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Frank C P Holstege

Publications and source records attributed to Frank C P Holstege.

At least 19 recordsLinked to original sources

Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae.

Defining protein complexes is critical to virtually all aspects of cell biology. Two recent affinity purification/mass spectrometry studies in Saccharomyces cerevisiae have vastly increased the available protein interaction data. The practical utility of such high throughput interaction sets, however, is substantially decreased by the presence of false positives. Here we created a novel probabilistic metric that takes advantage of the high density of these data, including both the presence and absence of individual associations, to provide a measure of the relative confidence of each potential protein-protein interaction. This analysis largely overcomes the noise inherent in high throughput immunoprecipitation experiments. For example, of the 12,122 binary interactions in the general repository of interaction data (BioGRID) derived from these two studies, we marked 7504 as being of substantially lower confidence. Additionally, applying our metric and a stringent cutoff we identified a set of 9074 interactions (including 4456 that were not among the 12,122 interactions) with accuracy comparable to that of conventional small scale methodologies. Finally we organized proteins into coherent multisubunit complexes using hierarchical clustering. This work thus provides a highly accurate physical interaction map of yeast in a format that is readily accessible to the biological community.

Cluster Analysis↗

Maintenance of head and neck tumor gene expression profiles upon lymph node metastasis.

Spread of cancer and development of solid metastases at distant sites is the main cause of cancer-related deaths. To understand and treat metastases, it is important to determine at which stages the most pivotal steps for development of metastases occur. In head and neck squamous cell carcinoma (HNSCC), metastasis nearly always occurs first in local lymph nodes before development of distant metastasis. Here, we have investigated gene expression patterns in HNSCC lymph node metastases using DNA microarrays. Several types of analyses show that the gene expression patterns in lymph node metastases are most similar to the corresponding primary tumors from which they arose, as long as samples contain sufficient proportions of tumor cells. Strikingly, gene expression patterns of metastatic primary HNSCC are largely maintained upon spread to the lymph node. Only a single gene, metastasis-associated gene 1 (MTA1), was found to show consistently changed expression between a large number of matched primary tumor-lymph node metastasis pairs. The maintained expression pattern includes the predictive signature for HNSCC lymph node metastasis. These results underscore the importance of the primary tumor gene expression profile for development and treatment of metastasis. The findings also agree with the concept that disseminated cancer cells alter the surrounding tissue into a metastatic environment that resembles the primary tumor microenvironment.

Adult↗

Genome-wide location of the coactivator mediator: Binding without activation and transient Cdk8 interaction on DNA.

Mediator is a general coactivator of RNA polymerase II (Pol II) transcription. Genomic location analyses of different Mediator subunits indicate a uniformly composed core complex upstream of active genes but unexpectedly also upstream of inactive genes and on the coding regions of some highly active genes. The repressive Cdk8 submodule is associated with core Mediator at all sites but with a lower degree of occupancy, indicating transient interaction, regardless of promoter activity. This suggests gene-specific regulation of Cdk8 activity, rather than regulated Cdk8 recruitment. Mediator presence is not necessarily linked to transcription. This goes beyond Cdk8-repressed genes, indicating that Mediator can mark some regulatory regions ahead of additional signals. Overlap with intergenic Pol II location in stationary phase points to a role as a binding platform for inactive Pol II during quiescence. These results shed light on Cdk8 repression, suggest additional roles for Mediator, and query models of recruitment-coupled regulation.

Binding Sites↗

Multiple robust signatures for detecting lymph node metastasis in head and neck cancer.

Genome-wide mRNA expression measurements can identify molecular signatures of cancer and are anticipated to improve patient management. Such expression profiles are currently being critically evaluated based on an apparent instability in gene composition and the limited overlap between signatures from different studies. We have recently identified a primary tumor signature for detection of lymph node metastasis in head and neck squamous cell carcinomas. Before starting a large multicenter prospective validation, we have thoroughly evaluated the composition of this signature. A multiple training approach was used for validating the original set of predictive genes. Based on different combinations of training samples, multiple signatures were assessed for predictive accuracy and gene composition. The initial set of predictive genes is a subset of a larger group of 825 genes with predictive power. Many of the predictive genes are interchangeable because of a similar expression pattern across the tumor samples. The head and neck metastasis signature has a more stable gene composition than previous predictors. Exclusion of the strongest predictive genes could be compensated by raising the number of genes included in the signature. Multiple accurate predictive signatures can be designed using various subsets of predictive genes. The absence of genes with strong predictive power can be compensated by including more genes with lower predictive power. Lack of overlap between predictive signatures from different studies with the same goal may be explained by the fact that there are more predictive genes than required to design an accurate predictor.

