genome tiling microarrays. ChIP-chip analysis indicates that the majority of the MBF occupancies are located at the intragenic regions. Deconvolution analysis using Rpb1 ChIP-chip results distinguishes the Cdc10 bindings at the Rpb1-poor loci (promoters) from those at the Rpb1-rich loci (intragenic sequences). Importantly, Res1 binding at the Rpb1-poor loci, but not at the Rpb1-rich loci, is dependent on the Cdc10 function, suggesting a distinct binding mechanism. Most Cdc10 promoter bindings at the Rpb1-poor loci are associated with the G 1 /S-phase genes. While Res1 or Res2 is found at both the Cdc10 promoter and intragenic binding sites, Rep2 appears to be absent at the Cdc10 promoter binding sites but present at the intragenic sites. Time course ChIP-chip analysis demonstrates that Rep2 is temporally accumulated at the coding region of the MBF target genes, resembling the RNAP-II occupancies. Taken together, our results show that deconvolution analysis of Cdc10 occupancies refines the functional subset of genomic binding sites. We propose that the MBF activator Rep2 plays a role in mediating the cell cycle-specific transcription through the recruitment of RNAP-II to the MBF-bound G 1 /S-phase genes.

Summary: The algorithm MGR enables the reconstruction of rearrangement phylogenies based on gene or synteny block order in multiple genomes. Although MGR has been successfully applied to study the evolution of different sets of species, its utilization has been hampered by the prohibitive running time for some applications. In the current work, we have designed new heuristics that significantly speed up the tool without compromising its accuracy. Moreover, we have developed a web server (webMGR) that includes elaborate web output to facilitate navigation through the results. Availability: webMGR can be accessed via http://www.gis.a-star.edu.sg/~bourque. The source code of the improved standalone version of MGR is also freely available from the web site. Contact: bourque@gis.a-star.edu.sg Supplementary information: Supplementary data are available at Bioinformatics online.

Careful analysis of microarray probe design should be an obligatory component of MicroArray Quality Control (MACQ) project [Patterson et al., 2006; Shi et al., 2006] initiated by the FDA (USA) in order to provide quality control tools to researchers of gene expression profiles and to translate the microarray technology from bench to bedside. The identification and filtering of unreliable probesets are important preprocessing steps before analysis of microarray data. These steps may result in an essential improvement in the selection of differentially expressed genes, gene clustering and construction of co-regulatory expression networks. We revised genome localization of the Affymetrix U133AB GeneChip initial (target) probe sequences, and evaluated the impact of erroneous and poorly annotated target sequences on the quality of gene expression data. We found about 25% of Affymetrix target sequences overlapping with interspersed repeats that could cause cross-hybridization effects. In total, discrepancies in target sequence annotation account for up to approximately 30% of 44692 Affymetrix probesets. We introduce a novel quality control algorithm based on target sequence mapping onto genome and GeneChip expression data analysis. To validate the quality of probesets we used expression data from large, clinically and genetically distinct groups of breast cancers (249 samples). For the first time, we quantitatively evaluated the effect of repeats and other sources of inadequate probe design on the specificity, reliability and discrimination ability of Affymetrix probesets. We propose that only functionally reliable Affymetrix probesets that passed our quality control algorithm (approximately 86%) for gene expression analysis should be utilized. The target sequence annotation and filtering is available upon request.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view about chromatin structure has emerged, including its interrelationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded novel mechanistic and evolutionary insights about the functional landscape of the human genome. Together, these studies are defining a path forward to pursue a more-comprehensive characterisation of human genome function.

