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1.  SCL, LMO1 and Notch1 Reprogram Thymocytes into Self-Renewing Cells 
PLoS Genetics  2014;10(12):e1004768.
The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network described here may be relevant to a majority of human T-ALL.
Author Summary
Deciphering the initiating events in lymphoid leukemia is important for the development of new therapeutic strategies. In this manuscript, we define oncogenic reprogramming as the process through which non-self-renewing progenitors are converted into pre-leukemic stem cells with sustained self-renewal capacities. We provide strong genetic evidence that this step is rate-limiting in leukemogenesis and requires the activation of a self-renewal program by oncogenic transcription factors, as exemplified by SCL and LMO1. Furthermore, NOTCH1 is a pathway that drives cell fate in the thymus. We demonstrate that homeostatic NOTCH1 levels that are highest in specific thymocyte subsets determine their susceptibilities to oncogenic reprogramming by SCL and LMO1. Our data provide novel insight into the acquisition of self-renewal as a critical first step in lymphoid cell transformation, requiring the synergistic interaction of oncogenic transcription factors with a cellular context controlled by high physiological NOTCH1.
PMCID: PMC4270438  PMID: 25522233
2.  Genome-Wide Discovery of Small RNAs in Mycobacterium tuberculosis 
PLoS ONE  2012;7(12):e51950.
Only few small RNAs (sRNAs) have been characterized in Mycobacterium tuberculosis and their role in regulatory networks is still poorly understood. Here we report a genome-wide characterization of sRNAs in M. tuberculosis integrating experimental and computational analyses. Global RNA-seq analysis of exponentially growing cultures of M. tuberculosis H37Rv had previously identified 1373 sRNA species. In the present report we show that 258 (19%) of these were also identified by microarray expression. This set included 22 intergenic sRNAs, 84 sRNAs mapping within 5′/3′ UTRs, and 152 antisense sRNAs. Analysis of promoter and terminator consensus sequences identified sigma A promoter consensus sequences for 121 sRNAs (47%), terminator consensus motifs for 22 sRNAs (8.5%), and both motifs for 35 sRNAs (14%). Additionally, 20/23 candidates were visualized by Northern blot analysis and 5′ end mapping by primer extension confirmed the RNA-seq data. We also used a computational approach utilizing functional enrichment to identify the pathways targeted by sRNA regulation. We found that antisense sRNAs preferentially regulated transcription of membrane-bound proteins. Genes putatively regulated by novel cis-encoded sRNAs were enriched for two-component systems and for functional pathways involved in hydrogen transport on the membrane.
PMCID: PMC3526491  PMID: 23284830
3.  Linking the Transcriptional Profiles and the Physiological States of Mycobacterium tuberculosis during an Extended Intracellular Infection 
PLoS Pathogens  2012;8(6):e1002769.
Intracellular pathogens such as Mycobacterium tuberculosis have evolved strategies for coping with the pressures encountered inside host cells. The ability to coordinate global gene expression in response to environmental and internal cues is one key to their success. Prolonged survival and replication within macrophages, a key virulence trait of M. tuberculosis, requires dynamic adaptation to diverse and changing conditions within its phagosomal niche. However, the physiological adaptations during the different phases of this infection process remain poorly understood. To address this knowledge gap, we have developed a multi-tiered approach to define the temporal patterns of gene expression in M. tuberculosis in a macrophage infection model that extends from infection, through intracellular adaptation, to the establishment of a productive infection. Using a clock plasmid to measure intracellular replication and death rates over a 14-day infection and electron microscopy to define bacterial integrity, we observed an initial period of rapid replication coupled with a high death rate. This was followed by period of slowed growth and enhanced intracellular survival, leading finally to an extended period of net growth. The transcriptional profiles of M. tuberculosis reflect these physiological transitions as the bacterium adapts to conditions within its host cell. Finally, analysis with a Transcriptional Regulatory Network model revealed linked genetic networks whereby M. tuberculosis coordinates global gene expression during intracellular survival. The integration of molecular and cellular biology together with transcriptional profiling and systems analysis offers unique insights into the host-driven responses of intracellular pathogens such as M. tuberculosis.
