Meta-analysis of gene expression profiles in breast cancer: toward a unified understanding of breast cancer subtyping and prognosis signatures.

INTRODUCTION: Breast cancer subtyping and prognosis have been studied extensively by gene expression profiling, resulting in disparate signatures with little overlap in their constituent genes. Although a previous study demonstrated a prognostic concordance among gene expression signatures, it was limited to only one dataset and did not fully elucidate how the different genes were related to one another nor did it examine the contribution of well-known biological processes of breast cancer tumorigenesis to their prognostic performance. METHOD: To address the above issues and to further validate these initial findings, we performed the largest meta-analysis of publicly available breast cancer gene expression and clinical data, which are comprised of 2,833 breast tumors. Gene coexpression modules of three key biological processes in breast cancer (namely, proliferation, estrogen receptor [ER], and HER2 signaling) were used to dissect the role of constituent genes of nine prognostic signatures. RESULTS: Using a meta-analytical approach, we consolidated the signatures associated with ER signaling, ERBB2 amplification, and proliferation. Previously published expression-based nomenclature of breast cancer 'intrinsic' subtypes can be mapped to the three modules, namely, the ER-/HER2- (basal-like), the HER2+ (HER2-like), and the low- and high-proliferation ER+/HER2- subtypes (luminal A and B). We showed that all nine prognostic signatures exhibited a similar prognostic performance in the entire dataset. Their prognostic abilities are due mostly to the detection of proliferation activity. Although ER- status (basal-like) and ERBB2+ expression status correspond to bad outcome, they seem to act through elevated expression of proliferation genes and thus contain only indirect information about prognosis. Clinical variables measuring the extent of tumor progression, such as tumor size and nodal status, still add independent prognostic information to proliferation genes. CONCLUSION: This meta-analysis unifies various results of previous gene expression studies in breast cancer. It reveals connections between traditional prognostic factors, expression-based subtyping, and prognostic signatures, highlighting the important role of proliferation in breast cancer prognosis.

Detection of plant-modulated alterations in antifungal gene expression in Pseudomonas fluorescens CHA0 on roots by flow cytometry.

The biocontrol activity of the root-colonizing Pseudomonas fluorescens strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the phlA and prnA genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of phlA-gfp and prnA-gfp in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the gfp reporters, the average gene expression per bacterial cell could be calculated. Levels of phlA and prnA expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered phlA expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.

Light-regulated interactions with SPA proteins underlie cryptochrome-mediated gene expression.

Cryptochromes are a class of photosensory receptors that control important processes in animals and plants primarily by regulating gene expression. How photon absorption by cryptochromes leads to changes in gene expression has remained largely elusive. Three recent studies, including Lian and colleagues (pp. 1023-1028) and Liu and colleagues (pp. 1029-1034) in this issue of Genes & Development, demonstrate that the interaction of light-activated Arabidopsis cryptochromes with a class of regulatory components of E3 ubiquitin ligase complexes leads to environmentally controlled abundance of transcriptional regulators.

Nonenzymatic lipid peroxidation reprograms gene expression and activates defense markers in Arabidopsis tocopherol-deficient mutants.

Tocopherols (vitamin E) are lipophilic antioxidants that are synthesized by all plants and are particularly abundant in seeds. Two tocopherol-deficient mutant loci in Arabidopsis thaliana were used to examine the functions of tocopherols in seedlings: vitamin e1 (vte1), which accumulates the pathway intermediate 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ); and vte2, which lacks all tocopherols and pathway intermediates. Only vte2 displayed severe seedling growth defects, which corresponded with massively increased levels of the major classes of nonenzymatic lipid peroxidation products: hydroxy fatty acids, malondialdehyde, and phytoprostanes. In the absence of pathogens, the phytoalexin camalexin accumulated in vte2 seedlings to levels 100-fold higher than in wild-type or vte1 seedlings. Similarly, gene expression profiling in wild-type, vte1, and vte2 seedlings indicated that increased levels of nonenzymatic lipid peroxidation in vte2 corresponded to increased expression of many defense-related genes, which were not induced in vte1. Both biochemical and transcriptional analyses of vte2 seedlings indicate that nonenzymatic lipid peroxidation plays a significant role in modulating plant defense responses. Together, these results establish that tocopherols in wild-type plants or DMPBQ in vte1 plants limit nonenzymatic lipid peroxidation during germination and early seedling development, thereby preventing the inappropriate activation of transcriptional and biochemical defense responses.

Copy number variants, diseases and gene expression.

