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Craig R Tomlinson

Publications and source records attributed to Craig R Tomlinson.

14 recordsLinked to original sources
PubMed2006

Intensity-based hierarchical Bayes method improves testing for differentially expressed genes in microarray experiments.

BACKGROUND: The small sample sizes often used for microarray experiments result in poor estimates of variance if each gene is considered independently. Yet accurately estimating variability of gene expression measurements in microarray experiments is essential for correctly identifying differentially expressed genes. Several recently developed methods for testing differential expression of genes utilize hierarchical Bayesian models to "pool" information from multiple genes. We have developed a statistical testing procedure that further improves upon current methods by incorporating the well-documented relationship between the absolute gene expression level and the variance of gene expression measurements into the general empirical Bayes framework. RESULTS: We present a novel Bayesian moderated-T, which we show to perform favorably in simulations, with two real, dual-channel microarray experiments and in two controlled single-channel experiments. In simulations, the new method achieved greater power while correctly estimating the true proportion of false positives, and in the analysis of two publicly-available "spike-in" experiments, the new method performed favorably compared to all tested alternatives. We also applied our method to two experimental datasets and discuss the additional biological insights as revealed by our method in contrast to the others. The R-source code for implementing our algorithm is freely available at http://eh3.uc.edu/ibmt. CONCLUSION: We use a Bayesian hierarchical normal model to define a novel Intensity-Based Moderated T-statistic (IBMT). The method is completely data-dependent using empirical Bayes philosophy to estimate hyperparameters, and thus does not require specification of any free parameters. IBMT has the strength of balancing two important factors in the analysis of microarray data: the degree of independence of variances relative to the degree of identity (i.e. t-tests vs. equal variance assumption), and the relationship between variance and signal intensity. When this variance-intensity relationship is weak or does not exist, IBMT reduces to a previously described moderated t-statistic. Furthermore, our method may be directly applied to any array platform and experimental design. Together, these properties show IBMT to be a valuable option in the analysis of virtually any microarray experiment.

Animals↗
PubMed2006

Genome-wide analyses show that nuclear and cytoplasmic RNA levels are differentially affected by dioxin.

The aryl hydrocarbon receptor (AHR) mounts the body's main molecular defense against environmental toxicants by inducing a battery of genes encoding xenobiotic metabolizing proteins. The AHR is activated by polycyclic aromatic hydrocarbon toxicants, including the pervasive teratogen and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). The TCDD-activated AHR significantly changes the cytoplasmic mRNA levels of hundreds of genes, but little is known of the mechanism by which the activated AHR causes such a strong effect on global gene expression. We used high-density microarrays to compare nuclear and cytoplasmic RNA levels from untreated and TCDD-treated mouse embryonic fibroblasts (MEF) to test the hypotheses that (1) TCDD has a large impact on nuclear RNA levels and (2) that cytoplasmic RNA levels are dependent on nuclear RNA levels. We found that nuclear RNA levels are strongly affected by TCDD, and that nuclear and cytoplasmic RNA levels are only weakly correlated, indicating that other regulatory mechanisms are controlling cytoplasmic RNA levels. The nuclear RNAs most affected by TCDD encode proteins involved in nuclear RNA processing and transcription. We conclude that although the AHR regulates key xenobiotic metabolizing genes at the transcriptional level, a larger impact of the TCDD-activated AHR may be at post-transcriptional levels.

Animals↗
PubMed2006

Gene expression and discovery during lens regeneration in mouse: regulation of epithelial to mesenchymal transition and lens differentiation.

PURPOSE: It has been shown that after extracapsular lens removal by anterior capsulotomy in the mouse, the lens can be regenerated. However, as the capsular bag is filled with fibers, epithelial to mesenchymal transition (EMT), an event which is common after cataract surgery as well, takes place during early stages. This study, using a unique mouse model, was undertaken to identify novel regulators and networks in order to more clearly understand secondary cataracts at the molecular level. METHODS: We examined global gene expression via microarray analysis of mouse lens regeneration after extracapsular surgery. Gene expression at different times after surgery was correlated with the processes of EMT, which is seen in the initial stages of regeneration, and lens fiber differentiation, which occurs later. RESULTS: Several notable patterns were observed from the gene clustering data. It was obvious from the analysis that initially there is a response to injury, extensive matrix remodeling, and severe downregulation of genes encoding lens structural proteins. The patterns returned gradually to normal three weeks after surgery. New genes were identified from the clustering results that might be potential regulators of EMT and lens differentiation. CONCLUSIONS: With this approach, we demonstrated the utility of a mouse model to study secondary cataracts at the molecular level. Extension of these studies in mice with known mutations affecting EMT or lens differentiation should allow the identification of the crucial molecular players that could lead to better treatments of secondary cataracts.

