Cancer transcriptomics: modeling metastasis.
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Cell Line, Tumor↗
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Transcriptome analysis reveals cyclobutane pyrimidine dimers as a major source of UV-induced DNA breaks.
Photolyase transgenic mice have opened new avenues to improve our understanding of the cytotoxic effects of ultraviolet (UV) light on skin by providing a means to selectively remove either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4) pyrimidone photoproducts. Here, we have taken a genomics approach to delineate pathways through which CPDs might contribute to the harmful effects of UV exposure. We show that CPDs, rather than other DNA lesions or damaged macromolecules, comprise the principal mediator of the cellular transcriptional response to UV. The most prominent pathway induced by CPDs is that associated with DNA double-strand break (DSB) signalling and repair. Moreover, we show that CPDs provoke accumulation of gamma-H2AX, P53bp1 and Rad51 foci as well as an increase in the amount of DSBs, which coincides with accumulation of cells in S phase. Thus, conversion of unrepaired CPD lesions into DNA breaks during DNA replication may comprise one of the principal instigators of UV-mediated cytotoxicity.
Animals↗
Transcriptome evolution - much ado about nothing?
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Animals↗
Comparative genomics: grasping human transcriptome evolution: what does it all mean?
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Animals↗
Comparison of medulloblastoma and normal neural transcriptomes identifies a restricted set of activated genes.
Over 1.4 million transcript tags expressed in 20 different human medulloblastomas were counted using serial analysis of gene expression. Digital gene expression profiles in the medulloblastoma were compared to multiple regions of the normal human brain, revealing 30 transcripts with high expression in multiple tumors and little or no expression in the normal cerebellum and other adult and pediatric brain regions. Using independent medulloblastoma samples and normal tissue, real-time PCR verified eight of nine selected genes as candidate tumor-associated antigens. Differential protein expression for CD24, prolactin and Topo2A was further confirmed by immunohistochemical analysis using medulloblastoma and normal brain sections and a tissue microarray. The genes highly expressed in the medulloblastoma include PRAME, a cancer-testis antigen and potential targets for immunotherapy.
Brain↗
Generation and analysis of melanoma SAGE libraries: SAGE advice on the melanoma transcriptome.
In this study, we generated three SAGE libraries from melanoma tissues. Using bioinformatics tools usually applied to microarray data, we identified several genes, including novel transcripts, which are preferentially expressed in melanoma. SAGE results converged with previous microarray analysis on the importance of intracellular calcium and G-protein signaling, and the Wnt/Frizzled family. We also examined the expression of CD74, which was specifically, albeit not abundantly, expressed in the melanoma libraries using a melanoma progression tissue microarray, and demonstrate that this protein is expressed by melanoma cells but not by benign melanocytes. Many genes involved in intracellular calcium and G-protein signaling were highly expressed in melanoma, results we had observed earlier from microarray studies (Bittner et al., 2000). One of the genes most highly expressed in our melanoma SAGE libraries was a calcium-regulated gene, calpain 3 (p94). Immunohistochemical analysis demonstrated that calpain 3 moves from the nuclei of non-neoplastic cells to the cytoplasm of malignant cells, suggesting activation of this intracellular proteinase. Our SAGE results and the clinical validation data demonstrate how SAGE profiles can highlight specific links between signaling pathways as well as associations with tumor progression. This may provide insights into new genes that may be useful for the diagnosis and therapy of melanoma.
Aged↗
Analysis of methylation-sensitive transcriptome identifies GADD45a as a frequently methylated gene in breast cancer.
Treatment of the breast cancer cell line, MDAMB468 with the DNA methylation inhibitor, 5-azacytidine (5-AzaC) results in growth arrest, whereas the growth of the normal breast epithelial line DU99 (telomerase immortalized) is relatively unaffected. Comparing gene expression profiles of these two lines after 5-AzaC treatment, we identified 36 genes that had relatively low basal levels in MDAMB468 cells compared to the DU99 line and were induced in the cancer cell line but not in the normal breast epithelial line. Of these genes, 33 have associated CpG islands greater than 300 bp in length but only three have been previously described as targets for aberrant methylation in human cancer. Northern blotting for five of these genes (alpha-Catenin, DTR, FYN, GADD45a, and Zyxin) verified the array results. Further analysis of one of these genes, GADD45a, showed that 5-AzaC induced expression in five additional breast cancer cell lines with little or no induction in three additional lines derived from normal breast epithelial cells. The CpG island associated with GADD45a was analysed by bisulfite sequencing, sampling over 100 CpG dinucleotides. We found that four CpG's, located approximately 700 bp upstream of the transcriptional start site are methylated in the majority of breast cancer cell lines and primary tumors but not in DNA from normal breast epithelia or matched lymphocytes from cancer patients. Therefore, this simple method of dynamic transcriptional profiling yielded a series of novel methylation-sensitive genes in breast cancer including the BRCA1 and p53 responsive gene, GADD45a.