Copy number variation (CNV) has recently gained considerable interest as a source of genetic variation likely to play a role in phenotypic diversity and evolution. Much effort has been put into the identification and mapping of regions that vary in copy number among seemingly normal individuals in humans and a number of model organisms, using bioinformatics or hybridization-based methods. These have allowed uncovering associations between copy number changes and complex diseases in whole-genome association studies, as well as identify new genomic disorders. At the genome-wide scale, however, the functional impact of CNV remains poorly studied. Here we review the current catalogs of CNVs, their association with diseases and how they link genotype and phenotype. We describe initial evidence which revealed that genes in CNV regions are expressed at lower and more variable levels than genes mapping elsewhere, and also that CNV not only affects the expression of genes varying in copy number, but also have a global influence on the transcriptome. Further studies are warranted for complete cataloguing and fine mapping of CNVs, as well as to elucidate the different mechanisms by which they influence gene expression.

Leishmania major: histone H1 gene expression from the sw3 locus.

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by the sw3 gene. Here we report that histone H1 variants exist in different Leishmania species and strains of L. major and that they are encoded by polymorphic genes. Amplification of the sw3 gene from the genome of three strains of L. major gave rise to different products in each strain, suggesting the presence of a multicopy gene family. In L. major, these genes were all restricted to a 50-kb Bg/II fragment found on a chromosomal band of 1.3 Mb (chromosome 27). The detection of RFLPs in this locus demonstrated its heterogeneity within several species and strains of Leishmania. Two different copies of sw3 (sw3.0 and sw3.1) were identified after screening a cosmid library containing L. major strain Friedlin genomic DNA. They were identical in their 5' UTRs and open reading frames, but differed in their 3' UTRs. With respect to the originally cloned copy of sw3 from L. major strain LV39, their open reading frames lacked a repeat unit of 9 amino acids. Immunoblots of L. guyanensis parasites transfected with these cosmids revealed that both copies could give rise to the histone H1 protein. The characterization of this locus will now make possible a detailed analysis of the function of histone H1 in Leishmania, as well as permit the dissection of the molecular mechanisms governing the developmental regulation of the sw3 gene.

Dynamics of multiple lin gene expression in Sphingomonas paucimobilis B90A in response to different hexachlorocyclohexane isomers.

Sphingomonas paucimobilis B90A is able to degrade the alpha-, beta-, gamma-, and delta-isomers of hexachlorocyclohexane (HCH). It contains the genes linA, linB, linC, linD, linE, and linR, which have been implicated in HCH degradation. In this study, dynamic expression of the lin genes was measured in chemostat-grown S. paucimobilis B90A by RNA dot blot hybridization and real-time reverse transcriptase PCR upon exposure to a pulse of different HCH isomers. Irrespective of the addition of HCH, linA, linB, and linC were all expressed constitutively. In contrast, linD and linE were induced with alpha-HCH (2 mg/liter) and gamma-HCH (7 mg/liter). A sharp increase in mRNA levels for linD and linE was observed from 10 to 45 min after the addition of alpha- or gamma-HCH. Induction of linD and linE was not detectable upon the addition of 0.7 mg of gamma-HCH per liter, although the compound was degraded by the cells. The addition of beta-HCH (5 mg/liter) or delta-HCH (20 mg/liter) did not lead to linE and linD induction, despite the fact that 50% of the compounds were degraded. This suggests that degradation of beta- and delta-HCH proceeds by a different pathway than that of alpha- and gamma-HCH.

Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation

The inflammatory response is a protective process of the body to counteract xenobiotic penetration and injury, although in disease this response can become deregulated. There are endogenous biochemical pathways that operate in the host to keep inflammation under control. Here we demonstrate that the counter-regulator annexin 1 (AnxA1) is critical for controlling experimental endotoxemia. Lipopolysaccharide (LPS) markedly activated the AnxA1 gene in epithelial cells, neutrophils, and peritoneal, mesenteric, and alveolar macrophages-cell types known to function in experimental endotoxemia. Administration of LPS to AnxA1-deficient mice produced a toxic response characterized by organ injury and lethality within 48 hours, a phenotype rescued by exogenous application of low doses of the protein. In the absence of AnxA1, LPS generated a deregulated cellular and cytokine response with a marked degree of leukocyte adhesion in the microcirculation. Analysis of LPS receptor expression in AnxA1-null macrophages indicated an aberrant expression of Toll-like receptor 4. In conclusion, this study has detailed cellular and biochemical alterations associated with AnxA1 gene deletion and highlighted the impact of this protective circuit for the correct functioning of the homeostatic response to sublethal doses of LPS. Copyright © American Society for Investigative Pathology.

Experimental hyperthyroidism decreases gene expression and serum levels of adipokines in obesity

Aims. To analyze the influence of hyperthyroidism on the gene expression and serum concentration of leptin, resistin, and adiponectin in obese animals. Main Methods. Male Wistar rats were randomly divided into two groups: control (C)fed with commercial chow ad libitumand obese (OB)fed with a hypercaloric diet. After group characterization, the OB rats continued receiving a hypercaloric diet and were randomized into two groups: obese animals (OB) and obese with 25g triiodothyronine (T3)/100BW (OT). The T3 dose was administered every day for the last 2 weeks of the study. After 30 weeks the animals were euthanized. Samples of blood and adipose tissue were collected for biochemical and hormonal analyses as well as gene expression of leptin, resistin, and adiponectin. Results. T3 treatment was effective, increasing fT3 levels and decreasing fT4 and TSH serum concentration. Administration of T3 promotes weight loss, decreases all fat deposits, and diminishes serum levels of leptin, resistin, and adiponectin by reducing their gene expression. Conclusions. Our results suggest that T3 modulate serum and gene expression levels of leptin, resistin, and adiponectin in experimental model of obesity, providing new insights regarding the relationship between T3 and adipokines in obesity. Copyright © 2012 Renata de Azevedo Melo Luvizotto et al.