Animals↗
PubMed2006

A new method to remove hybridization bias for interspecies comparison of global gene expression profiles uncovers an association between mRNA sequence divergence and differential gene expression in Xenopus.

The recent sequencing of a large number of Xenopus tropicalis expressed sequences has allowed development of a high-throughput approach to study Xenopus global RNA gene expression. We examined the global gene expression similarities and differences between the historically significant Xenopus laevis model system and the increasingly used X.tropicalis model system and assessed whether an X.tropicalis microarray platform can be used for X.laevis. These closely related species were also used to investigate a more general question: is there an association between mRNA sequence divergence and differences in gene expression levels? We carried out a comprehensive comparison of global gene expression profiles using microarrays of different tissues and developmental stages of X.laevis and X.tropicalis. We (i) show that the X.tropicalis probes provide an efficacious microarray platform for X.laevis, (ii) describe methods to compare interspecies mRNA profiles that correct differences in hybridization efficiency and (iii) show independently of hybridization bias that as mRNA sequence divergence increases between X.laevis and X.tropicalis differences in mRNA expression levels also increase.

Animals↗
PubMed2005

The role of metallothionein in the pathogenesis of acute lung injury.

Often fatal, acute lung injury has a complicated etiology. Previous studies from our laboratory in mice have demonstrated that survival during acute lung injury is a complex trait governed by multiple loci. We also found that the increase in metallothionein (MT) is one of the greatest noted in transcriptome-wide analyses of gene expression. To assess the role of MT in nickel-induced acute lung injury, the survival of Mt-transgenic, Mt1/2(+/+), and Mt1/2(-/-) mice was compared. Pulmonary inflammation and global gene expression were compared in Mt1/2(+/+) and Mt1/2(-/-) mice. Gene-targeted Mt1/2(-/-) mice were more susceptible than Mt1/2(+/+) mice to nickel-induced inflammation, surfactant-associated protein B transcript loss, and lethality. Similarly, Mt-transgenic mice exhibited increased survival. MAPPFinder analyses also noted significant decreases in genes involved in protein processing (e.g., ubiquitination, folding), which were greater in Mt1/2(-/-) mice as compared with Mt1/2(+/+) mice early in the progression of acute lung injury, possibly due to a zinc-mediated transcript destabilization. In contrast, transcript levels of genes associated with the inflammatory response, extracellular matrix regulation, and coagulation/fibrinolysis were increased more in Mt1/2(-/-) mice as compared with Mt1/2(+/+) mice late in the development of acute lung injury. Thus, MT ultimately improves survival in the progression of acute lung injury in mice. Transcriptome-wide analysis suggests that this survival may be mediated through changes in the destabilization of transcripts associated with protein processing, the subsequent augmentation of transcripts controlling inflammation, extracellular matrix regulation, coagulation/fibrinolysis, and disruption of surfactant homeostasis.

Animals↗
PubMed2005

Gene expression changes during the development of acute lung injury: role of transforming growth factor beta.

RATIONALE: Acute lung injury can occur from multiple causes, resulting in high mortality. The pathophysiology of nickel-induced acute lung injury in mice is remarkably complex, and the molecular mechanisms are uncertain. OBJECTIVES: To integrate molecular pathways and investigate the role of transforming growth factor beta (TGF-beta) in acute lung injury in mice. METHODS: cDNA microarray analyses were used to identify lung gene expression changes after nickel exposure. MAPPFinder analysis of the microarray data was used to determine significantly altered molecular pathways. TGF-beta1 protein in bronchoalveolar lavage fluid, as well as the effect of inhibition of TGF-beta, was assessed in nickel-exposed mice. The effect of TGF-beta on surfactant-associated protein B (Sftpb) promoter activity was measured in mouse lung epithelial cells. MEASUREMENTS AND MAIN RESULTS: Genes that decreased the most after nickel exposure play important roles in lung fluid absorption or surfactant and phospholipid synthesis, and genes that increased the most were involved in TGF-beta signaling. MAPPFinder analysis further established TGF-beta signaling to be significantly altered. TGF-beta-inducible genes involved in the regulation of extracellular matrix function and fibrinolysis were significantly increased after nickel exposure, and TGF-beta1 protein was also increased in the lavage fluid. Pharmacologic inhibition of TGF-beta attenuated nickel-induced protein in bronchoalveolar lavage. In addition, treatment with TGF-beta1 dose-dependently repressed Sftpb promoter activity in vitro, and a novel TGF-beta-responsive region in the Sftpb promoter was identified. CONCLUSIONS: These data suggest that TGF-beta acts as a central mediator of acute lung injury through the alteration of several different molecular pathways.

Animals↗
PubMed2004

Ah receptor signals cross-talk with multiple developmental pathways.