SummaryEpigenetic modifications are crucial for proper lineage specification and embryo development. To explore the chromatin modification landscapes in human ES cells, we profiled two histone modifications, H3K4me3 and H3K27me3, by ChIP coupled with the paired-end ditags sequencing strategy. H3K4me3 was found to be a prevalent mark and occurred in close proximity to the promoters of two-thirds of total human genes. Among the H3K27me3 loci identified, 56% are associated with promoters and the vast majority of them are comodified by H3K4me3. By deep-transcript digital counting, 80% of H3K4me3 and 36% of comodified promoters were found to be transcribed. Remarkably, we observed that different combinations of histone methylations are associated with genes from distinct functional categories. These global histone methylation maps provide an epigenetic framework that enables the discovery of novel transcriptional networks and delineation of different genetic compartments of the pluripotent cell genome.

Unequivocal demonstration of the therapeutic utility of γ-retroviral vectors for gene therapy applications targeting the hematopoietic system was accompanied by instances of insertional mutagenesis. These events stimulated the ongoing development of putatively safer integrating vector systems and analysis methods to characterize and compare integration site (IS) biosafety profiles. Continuing advances in next-generation sequencing technologies are driving the generation of ever-more complex IS datasets. Available bioinformatic tools to compare such datasets focus on the association of integration sites (ISs) with selected genomic and epigenetic features, and the choice of these features determines the ability to discriminate between datasets. We describe the scalable application of point-process coherence analysis (CA) to compare patterns produced by vector ISs across genomic intervals, uncoupled from association with genomic features. To explore the utility of CA in the context of an unresolved question, we asked whether the differing transduction conditions used in the initial Paris and London SCID-X1 gene therapy trials result in divergent genome-wide integration profiles. We tested a transduction carried out under each condition, and showed that CA could indeed resolve differences in IS distributions. Existence of these differences was confirmed by the application of established methods to compare integration datasets.

The use of tyrosine kinase inhibitors (TKI) to target oncogenic kinases has led to remarkable responses in patients with chronic myeloid leukemia (CML) and EGFR-mutated non-small cell lung cancer (EGFR NSCLC). However, a significant subset of patients have a minimal or very brief response. It has been suggested that germline polymorphisms may account for this upfront TKI resistance, and that identifying such polymorphisms will allow personalization of targeted therapy to achieve optimal responses in patients. Using paired-end DNA sequencing, we discovered a common (12.3% carrier rate) deletion polymorphism in intron 2 of the BIM gene. BIM is a pro-apoptotic member of the BCL2 family of proteins, and is required for TKIs to induce apoptosis in many cancers. We investigated the effects of the polymorphism on BIM function and TKI resistance in CML and EGFR NSCLC. Inspection of BIM gene structure suggested the polymorphism would result in mutually exclusive splicing of exon 3 (E3) and 4 (E4). Importantly, such an event is predicted to affect TKI sensitivity, since the pro-apoptotic BH3 domain is found only in E4. Using minigenes, we confirmed the deletion favored splicing of E3 over E4 by 5-fold (p=0.008), and that the deletion contained a cis-acting splicing suppressor. Next, using Zn finger nuclease-editing, we recreated the polymorphism in TKI sensitive CML (K562) and EGFR NSCLC (PC9) cell lines. Polymorphism-containing subclones had increased E3/E4 transcript ratios, decreased expression of BH3-containing BIM protein and defective apoptotic signaling, and were intrinsically TKI resistant. Importantly, while manipulation of E3-containing transcript levels did not alter the resistance phenotype, pharmacologic restoration of BH3 function (using a BH3 mimetic ABT-737) restored apoptotic signaling as well as TKI-sensitivity. Finally, we determined if the polymorphism predicted for inferior clinical responses in TKI-treated CML and EGFR NSCLC patients. In 203 CML patients, the polymorphism predicted inferior imatinib responses (defined by EuropeanLeukemiaNet criteria) among those with the polymorphism vs those without (odds ratio=2.94, p=0.02, 95% CI 1.17-7.43). In 141 EGFR NSCLC patients, the polymorphism predicted a shorter PFS of 6.6 vs 11.9 months (p=0.0027), and was independently prognostic for poorer PFS (hazard ratio=2.14, p=0.0026, 95% CI 1.30-3.50). In summary, by altering BIM splicing, the BIM polymorphism is sufficient to cause intrinsic TKI resistance in vitro, and predicts inferior TKI responses in patients. Upfront testing of CML and EGFR NSCLC patients for the BIM polymorphism may identify individuals at risk for developing clinical TKI resistance. Our results also offer an explanation for the heterogeneity of TKI responses among CML and EGFR NSCLC patients, and suggest the possibility of personalizing therapy with BH3 mimetics to improve TKI responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1911. doi:1538-7445.AM2012-1911