Azacitidine↗
Cell cycle-dependent transcription in yeast: promoters, transcription factors, and transcriptomes.
In the budding yeast, Saccharomyces cerevisiae, a significant fraction of genes (>10%) are transcribed with cell cycle periodicity. These genes encode critical cell cycle regulators as well as proteins with no direct connection to cell cycle functions. Cell cycle-regulated genes can be organized into 'clusters' exhibiting similar patterns of regulation. In most cases periodic transcription is achieved via both repressive and activating mechanisms. Fine-tuning appears to have evolved by the juxtaposition of regulatory motifs characteristic of more than one cluster within the same promoter. Recent reports have provided significant new insight into the role of the cyclin-dependent kinase Cdk1 (Cdc28) in coordination of transcription with cell cycle events. In early G1, the transcription factor complex known as SBF is maintained in a repressed state by association of the Whi5 protein. Phosphorylation of Whi5 by Cdk1 in late G1 leads to dissociation from SBF and transcriptional derepression. G2/M-specific transcription is achieved by converting the repressor Fkh2 into an activator. Fkh2 serves as a repressor during most of the cell cycle. However, phosphorylation of a cofactor, Ndd1, by Cdk1 late in the cell cycle promotes binding to Fkh2 and conversion into a transcriptional activator. Such insights derived from analysis of specific genes when combined with genome-wide analysis provide a more detailed and integrated view of cell cycle-dependent transcription.
Cell Cycle↗
Transcriptome analysis of microdissected pancreatic intraepithelial neoplastic lesions.
Pancreatic ductal adenocarcinoma (PDAC) carries the most dismal prognosis of all solid tumours. Both the late clinical presentation of patients, due to lack of early symptoms, as well as the rapid and aggressive course of the disease contribute to the extremely high mortality of this malignancy. Recently, a multistep progression model for PDAC integrating morphological, clinical and molecular evidence has been proposed. Putative precursor lesions, termed pancreatic intraepithelial neoplasia (PanIN), are classified into three different grades (PanIN-1 through -3) based on the degree of cellular atypia they display. We have conducted large-scale expression profiling analyses of microdissected cells from normal pancreatic ducts, PanINs of different grades and PDACs using whole-genome oligonucleotide microarrays. Verification of hybridisation results for selected genes was performed using quantitative real-time PCR and immunohistochemical analyses on PanIN tissue microarrays. Comparison of the expression profiles demonstrated that the greatest changes in gene expression occur between PanIN stages 1B and 2, suggesting that PanIN-2 may represent the first truly preneoplastic stage in PDAC progression. Our results identify a large number of potential target genes for the development of novel molecular diagnostic and therapeutic tools for the prevention and early diagnosis of PDAC and provide novel insights into the pathophysiological mechanisms involved in tumour progression in the pancreas.
Base Sequence↗
Transcriptomic and proteomic analysis of liver and muscle alterations caused by surgical stress in rats.
The metabolic response to injury includes major alterations in protein metabolism; however, little is known about alterations in the synthesis of individual proteins and their role in the stress response. Our aim was to study how individual proteins in liver and muscle are altered by abdominal surgery. Changes produced in mRNA and proteins by abdominal surgery were studied in rats using RAP (random arbitrary priming)-PCR, to investigate mRNA alterations, and standard or isotopic (with in vivo radioactive labelling of proteins) two-dimensional electrophoresis/MS proteomic analyses, to study differential expression of proteins. Many of the differentially expressed proteins identified in blood were specifically synthesized by the liver to participate in the stress response. The hepatic proteins (antioxidant proteins, serine protease inhibitors, acute-phase proteins and transport proteins) were secreted into the bloodstream to produce a systemic action, indicating the central role of the liver in the stress response. Overexpressed proteins identified in liver were associated with the glycolytic processes and the folding of nascent proteins, confirming the high metabolic activity of the liver after surgery. The role of skeletal muscle protein as an amino acid donor to fuel the processes involved in the stress response was shown by the decrease in high-molecular-mass myofibrillar proteins. Combined use of the three techniques studied, differential RAP-PCR and standard and isotopic proteome analysis, provided complementary information on the differentially expressed proteins in a rat model of surgical stress.
Abdomen↗
Tuning in the transcriptome: basins of attraction in the yeast cell cycle.