Author Summary
The impact of Mycobacterium tuberculosis on global health is undeniable, with ∼2 million deaths and ∼9 million new cases of tuberculosis each year. A key to the success of M. tuberculosis as a persistent, intracellular pathogen is its ability to survive for extended periods within professional phagocytes. Sustained growth within macrophage phagosomes requires avoiding or resisting antimicrobial mechanisms and adapting to replicate in a stressful, nutrient-restricted environment. Our understanding of the survival strategies, metabolism, and physiology of M. tuberculosis during intracellular growth remains incomplete. We employed multi-disciplinary approaches to gain new insights into adaptive responses that M. tuberculosis mobilizes to secure a productive infection. We simultaneously quantified replication and death rates, used electron microscopy to evaluate bacterial integrity, and determined the temporal changes in bacterial gene expression during a 14-day infection. By overlaying this temporal transcriptome dataset onto an extended Transcriptional Regulatory Network model, we identified regulatory pathways, stress responses, and metabolic adaptations activated during key physiological transitions over the 14 days of infection.
PMCID: PMC3380936  PMID: 22737072
4.  Network inference and network response identification: moving genome-scale data to the next level of biological discovery 
Molecular bioSystems  2009;6(3):469-480.
The escalating amount of genome-scale data demands a pragmatic stance from the research community. How can we utilize this deluge of information to better understand biology, cure diseases, or engage cells in bioremediation or biomaterial production for various purposes? A research pipeline moving new sequence, expression and binding data towards practical end goals seems to be necessary. While most individual researchers are not motivated by such well-articulated pragmatic end goals, the scientific community has already self-organized itself to successfully convert genomic data into fundamentally new biological knowledge and practical applications. Here we review two important steps in this workflow: network inference and network response identification, applied to transcriptional regulatory networks. Among network inference methods, we concentrate on relevance networks due to their conceptual simplicity. We classify and discuss network response identification approaches as either data-centric or network-centric. Finally, we conclude with an outlook on what is still missing from these approaches and what may be ahead on the road to biological discovery.
PMCID: PMC3087299  PMID: 20174676
5.  Exposing the cancer genome atlas as a SPARQL endpoint 
Journal of biomedical informatics  2010;43(6):998-1008.
The Cancer Genome Atlas (TCGA) is a multidisciplinary, multi-institutional effort to characterize several types of cancer. Datasets from biomedical domains such as TCGA present a particularly challenging task for those interested in dynamically aggregating its results because the data sources are typically both heterogeneous and distributed. The Linked Data best practices offer a solution to integrate and discover data with those characteristics, namely through exposure of data as Web services supporting SPARQL, the Resource Description Framework query language. Most SPARQL endpoints, however, cannot easily be queried by data experts. Furthermore, exposing experimental data as SPARQL endpoints remains a challenging task because, in most cases, data must first be converted to Resource Description Framework triples. In line with those requirements, we have developed an infrastructure to expose clinical, demographic and molecular data elements generated by TCGA as a SPARQL endpoint by assigning elements to entities of the Simple Sloppy Semantic Database (S3DB) management model. All components of the infrastructure are available as independent Representational State Transfer (REST) Web services to encourage reusability, and a simple interface was developed to automatically assemble SPARQL queries by navigating a representation of the TCGA domain. A key feature of the proposed solution that greatly facilitates assembly of SPARQL queries is the distinction between the TCGA domain descriptors and data elements. Furthermore, the use of the S3DB management model as a mediator enables queries to both public and protected data without the need for prior submission to a single data source.
PMCID: PMC3071752  PMID: 20851208
TCGA; SPARQL; RDF; Linked Data; Data integration
6.  A Semantic Web Management Model for Integrative Biomedical Informatics 
PLoS ONE  2008;3(8):e2946.
Data, data everywhere. The diversity and magnitude of the data generated in the Life Sciences defies automated articulation among complementary efforts. The additional need in this field for managing property and access permissions compounds the difficulty very significantly. This is particularly the case when the integration involves multiple domains and disciplines, even more so when it includes clinical and high throughput molecular data.
Methodology/Principal Findings
The emergence of Semantic Web technologies brings the promise of meaningful interoperation between data and analysis resources. In this report we identify a core model for biomedical Knowledge Engineering applications and demonstrate how this new technology can be used to weave a management model where multiple intertwined data structures can be hosted and managed by multiple authorities in a distributed management infrastructure. Specifically, the demonstration is performed by linking data sources associated with the Lung Cancer SPORE awarded to The University of Texas MDAnderson Cancer Center at Houston and the Southwestern Medical Center at Dallas. A software prototype, available with open source at, was developed and its proposed design has been made publicly available as an open source instrument for shared, distributed data management.
The Semantic Web technologies have the potential to addresses the need for distributed and evolvable representations that are critical for systems Biology and translational biomedical research. As this technology is incorporated into application development we can expect that both general purpose productivity software and domain specific software installed on our personal computers will become increasingly integrated with the relevant remote resources. In this scenario, the acquisition of a new dataset should automatically trigger the delegation of its analysis.
PMCID: PMC2491554  PMID: 18698353

Results 1-6 (6)