Michael P. Cooke

Large-scale analysis of the human and mouse transcriptomes

High-throughput gene expression profiling has become an important tool for investigating transcriptional activity in a variety of biological samples. To date, the vast majority of these experiments have focused on specific biological processes and perturbations. Here, we have generated and analyzed gene expression from a set of samples spanning a broad range of biological conditions. Specifically, we profiled gene expression from 91 human and mouse samples across a diverse array of tissues, organs, and cell lines. Because these samples predominantly come from the normal physiological state in the human and mouse, this dataset represents a preliminary, but substantial, description of the normal mammalian transcriptome. We have used this dataset to illustrate methods of mining these data, and to reveal insights into molecular and physiological gene function, mechanisms of transcriptional regulation, disease etiology, and comparative genomics. Finally, to allow the scientific community to use this resource, we have built a free and publicly accessible website (http://expression.gnf.org) that integrates data visualization and curation of current gene annotations.

Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells

Stem Cell Expansion The ability to expand hematopoietic stem cells (HSCs) during ex-vivo culture has been an important goal for over 20 years. Using a high-throughput chemical screen, Boitano et al. (p. 1345, published online 5 August; see the Perspective by Sauvageau and Humphries) found that a purine derivative, StemRegenin1 (SR1), promoted the expansion of human HSCs. Treatment of HSCs with SR1 (which blocked the activity of the aryl hydrocarbon receptor) led to the expansion of CD34+ cells and a 12 to 17-fold increase in the number of HSCs that engraft immune deficient mice. The identification of a mechanism for ex vivo amplification may facilitate clinical application of hematopoietic stem cell therapies. Although practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative, StemRegenin 1 (SR1), that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy.

Elimination of self-reactive B lymphocytes proceeds in two stages: Arrested development and cell death

In transgenic mice, self-reactive B lymphocytes are eliminated if they encounter membrane-bound self antigens during their development within the bone marrow. We show here that two separate and sequential events, arrested development and cell death, bring about B cell elimination. Developmental arrest is an early outcome of antigen binding in immature B cells, blocks acquisition of adhesion molecules and receptors important for B cell migration and activation, and is rapidly reversible by removal of antigen. Death of the arrested B cells occurs within 1 to 3 days and can be delayed by expression of a bcl-2 transgene, which results in escape of large numbers of self-reactive B cells from the bone marrow but fails to override the developmental arrest. These findings define a novel pathway for B cell elimination, involving an initial stage vulnerable to breakdown in autoimmune disease.

Uncovering regulatory pathways that affect hematopoietic stem cell function using 'genetical genomics'.

We combined large-scale mRNA expression analysis and gene mapping to identify genes and loci that control hematopoietic stem cell (HSC) function. We measured mRNA expression levels in purified HSCs isolated from a panel of densely genotyped recombinant inbred mouse strains. We mapped quantitative trait loci (QTLs) associated with variation in expression of thousands of transcripts. By comparing the physical transcript position with the location of the controlling QTL, we identified polymorphic cis-acting stem cell genes. We also identified multiple trans-acting control loci that modify expression of large numbers of genes. These groups of coregulated transcripts identify pathways that specify variation in stem cells. We illustrate this concept with the identification of candidate genes involved with HSC turnover. We compared expression QTLs in HSCs and brain from the same mice and identified both shared and tissue-specific QTLs. Our data are accessible through WebQTL, a web-based interface that allows custom genetic linkage analysis and identification of coregulated transcripts.

Identification of Modulators of TRAIL-Induced Apoptosis via RNAi-Based Phenotypic Screening

New opportunities in mammalian functional genomics are emerging through the combination of high throughput technology and methods that allow manipulation of gene expression in living cells. Here we describe the application of an RNAi-based forward genomics approach toward understanding the biology and mechanism of TRAIL-induced apoptosis. TRAIL is a TNF superfamily member that induces selective cytotoxicity of tumor cells when bound to its cognate receptors. In addition to detecting well-characterized genes in the apoptosis pathway, we uncover several modulators including DOBI, a gene required for progression of the apoptotic signal through the intrinsic mitochondrial cell death pathway, and MIRSA, a gene that acts to limit TRAIL-induced apoptosis. Moreover, our data suggest a role for MYC and the WNT pathway in maintaining susceptibility to TRAIL. Collectively, these observations offer several insights on how TRAIL mediates the selective killing of tumor cells and demonstrate the utility of large-scale RNAi screens in mammalian cells.

Self-tolerance checkpoints in B lymphocyte development.

Publisher Summary The chapter discusses humoral self-tolerance as the cumulative action of a series of separate checkpoints placed along the B cell lineage. The strategy of employing multiple and distinct checkpoints presumably minimizes the risk that one or two inherited or somatically acquired mutations leads to uncontrolled autoantibody production, analogous to the role of checkpoints in the cell cycle that minimize neoplasia. Each checkpoint may depend on distinct sets of genes and molecules. From a genetic perspective, the use of broad terms such as “deletion” or “anergy” can be used to describe several distinct checkpoint mechanisms that likely involve discrete molecular pathways. The chapter discusses self-tolerance checkpoints acting (1) during formation of the pre-immune B cell repertoire and (2) during subsequent formation of an immune repertoire. A series of cellular phenomena potentially censor and remove autoantibody-bearing B cells from the pathway to antibody secretion. Each of these checkpoints has triggering thresholds that depend on the local auto-antigen concentration, auto-antigen valency, and the B cells binding affinity for elimination of auto-reactive B cells in the bone marrow. These thresholds impose limits on the extent of tolerance.