For many years, the Ah receptor (AHR) has been a favorite of toxicologists and molecular biologists studying the connections between genes and the changes in the control of gene expression resulting from environmental exposures. Much of the attention given to the Ah receptor has focused on the nature of its ligands, many of which are known or suspected carcinogens, and on the role that its best studied regulatory product, the CYP1A1 enzyme, plays in toxic responses and carcinogen activation. This understandable bias has resulted in a disproportionate amount of Ah receptor research being directed at toxicological or adaptive end points. In recent times, it has become evident that Ah receptor functions are also involved in molecular cascades that lead to inhibition of proliferation, promotion of differentiation, or apoptosis, with an important bearing in development. Developmental and toxicological AHR functions may not always be related. The ancestral AHR protein in invertebrates directs the developmental fate of a few specific neurons and does not bind xenobiotic ligands. The mammalian AHR maintains normal liver function in the absence of exogenous ligands and, when activated by dioxin, cross-talks with morphogenetic and developmental signals. Toxic end points, such as the induction of cleft palate by dioxin in mice embryos, might be at the crossroads of these signals and provide important clues as to the developmental role of the AHR.

Animals↗
PubMed2004

A role for Saccharomyces cerevisiae Chk1p in the response to replication blocks.

Replication blocks and DNA damage incurred during S phase activate the S-phase and intra-S-phase checkpoint responses, respectively, regulated by the Atrp and Chk1p checkpoint kinases in metazoans. In Saccharomyces cerevisiae, these checkpoints are regulated by the Atrp homologue Mec1p and the kinase Rad53p. A conserved role of these checkpoints is to block mitotic progression until DNA replication and repair are completed. In S. cerevisiae, these checkpoints include a transcriptional response regulated by the kinase Dun1p; however, dun1Delta cells are proficient for the S-phase-checkpoint-induced anaphase block. Yeast Chk1p kinase regulates the metaphase-to-anaphase transition in the DNA-damage checkpoint pathway via securin (Pds1p) phosphorylation. However, like Dun1p, yeast Chk1p is not required for the S-phase-checkpoint-induced anaphase block. Here we report that Chk1p has a role in the intra-S-phase checkpoint activated when yeast cells replicate their DNA in the presence of low concentrations of hydroxyurea (HU). Chk1p was modified and Pds1p was transiently phosphorylated in this response. Cells lacking Dun1p were dependent on Chk1p for survival in HU, and chk1Delta dun1Delta cells were defective in the recovery from replication interference caused by transient HU exposure. These studies establish a relationship between the S-phase and DNA-damage checkpoint pathways in S. cerevisiae and suggest that at least in some genetic backgrounds, the Chk1p/securin pathway is required for the recovery from stalled or collapsed replication forks.

Base Sequence↗
PubMed2004

Expression of genes in the TGF-beta signaling pathway is significantly deregulated in smooth muscle cells from aorta of aryl hydrocarbon receptor knockout mice.

The molecular basis for the adverse biological effects of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD), a pervasive environmental toxin, is largely unknown. TCDD is a ligand for the cytosolic aromatic hydrocarbon receptor (AHR) which mediates the transcriptional induction of the xenobiotic metabolizing genes in the CYP1 family of cytochromes P450. Previous studies have suggested that the AHR may carry out important functions in the cell in addition to metabolizing toxins. We present gene expression profiles of smooth muscle cells from wild type and Ahr(-/-) mice that show significant changes in the RNA levels of the transforming growth factor-beta3 (Tgfb3) gene and genes involved in the modulation and processing of TGF-beta. The RNA expression profiles support a hypothesis that in the wild type, the AHR represses Tgfb gene expression and affects the gene expression of several TGF-beta-modulating and processing genes. We also observed that RNA levels increased for TGF-beta2, CYP1b1, and TGF-beta-related genes in Ahr(-/-) smooth muscle cells exposed to TCDD. These data are consistent with a hypothesis that TCDD stimulates the TGF-beta2 signaling pathway in the absence of the AHR to activate the Cyp1b1 gene. The above results provide a possible explanation for some of the multiple biological effects of TCDD and the physiological role played by the AHR in the absence of environmental agents.

Animals↗
PubMed2004

Critical regulation of genes for tumor cell migration by AP-1.