Carcinoma, Squamous Cell↗

Transcription factor target practice.

In this issue of Cell, Hallikas et al. (2006) and Wei et al. (2006) describe different ways of identifying direct targets of transcription factors and their corresponding regulatory sequences in the genome. Although still under development, these studies provide an efficient way to decipher regulatory networks.

Animals↗

Dissection of a metastatic gene expression signature into distinct components.

BACKGROUND: Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures. RESULTS: For a head-neck squamous cell carcinoma (HNSCC) primary tumor expression signature that predicts the presence of lymph node metastasis, we first show that reduced proportions of tumor cells results in decreased predictive accuracy. To determine the influence of stroma on the predictive signature and to investigate the interaction between tumor cells and the surrounding microenvironment, we used laser capture microdissection to divide the metastatic signature into six distinct components based on tumor versus stroma expression and on association with the metastatic phenotype. A strikingly skewed distribution of metastasis associated genes is revealed. CONCLUSION: Dissection of predictive signatures into different components has implications for design of expression signatures and for our understanding of the metastatic process. Compared to primary tumors that have not formed metastases, primary HNSCC tumors that have metastasized are characterized by predominant down-regulation of tumor cell specific genes and exclusive up-regulation of stromal cell specific genes. The skewed distribution agrees with poor signature performance on samples that contain less than 50% tumor cells. Methods for reducing tumor composition bias that lead to greater predictive accuracy and an increase in the types of samples that can be included are presented.

Carcinoma, Squamous Cell↗

Mediator expression profiling epistasis reveals a signal transduction pathway with antagonistic submodules and highly specific downstream targets.

Mediator is an evolutionarily conserved coregulator of RNA polymerase II transcription. Microarray structure-function analysis of S. cerevisiae Mediator reveals functional antagonism between the cyclin-dependent kinase (Cdk) submodule and components from the Tail (Med15, Med2, Med3), Head (Med20, Med18), and Middle (Med31). Certain genes exhibit increased or decreased expression, depending on which subunit is deleted. Epistasis analysis with expression-profile phenotypes shows that MED2 and MED18 are downstream of CDK8. Strikingly, Cdk8-mediated modification of a single amino acid within Mediator represses the regulon of a single transcription factor, Rcs1/Aft1. Highly specific gene regulation is thought to be determined by activators and combinatorial use of cofactors. Here, subtle modification of the general transcription machinery through one of its own components is shown to determine highly specific expression patterns. Expression profiling can therefore precisely map regulatory cascades, and our findings support a role for Mediator as a direct processor of signaling pathways for determining specificity.

Chromosomal Proteins, Non-Histone↗

Tumor profiling turmoil.

Tumor profiling aims to determine gene expression signatures that can discriminate between different sub-types of tumors. We have recently discovered a signature that can reliably detect which primary head-neck tumors have metastasized to local lymph nodes. This signature has great potential for clinical application and also offers unique insights into how metastasis occurs. Despite these obvious advances, discussed here alongside several other findings, such tumor profiling studies are currently receiving harsh criticism. We make clear that such evaluations can themselves also be critically evaluated. The separation between the two factions actually shows that it is too early to either dismiss or exalt tumor profiling studies. Final judgment requires waiting for the results of larger prospective studies carried out in parallel with current clinical practice.

Carcinoma, Squamous Cell↗

Gene expression profiling and phenotype analyses of S. cerevisiae in response to changing copper reveals six genes with new roles in copper and iron metabolism.

Exhaustive microarray time course analyses of Saccharomyces cerevisiae during copper starvation and copper excess reveal new aspects of metal-induced gene regulation. Aside from identifying targets of established copper- and iron-responsive transcription factors, we find that genes encoding mitochondrial proteins are downregulated and that copper-independent iron transport genes are preferentially upregulated, both during prolonged copper deprivation. The experiments also suggest the presence of a small regulatory iron pool that links copper and iron responses. One hundred twenty-eight genes with putative roles in metal metabolism were further investigated by several systematic phenotype screens. Of the novel phenotypes uncovered, hsp12-Delta and arn1-Delta display increased sensitivity to copper, cyc1-Delta and crr1-Delta show resistance to high copper, vma13-Delta exhibits increased sensitivity to iron deprivation, and pep12-Delta results in reduced growth in high copper and low iron. Besides revealing new components of eukaryotic metal trafficking pathways, the results underscore the previously determined intimate links between iron and copper metabolism and mitochondrial and vacuolar function in metal trafficking. The analyses further suggest that copper starvation can specifically lead to downregulation of respiratory function to preserve iron and copper for other cellular processes.