Genome-wide single-nucleotide polymorphism analysis defines haplotype patterns in mouse

The nature and organization of polymorphisms, or differences, between genomes of individuals are of great interest, because these variations can be associated with or even underlie phenotypic traits, including disease susceptibility. To gain insight into the genetic and evolutionary factors influencing such biological variation, we have examined the arrangement (haplotype) of single-nucleotide polymorphisms across the genomes of eight inbred strains of mice. These analyses define blocks of high or low diversity, often extending across tens of megabases that are delineated by abrupt transitions. These observations provide a striking contrast to the haplotype structure of the human genome.

Interleukin-15 rescues tolerant CD8 + T cells for use in adoptive immunotherapy of established tumors

CD8+ T cells can mediate eradication of established tumors, and strategies to amplify tumor-reactive T-cell numbers by immunization or ex vivo expansion followed by adoptive transfer are currently being explored in individuals with cancer. Generating effective CD8+ T cell–mediated responses to tumors is often impeded by T-cell tolerance to relevant tumor antigens, as most of these antigens are also expressed in normal tissues. We examined whether such tolerant T cells could be rescued and functionally restored for use in therapy of established tumors. We used a transgenic T-cell receptor (TCR) mouse model in which peripheral CD8+ T cells specific for a candidate tumor antigen also expressed in liver are tolerant, failing to proliferate or secrete interleukin (IL)-2 in response to antigen. Molecular and cellular analysis showed that these tolerant T cells expressed the IL-15 receptor α chain, and could be induced to proliferate in vitro in response to exogenous IL-15. Such proliferation abrogated tolerance and the rescued cells became effective in treating leukemia. Therefore, high-affinity CD8+ T cells are not necessarily deleted by encounter with self-antigen in the periphery, and can potentially be rescued and expanded for use in tumor immunotherapy.

A Comparison of the Celera and Ensembl Predicted Gene Sets Reveals Little Overlap in Novel Genes

genes, but the novel genes predicted by both groups are largely nonoverlapping. To validate the existence of the transcript predictions, we used RNA expression profiling and a bank of 13 diverse human tissues. The commercial high-density oligonucleo-The recent description of the human genome and the tide arrays used are based on Expressed Sequence subsequent annotation of putative novel genes has Tags (ESTs) represented in Unigene (release 95). ushered in a new era in biology. One of the revelations of BLASTN was used to assign the transcript predictions the human genome project was the remarkably consistent to a Unigene cluster, and the RNA expression pattern prediction that the genome harbors around 30,000 genes. was determined for the 8,000 known and 5,000 novel This observation was based on independent analyses predicted genes with a corresponding Unigene cluster done by a public genome consortium (29,691 transcripts, on the arrays (see legend to Figure 2 for details). Using Ensembl v0.8) (Lander et al., 2001), by work done at Celera these methods, we found evidence of expression for Genomics (39,114 transcripts) (Venter et al., 2001), and by more than 80% of the known genes in at least one of Green and colleagues using expressed sequence tag the tissue samples analyzed (Figure 2A). Similarly, more (EST) clustering incorporating quality scores (35,000 than 80% of the novel predicted transcripts were genes) (Ewing and Green, 2000). This conclusion was detected as expressed in at least one of the 13 human surprising for two reasons. First, less complex organisms tissues. Hierarchical clustering and visualization of like Arabidopsis (25,000) and C. elegans (19,000) have these expression data revealed a similar fraction of approximately the same number of genes (C. elegans tissue-restricted transcripts for both known and novel Sequencing Consortium, 1998; Arabidopsis Genome genes (Figure 2B). These data support the view that the Initiative, 2000). Second, earlier estimates of gene number novel transcripts predicted by both groups encode bona based on EST clustering and detailed chromosomal fide differentially expressed mRNAs. Since many of analysis were much higher, ranging from 45,000 to 140,000 these verified transcripts were contained in only one of the two predicted transcriptomes, we conclude that the Scott, 1999). While the Celera and Ensembl annotation computational methods used for gene prediction by efforts predicted approximately the same number of either group are inadequate and that the respective genes, a direct comparison of the predicted transcript sets transcriptomes are individually …

Rapid Expansion of Human Hematopoietic Stem Cells by Automated Control of Inhibitory Feedback Signaling

Clinical hematopoietic transplantation outcomes are strongly correlated with the numbers of cells infused. Anticipated novel therapeutic implementations of hematopoietic stem cells (HSCs) and their derivatives further increase interest in strategies to expand HSCs ex vivo. A fundamental limitation in all HSC-driven culture systems is the rapid generation of differentiating cells and their secreted inhibitory feedback signals. Herein we describe an integrated computational and experimental strategy that enables a tunable reduction in the global levels and impact of paracrine signaling factors in an automated closed-system process by employing a controlled fed-batch media dilution approach. Application of this system to human cord blood cells yielded a rapid (12-day) 11-fold increase of HSCs with self-renewing, multilineage repopulating ability. These results highlight the marked improvements that control of feedback signaling can offer primary stem cell culture and demonstrate a clinically relevant rapid and relatively low culture volume strategy for ex vivo HSC expansion.