BACKGROUND: Gastric cancer is the second highest cause of global cancer mortality. To explore the complete repertoire of somatic alterations in gastric cancer, we combined massively parallel short read and DNA paired-end tag sequencing to present the first whole-genome analysis of two gastric adenocarcinomas, one with chromosomal instability and the other with microsatellite instability. RESULTS: Integrative analysis and de novo assemblies revealed the architecture of a wild-type KRAS amplification, a common driver event in gastric cancer. We discovered three distinct mutational signatures in gastric cancer--against a genome-wide backdrop of oxidative and microsatellite instability-related mutational signatures, we identified the first exome-specific mutational signature. Further characterization of the impact of these signatures by combining sequencing data from 40 complete gastric cancer exomes and targeted screening of an additional 94 independent gastric tumors uncovered ACVR2A, RPL22 and LMAN1 as recurrently mutated genes in microsatellite instability-positive gastric cancer and PAPPA as a recurrently mutated gene in TP53 wild-type gastric cancer. CONCLUSIONS: These results highlight how whole-genome cancer sequencing can uncover information relevant to tissue-specific carcinogenesis that would otherwise be missed from exome-sequencing data.

Nat. Genet. 43, 630–638 (2011); published online 19 June; corrected after print 11 July 2011 In the version of this article initially published, the accession codes section contained inaccuracies. The raw sequences and processed data generated from this study can be downloaded with accession number GSE28247.

SummaryThe ability to derive a whole-genome map of transcription-factor binding sites (TFBS) is crucial for elucidating gene regulatory networks. Herein, we describe a robust approach that couples chromatin immunoprecipitation (ChIP) with the paired-end ditag (PET) sequencing strategy for unbiased and precise global localization of TFBS. We have applied this strategy to map p53 targets in the human genome. From a saturated sampling of over half a million PET sequences, we characterized 65,572 unique p53 ChIP DNA fragments and established overlapping PET clusters as a readout to define p53 binding loci with remarkable specificity. Based on this information, we refined the consensus p53 binding motif, identified at least 542 binding loci with high confidence, discovered 98 previously unidentified p53 target genes that were implicated in novel aspects of p53 functions, and showed their clinical relevance to p53-dependent tumorigenesis in primary cancer samples.

SummaryTranscription factors (TFs) and their specific interactions with targets are crucial for specifying gene-expression programs. To gain insights into the transcriptional regulatory networks in embryonic stem (ES) cells, we use chromatin immunoprecipitation coupled with ultra-high-throughput DNA sequencing (ChIP-seq) to map the locations of 13 sequence-specific TFs (Nanog, Oct4, STAT3, Smad1, Sox2, Zfx, c-Myc, n-Myc, Klf4, Esrrb, Tcfcp2l1, E2f1, and CTCF) and 2 transcription regulators (p300 and Suz12). These factors are known to play different roles in ES-cell biology as components of the LIF and BMP signaling pathways, self-renewal regulators, and key reprogramming factors. Our study provides insights into the integration of the signaling pathways into the ES-cell-specific transcription circuitries. Intriguingly, we find specific genomic regions extensively targeted by different TFs. Collectively, the comprehensive mapping of TF-binding sites identifies important features of the transcriptional regulatory networks that define ES-cell identity.