The AP-1 transcription factor plays a critical role in regulating tumor cell proliferation and has been implicated in controlling a program of gene expression that mediates cell motility and invasion in vitro. We have utilized two dominant negative AP-1 constructs, TAM67 and aFos, each fused to GFP, to investigate the role of AP-1 complexes in an invasive, clinically derived human tumor cell line, HT-1080. As expected, high levels of both GFP-TAM67 and GFP-aFos arrested HT-1080 cells in the G1 phase of the cell cycle. Strikingly, at low levels GFP-aFos, but not GFP-TAM67, caused a change in colony morphology, impairment of directional motility in a monolayer wound healing assay, as well as inhibition of chemotaxis and haptotaxis. Microarray analysis identified a novel set of AP-1 target genes, including the tumor suppressor TSCL-1 and regulators of actin cytoskeletal dynamics, including the gelsolin-like actin capping protein CapG. The demonstration that AP-1 regulates the expression of genes involved in tumor cell motility and cytoskeletal dynamics in a clinically derived human tumor cell line identifies new pathways of control for tumor cell motility.

Cell Adhesion Molecule-1↗
PubMed2004

Different global gene expression profiles in benzo[a]pyrene- and dioxin-treated vascular smooth muscle cells of AHR-knockout and wild-type mice.

Benzo[a]pyrene (B[a]P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are potent ligands for the aryl hydrocarbon receptor (AHR). High-density oligonucleotide microarrays were used to generate global gene expression profiles of wild-type and Ahr(-/-) vascular smooth muscle cells (SMCs) from mouse aorta. To determine whether there are signaling pathways other than the AHR involved in B[a]P metabolism, wild-type and AHR knockout (Ahr(-/-) SMCs were exposed to B[a]P. Two signaling pathways, represented by TGF-beta2 and IGF-1, were identified as potential candidates of an AHR alternate pathway for cells to respond to B[a]P. The wild-type SMCs responded similarly to B[a]P and TCDD in the regulation of a small set of common genes known to respond to the activated AHR (e.g., glutamine S-transferase). However, wild-type SMCs responded in a way that involves many additional genes, suggesting that a very divergent cellular response may be involved when SMCs are exposed to the two classic inducers of the AHR. In contrast, many more genes in the Ahr(-/-) cells responded similarly to B[a]P and TCDD, including Cyp1b1, than responded differently, which indicates that eliminating the AHR is effective for investigating potential alternate cellular mechanisms that respond to B[a]P and TCDD.

Animals↗
PubMed2004

Gene expression profiles of mouse aorta and cultured vascular smooth muscle cells differ widely, yet show common responses to dioxin exposure.

Exposure to environmental toxicants may play a role in the onset and progression of cardiovascular disease. Many environmental agents, such as dioxin, are risk factors for atherosclerosis because they may exacerbate an underlying disease by altering gene expression patterns. Expression profiling of vascular tissues allows the simultaneous analysis of thousands of genes and may provide predictive information particularly useful in early disease stages. Often, however, in vivo experiments are unfeasible for material or ethical reasons, and data from cultured cells must be used instead, even though it may not be known whether cultured cells and live tissues share common global responses to the same toxicant. In a search for genes responsive to dioxin exposure, we used oligonucleotide microarrays with DNA sequences from 13,433 genes to compare global gene expression profiles of C57BL/6 mice aortas with cultured vascular smooth muscle cells (vSMCs) of the same mice. Aorta segments and vSMCs differed in the expression of more than 4500 genes, many showing expression differences greater than 1000-fold. Integration of microarray data into Gene Ontology Project annotations showed that many of the genes differentially expressed belonged to the same biological process or metabolic pathway. Notwithstanding these results, a subset of 35 genes responded in the same fashion to dioxin exposure in both systems. Genes in this subset encoded phase I and phase II detoxification enzymes, signal transduction kinases and phosphatases, and proteins involved in DNA repair and the cell cycle. We conclude that vSMCS may be useful aorta surrogates to study early gene expression responses to dioxin exposure, provided that analyses focus on this subset of genes.

Animals↗
PubMed2004

Microarray results improve significantly as hybridization approaches equilibrium.

Dual-channel long oligonucleotide microarrays are in widespread use. Although much attention has been given to proper experimental design and analysis regarding long oligonucleotide microarrays, relatively little information is available concerning the optimization of protocols. We carried out a series of microarray experiments designed to investigate the effects of different levels of target concentration and hybridization times using a long oligonucleotide library. Based on principles developed from nucleic acid renaturation kinetics studies, we show that increasing the time of hybridization from 18 h to 42 h and 66 h, especially when lower than optimal concentrations of target were used, significantly improved the quality of the microarray results. Longer hybridization times significantly increased the number of spots detected, signal-to-noise ratios, and the number of differentially expressed genes and correlations among replicate arrays. We conclude that at 18 h of incubation, target-to-probe hybridization has not reached equilibrium and that a relatively high proportion of nonspecific hybridization occurs. This result is striking, given that most, if not all, published microarray protocols stipulate 8-24 h for hybridization. Using shorter than optimal hybridization times (i.e., not allowing hybridization to reach equilibrium) has the consequence of underestimating the fold change of differentially expressed genes and of missing less represented sequences.

Algorithms↗