Amino Acid Motifs↗

Gene expression of energy and protein metabolism in hearts of hypertensive nitric oxide- or GSH-depleted mice.

Hypertension demands cardiac synthetic and metabolic adaptations to increased afterload. We studied gene expression in two models of mild hypertension without overt left ventricular hypertrophy using the NO synthase inhibitor nitro-L-arginine (L-NNA) and the glutathione depletor buthionine-S,R-sulfoximine (BSO). Mice were administered L-NNA, BSO, or water for 8 weeks. RNA of left ventricles was pooled per group, reverse transcribed, Cy3 and Cy5 labeled, and hybridized to cDNA microarrays. Normalized log(2) Cy3/Cy5 ratios of > or =0.7 or < or =-0.7 were considered significant. L-NNA and BSO both caused hypertension. Gene expression was regulated in cytoskeletal components in both models, protein synthesis in L-NNA-treated mice, and energy metabolism in BSO-treated mice. Energy metabolism genes shared several common transcription factor-binding sites such as Coup-Tf2, of which gene expression was increased in BSO-treated mice, and COMP-1. Characterization of the left ventricular adaptations as assessed with gene expression profiles reveals differential expression in energy and protein metabolism related to the pathogenetic background of the hypertension.

Animals↗

Genome-wide analyses reveal RNA polymerase II located upstream of genes poised for rapid response upon S. cerevisiae stationary phase exit.

The resting state of eukaryotic cells (G0) is relatively uncharacterized. We have applied DNA microarray expression profiling of S. cerevisiae to reveal multiple transitions during a complete 9-day growth cycle between stationary phase (SP) exit and entry. The findings include distinct waves of transcription after the diauxic shift (DS), identification of genes active in SP, and upregulation of over 2500 genes during the first minutes of lag phase. This provides a framework for analyzing large-scale reprogramming of gene expression. Despite global repression, the general transcription machinery is found to be present in quiescent cells but is largely inactive. Genome-wide location analysis by chromatin immunoprecipitation (ChIP on chip) reveals that RNA polymerase II is more predominantly bound at intergenic regions in SP, upstream of hundreds of genes immediately induced upon exit. In contrast to current models of activation-coupled recruitment, the results show that RNA polymerase II is located and maintained upstream of many inactive genes in quiescence.

Chromatin Immunoprecipitation↗

An expression profile for diagnosis of lymph node metastases from primary head and neck squamous cell carcinomas.

Metastasis is the process by which cancers spread to distinct sites in the body. It is the principal cause of death in individuals suffering from cancer. For some types of cancer, early detection of metastasis at lymph nodes close to the site of the primary tumor is pivotal for appropriate treatment. Because it can be difficult to detect lymph node metastases reliably, many individuals currently receive inappropriate treatment. We show here that DNA microarray gene-expression profiling can detect lymph node metastases for primary head and neck squamous cell carcinomas that arise in the oral cavity and oropharynx. The predictor, established with an 82-tumor training set, outperforms current clinical diagnosis when independently validated. The 102 predictor genes offer unique insights into the processes underlying metastasis. The results show that the metastatic state can be deciphered from the primary tumor gene-expression pattern and that treatment can be substantially improved.

Carcinoma, Squamous Cell↗

Predicting gene function through systematic analysis and quality assessment of high-throughput data.

MOTIVATION: Determining gene function is an important challenge arising from the availability of whole genome sequences. Until recently, approaches based on sequence homology were the only high-throughput method for predicting gene function. Use of high-throughput generated experimental data sets for determining gene function has been limited for several reasons. RESULTS: Here a new approach is presented for integration of high-throughput data sets, leading to prediction of function based on relationships supported by multiple types and sources of data. This is achieved with a database containing 125 different high-throughput data sets describing phenotypes, cellular localizations, protein interactions and mRNA expression levels from Saccharomyces cerevisiae, using a bit-vector representation and information content-based ranking. The approach takes characteristic and qualitative differences between the data sets into account, is highly flexible, efficient and scalable. Database queries result in predictions for 543 uncharacterized genes, based on multiple functional relationships each supported by at least three types of experimental data. Some of these are experimentally verified, further demonstrating their reliability. The results also generate insights into the relative merits of different data types and provide a coherent framework for functional genomic datamining. AVAILABILITY: Free availability over the Internet. CONTACT: f.c.p.holstege@med.uu.nl SUPPLEMENTARY INFORMATION: http://www.genomics.med.uu.nl/pub/pk/comb_gen_network.