Maternal protein restriction during pregnancy affects gene expression and immunolocalization of intestinal nutrient transporters in rats

Intrauterine dietary restriction may cause changes in the functioning of offspring organs and systems later in life, an effect known as fetal programming. The present study evaluated mRNA abundance and immunolocalization of nutrient transporters as well as enterocytes proliferation in the proximal, median and distal segments of small intestine of rats born to protein-restricted dams. Pregnant rats were fed hypoproteic (6% protein) or control (17% protein) diets, and offspring rats were evaluated at 3 and 16 weeks of age. The presence of SGLT1 (sodium-glucose co-transporter 1), GLUT2 (glucose transporter 2), PEPT1 (peptide transporter 1) and the intestinal proliferation were evaluated by immunohistochemical techniques and the abundance of specific mRNA for SGLT1, GLUT2 and PEPT1 was assessed by the real-time PCR technique. Rats born to protein-restricted dams showed higher cell proliferation in all intestinal segments and higher gene expression of SGLT1 and PEPT1 in the duodenum. Moreover, in adult animals born to protein-restricted dams the immunoreactivity of SGLT1, GLUT2 and PEPT1in the duodenum was more intense than in control rats. Taken together, the results indicate that changes in the small intestine observed in adulthood can be programmed during the gestation. In addition, they show that this response is caused by both up-regulation in transporter gene expression, a specific adaptation mechanism, and intestinal proliferation, an unspecific adaptation mechanism.

Insulin, CCAAT/enhancer-binding proteins and lactate regulate the human 11β-hydroxysteroid dehydrogenase type 2 gene expression in colon cancer cell lines.

11β-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon.

Controlling Hox gene expression and activity to build the vertebrate axial skeleton

It has long been known that Hox genes are central players in patterning the vertebrate axial skeleton. Extensive genetic studies in the mouse have revealed that the combinatorial activity of Hox genes along the anterior-posterior body axis specifies different vertebral identities. In addition, Hox genes were instrumental for the evolutionary diversification of the vertebrate body plan. In this review, we focus on fundamental questions regarding the intricate mechanisms controlling Hox gene activity. In particular, we discuss the functional relevance of the precise timing of Hox gene activation in the embryo. Moreover, we provide insight into the epigenetic regulatory mechanisms that are likely to control this process and are responsible for the maintenance of spatially restricted Hox expression domains throughout embryonic development. We also analyze how specific features of each Hox protein may contribute to the functional diversity of Hox family. Altogether, the work reviewed here further supports the notion that the Hox program is far more complex than initially assumed. Exciting new findings will surely emerge in the years ahead.

von Hippel Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin-proteasome pathway. Here, we identify von Hippel-Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel-Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin-VHL-proteasome pathway in the integration-transcription transition of the viral replication cycle.

Whole Genome Analysis of Gene Expression Reveals Coordinated Activation of Signaling and Metabolic Pathways during Pollen-Pistil Interactions in Arabidopsis

Plant reproduction depends on the concerted activation of many genes to ensure correct communication between pollen and pistil. Here, we queried the whole transcriptome of Arabidopsis (Arabidopsis thaliana) in order to identify genes with specific reproductive functions. We used the Affymetrix ATH1 whole genome array to profile wild-type unpollinated pistils and unfertilized ovules. By comparing the expression profile of pistils at 0.5, 3.5, and 8.0 h after pollination and applying a number of statistical and bioinformatics criteria, we found 1,373 genes differentially regulated during pollen-pistil interactions. Robust clustering analysis grouped these genes in 16 time-course clusters representing distinct patterns of regulation. Coregulation within each cluster suggests the presence of distinct genetic pathways, which might be under the control of specific transcriptional regulators. A total of 78% of the regulated genes were expressed initially in unpollinated pistil and/or ovules, 15% were initially detected in the pollen data sets as enriched or preferentially expressed, and 7% were induced upon pollination. Among those, we found a particular enrichment for unknown transcripts predicted to encode secreted proteins or representing signaling and cell wall-related proteins, which may function by remodeling the extracellular matrix or as extracellular signaling molecules. A strict regulatory control in various metabolic pathways suggests that fine-tuning of the biochemical and physiological cellular environment is crucial for reproductive success. Our study provides a unique and detailed temporal and spatial gene expression profile of in vivo pollen-pistil interactions, providing a framework to better understand the basis of the molecular mechanisms operating during the reproductive process in higher plants.