Abstract 1666 The use of the tyrosine kinase inhibitor (TKI), imatinib (IM), to target the oncogenic BCR-ABL kinase has resulted in profound responses in patients with chronic phase (CP) chronic myelogenous leukemia (CML). However, a subset of patients do not respond to TKIs, and are deemed to have primary resistance. Importantly, patients with European LeukemiaNet (ELN)-defined ‘failure’ or ‘suboptimal response’ are at increased risk of poorer long-term outcomes. Little is known about mechanisms underlying primary resistance, where only a minority of patients have BCR-ABL kinase domain (KD) mutations. Interestingly, East-Asian CML patients are reported to have lower complete cytogenetic response rates compared to the West (∼50 vs 80% respectively, Au et al. 2009). We used massively parallel DNA sequencing of paired-end ditags to identify genetic factors associated with resistance in CML patient samples. We discovered a novel deletion polymorphism in the BIM gene that correlated with resistance, and which represented a common polymorphism in normal East-Asian (12.3% carriers), but not African or Caucasian (0%), populations (n=2465). BIM is a pro-apoptotic BCL2 family member, and plays a central role in CML pathophysiology. Here, BCR-ABL suppresses FoxO3a-mediated BIM transcription to maintain a survival advantage, while preventing BIM expression following BCR-ABL inhibition results in TKI resistance. Inspection of BIM gene structure suggested the polymorphism would result in mutually exclusive splicing of exon 3 (E3) vs 4 (E4), leading to decreased expression of BIM transcripts encoding the pro-apoptotic BH3 domain (found only in E4). To test this hypothesis, we constructed a minigene to measure E3 vs E4 splicing, and found the polymorphism decreased splicing to E4 over E3 by >5-fold. Importantly, primary CML cells exhibited the same phenomenon, since polymorphism-containing samples expressed lower levels of E4- vs E3-containing transcripts (p=0.008), while general BIM transcription was unaffected. Our observations suggested a novel mechanism for intrinsic TKI resistance. Here, upon IM exposure, polymorphism-containing CML cells would favor induction of E3- vs E4-containing BIM transcripts, decreased expression of BH3-containing BIM isoforms, and impaired apoptosis. To facilitate these studies, we identified a Japanese CML cell line, KCL22, which contained the polymorphism, and confirmed it had a decreased E4/E3 transcript ratio compared to cells without the polymorphism. KCL22 cells also had decreased induction of E4-containing transcripts following IM, as well as lower levels of BIMEL protein, a major BH3-containing BIM isoform. Consistent with prior reports, KCL22 cells were resistant to IM, despite effective BCR-ABL inhibition, and had impaired apoptotic signalling upon IM exposure. Importantly, and as predicted by our model, pharmacologic restoration of BH3 activity (using the BH3-mimetic drug, ABT-737) sensitized cells to IM-induced death. Next, we used zinc finger nuclease-facilitated gene targeting to precisely create the polymorphism in the BIM gene of IM-sensitive K562 CML cells. We generated subclones that were heterozygous or homozygous for the polymorphism, and confirmed a decreased E4/E3 ratio in these cells in a polymorphism-dosage-dependent manner. Polymorphism-containing cells exhibited decreased induction of E4-containing transcripts following IM exposure, as well as impaired upregulation of BIMEL protein, and diminished apoptotic cell death. As in KCL22 cells, ABT-737 enhanced the ability of IM to activate apoptosis in polymorphism-containing cells. Using an expanded East-Asian CML cohort (n=203), we found the polymorphism correlated with TKI resistance (defined as ‘failure’ or ‘suboptimal’ per ELN criteria) in CP patients treated with 400 mg IM daily (p=0.02). Further, patients with the polymorphism were more likely to have resistance in the absence of a KD mutation than those without (OR=2.24, 95% CI of 1.22–4.12). In sum, we have found an East-Asian polymorphism in BIM that is associated with intrinsic resistance to TKIs. Screening for this polymorphism may be useful in identifying patients at risk of TKI resistance; a resistance we show can be overcome by BH3 mimetics. Our findings may also apply to other cancers and proliferative disorders in which drug-sensitivity is BIM-dependent. Disclosures: No relevant conflicts of interest to declare.