Image processing techniques and wavelet analyses have been applied to the yeast cell cycle expression microchip data to reveal large-scale temporally coherent structures and high frequency oscillations in mRNA levels through the cycle. Because transitions in expression frequently occur in phase, they appear as peaks or troughs in colour maps and contour plots of expression levels. Although apparent in the untreated data, these transitions were identified and enhanced by convolution of a Laplacian kernel with the expression arrays of the first 4096 genes. Transitions associated with maximum up- or down-regulation of mRNA levels appear as bands at 30-40 min intervals through two cell cycles. Time-frequency analyses using wavelet transforms support these visualization techniques and lead to the conclusion that, with respect to gene expression, the dominant period is not the cell cycle (90-120 min) but, more commonly, the higher frequency 30-40 minute submultiple of the cycle period.
Cell Cycle↗
Transcriptome analysis of all two-component regulatory system mutants of Escherichia coli K-12.
We have systematically examined the mRNA profiles of 36 two-component deletion mutants, which include all two-component regulatory systems of Escherichia coli, under a single growth condition. DNA microarray results revealed that the mutants belong to one of three groups based on their gene expression profiles in Luria-Bertani broth under aerobic conditions: (i) those with no or little change; (ii) those with significant changes; and (iii) those with drastic changes. Under these conditions, the anaeroresponsive ArcB/ArcA system, the osmoresponsive EnvZ/OmpR system and the response regulator UvrY showed the most drastic changes. Cellular functions such as flagellar synthesis and expression of the RpoS regulon were affected by multiple two-component systems. A high correlation coefficient of expression profile was found between several two-component mutants. Together, these results support the view that a network of functional interactions, such as cross-regulation, exists between different two-component systems. The compiled data are avail-able at our website (http://ecoli.aist-nara.ac.jp/xp_analysis/ 2_components).
Bacterial Proteins↗
Transcriptome analysis of Listeria monocytogenes identifies three groups of genes differently regulated by PrfA.
PrfA is the major regulator of Listeria virulence gene expression. This protein is a member of the Crp/Fnr family of transcription regulators. To gain a deeper understanding of the PrfA regulon, we constructed a whole-genome array based on the complete genome sequence of Listeria monocytogenes strain EGDe and evaluated the expression profiles of the wild-type EGDe and a prfA-deleted mutant (EGDe Delta prfA). Both strains were grown at 37 degrees C in brain-heart infusion broth (BHI) and BHI supplemented with either activated charcoal, a compound known to enhance virulence gene expression, or cellobiose, a sugar reported to downregulate virulence gene expression in spite of full expression of PrfA. We identified three groups of genes that are regulated differently. Group I comprises, in addition to the 10 already known genes, two new genes, lmo2219 and lmo0788, both positively regulated and preceded by a putative PrfA box. Group II comprises eight negatively regulated genes: lmo0278 is preceded by a putative PrfA box, and the remaining seven genes (lmo0178-lmo0184) are organized in an operon. Group III comprises 53 genes, of which only two (lmo0596 and lmo2067) are preceded by a putative PrfA box. Charcoal addition induced upregulation of group I genes but abolished regulation by PrfA of most group III genes. In the presence of cellobiose, all the group I genes were downregulated, whereas group III genes remained fully activated. Group II genes were repressed in all conditions tested. A comparison of the expression profiles between a second L. monocytogenes strain (P14), its spontaneous mutant expressing a constitutively active PrfA variant (P14prfA*) and its corresponding prfA-deleted mutant (P14 Delta prfA) and the EGDe strain revealed interesting strain-specific differences. Sequences strongly similar to a sigma B-dependent promoter were identified upstream of 22 group III genes. These results suggest that PrfA positively regulates a core set of 12 genes preceded by a PrfA box and probably expressed from a sigma A-dependent promoter. In contrast, a second set of PrfA-regulated genes lack a PrfA box and are expressed from a sigma B-dependent promoter. This study reveals that PrfA can act as an activator or a repressor and suggests that PrfA may directly or indirectly activate different sets of genes in association with different sigma factors.
Bacterial Proteins↗
Differential transcriptome profiling identifies Plasmodium genes encoding pre-erythrocytic stage-specific proteins.
Invasive sporozoite and merozoite stages of malaria parasites that infect mammals enter and subsequently reside in hepatocytes and red blood cells respectively. Each invasive stage may exhibit unique adaptations that allow it to interact with and survive in its distinct host cell environment, and these adaptations are likely to be controlled by differential gene expression. We used suppression subtractive hybridization (SSH) of Plasmodium yoelii salivary gland sporozoites versus merozoites to identify stage-specific pre-erythrocytic transcripts. Sequencing of the SSH library and matching the cDNA sequences to the P. yoelii genome yielded 25 redundantly tagged genes including the only two previously characterized sporozoite-specific genes encoding the circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Twelve novel genes encode predicted proteins with signal peptides, indicating that they enter the secretory pathway of the sporozoite. We show that one novel protein bearing a thrombospondin type 1 repeat (TSR) exhibits an expression pattern that suggests localization in the sporozoite secretory rhoptry organelles. In addition, we identified a group of four genes encoding putative low-molecular-mass proteins. Two proteins in this group exhibit an expression pattern similar to TRAP, and thus possibly localize in the sporozoite secretory micronemes. Proteins encoded by the differentially expressed genes identified here probably mediate specific interactions of the sporozoite with the mosquito vector salivary glands or the mammalian host hepatocyte and are not used during merozoite-red blood cell interactions.
Amino Acid Sequence↗
Transcriptome analysis of sulfur depletion in Arabidopsis thaliana: interlacing of biosynthetic pathways provides response specificity.
Higher plants assimilate inorganic sulfate into cysteine, which is subsequently converted to methionine, and into a variety of other sulfur-containing organic compounds. To resist sulfur deficiency, plants must demonstrate physiological flexibility: the expression of an extensive set of genes and gene regulators that act in the affected pathways or signalling cascades must be delicately tuned in response to environmental challenges. To elucidate this network of interactions, we have applied an array hybridisation/transcript profiling method to Arabidopsis plants subjected to 6, 10 and 13 days of constitutive and induced sulfur starvation. The temporal expression behaviour of approximately 7200 non-redundant genes was analysed simultaneously. The experiment was designed in a way to identify statistically significant changes of gene expression based on sufficient numbers of repeated hybridisations performed with five uniform pools of plant material. The expression profiles were processed to select differentially expressed genes. Among the 1507 sulfur-responsive clones implicated in this way, 632 genes responded specifically to sulfur deficiency by significant over-expression. The sulfur-responsive genes were grouped according to functional categories or biosynthetic pathways. As expected, genes of the sulfur assimilation pathway were altered in expression. Furthermore, genes involved in flavonoid, auxin, and jasmonate biosynthesis pathways were upregulated in conditions of sulfur deficiency. Based on the correlative analysis of gene expression patterns, we suggest that a complex co-ordination of systematic responses to sulfur depletion is provided via integration of flavonoid, auxin and jasmonate pathway elements. Plait concept for transduction of specificity via the main non-specific signalling stream is proposed.
Adaptation, Physiological↗
Towards deciphering phloem: a transcriptome analysis of the phloem of Apium graveolens.
Events occurring in the phloem tissue are key to understanding a wide range of developmental and physiological processes in vascular plants. While a considerable amount of molecular information on phloem proteins has emerged in the past decade, a unified picture of the molecular mechanisms involved in phloem differentiation and function is still lacking. New models to increase our understanding of this complex tissue can be created by the development of global approaches such as genomic analysis. In order to obtain a comprehensive overview of the molecular biology of the phloem tissue, we developed a genomic approach using Apium graveolens as a model. cDNA libraries were constructed from mRNAs extracted from isolated phloem of petioles. Expression data obtained from the analysis of 989 expressed sequence tags (ESTs) and the transcript profile deduced from a cDNA macroarray of 1326 clones were combined to identify genes showing distinct expression patterns in the vascular tissues. Comparisons of expression profiles obtained from the phloem, xylem and storage parenchyma tissues uncovered tissue-specific differential expression patterns for given sets of genes. The major classes of mRNAs predominantly found in the phloem encode proteins related to phloem structure, metal homeostasis or distribution, stress responses and degradation or turnover of proteins. Of great interest for future studies are the genes we found to be specifically expressed in the phloem but for which the function is still unknown, and also those genes described in previous reports to be up or downregulated by specific interactions. From a broader prospective, our results also clearly demonstrate that cDNA macroarray technology can be used to identify the key genes involved in various physiological and developmental processes in the phloem.
Apium↗
Transcriptome analysis of Arabidopsis colonized by a plant-growth promoting rhizobacterium reveals a general effect on disease resistance.
RNA transcript levels of Arabidopsis plants, infected by the rhizobacterium Pseudomonas thivervalensis (strain MLG45), and axenic control plants were compared using cDNA microarrays representing approximately 14 300 genes. The analysis revealed an increase of defence-related transcripts in the shoots of bacterized plants relative to control (axenic) plants. These modifications of transcript levels were confirmed by physiological experiments. Plants infected with P. thivervalensis were more resistant to subsequent infections by the virulent pathogen P. syringae pv. tomato (strain DC3000) than control plants. In addition, photosynthesis rates were repressed consistently with the reduced growth of plants colonized by P. thivervalensis. These results highlight the value of molecular phenotyping to predict physiological changes.
Arabidopsis↗
[Salmonella transcriptome during macrophage infection].
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DNA, Bacterial↗