Computer Simulation↗

A high resolution protein interaction map of the yeast Mediator complex.

Mediator is a large, modular protein complex remotely conserved from yeast to man that conveys regulatory signals from DNA-binding transcription factors to RNA polymerase II. In Saccharomyces cerevisiae, Mediator is thought to be composed of 24 subunits organized in four sub-complexes, termed the head, middle, tail and Cdk8 (Srb8-11) modules. In this work, we have used screening and pair-wise two-hybrid approaches to investigate protein-protein contacts between budding yeast Mediator subunits. The derived interaction map includes the delineation of numerous interaction domains between Mediator subunits, frequently corresponding to segments that have been conserved in evolution, as well as novel connections between the Cdk8 (Srb8-11) and head modules, the head and middle modules, and the middle and tail modules. The two-hybrid analysis, together with co-immunoprecipitation studies and gel filtration experiments revealed that Med31 (Soh1) is associated with the yeast Mediator that therefore comprises 25 subunits. Finally, analysis of the protein interaction network within the Drosophila Mediator middle module indicated that the structural organization of the Mediator complex is conserved from yeast to metazoans. The resulting interaction map provides a framework for delineating Mediator structure-function and investigating how Mediator function is regulated.

Animals↗

Nitric oxide donor induces temporal and dose-dependent reduction of gene expression in human endothelial cells.

The present study tested the hypothesis that acute increases in nitric oxide (NO) exert substantial influences on gene transcription in endothelial cells (ECs) via guanylyl cyclase (GC). Human umbilical veins ECs (HUVECs) were exposed to 0.1, 1, and 10 mM of sodium nitroprusside (SNP) for 4 h and to 1 mM SNP or 250 microM of (Z)-1[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) for 2, 4, 8, and 24 h. Also, cells were exposed to DETA-NONOate in the presence and absence of the GC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 10 microM) for 4 h. RNA was isolated, reverse transcribed, Cy3 and Cy5 labeled, and analyzed using cDNA microarrays. Increasing doses of SNP predominantly depressed gene expression in HUVECs. Gene function was related to growth, adhesion, and cell structure. DETA-NONOate evoked a wave of expression changes (maximum at 4 h), with a remarkable downregulation of the transcription factors MSX1, RELB, and Egr-1. Both SNP- and DETA-NONOate-induced gene expression had faded after 24 h, despite continued elevation of cGMP in the medium. Coadministration of ODQ decreased many, but not all, of the transcriptional responses to DETA-NONOate. NO pronouncedly depressed EC gene expression, in particular of transcription factors. The observation that many, but not all, transcriptional changes induced by NO could be inhibited by inhibition of GC indicates the presence of GC-independent NO actions on gene expression. Thus EC gene expression responds to NO; however, the transcriptional response fades during prolonged exposure. This could allow the EC to respond to increased shear, without vigorous changes in gene expression.

Cell Survival↗

Broadly altered gene expression in blood leukocytes in essential hypertension is absent during treatment.

We assessed whether large-scale expression profiling of leukocytes of patients with essential hypertension reflects characteristics of systemic disease and whether such changes are responsive to antihypertensive therapy. Total RNA from leukocytes were obtained from untreated (n=6) and treated (n=6) hypertensive patients without apparent end-organ damage and from normotensive controls (n=9). RNA was reverse-transcribed and labeled and gene expression analyzed using a 19-K oligonucleotide microarray using dye swaps. Samples of untreated and of treated patients were pooled for each sex and compared with age- and sex-matched controls. In untreated patients, 680 genes were differentially regulated (314 up and 366 down). In the treated patients, these changes were virtually absent (4 genes up, 3 genes down). A myriad of changes was observed in pathways involved in inflammation. Inflammation-dampening interleukin receptors were decreased in expression. Intriguingly, inhibitors of cytokine signaling (the PIAS family of proteins) were differentially expressed. The expression of several genes that are involved in regulation of blood pressure were also differentially expressed: angiotensin II type 1 receptor, ANP-A receptor, endothelin-2, and 3 of the serotonin receptors were increased, whereas endothelin-converting enzyme-1 was decreased. Strikingly, virtually no changes in gene expression could be detected in hypertensive patients who had become normotensive with treatment. This observation substantiates the long-standing idea that hypertension is associated with a complex systemic response involving inflammation-related genes. Furthermore, leukocytes display differential gene expression that is of importance in blood pressure control. Importantly, treatment of blood pressure to normal values can virtually correct such disturbances.

Adult↗

Fibronectin is a hypoxia-independent target of the tumor suppressor VHL.

The von Hippel-Lindau (VHL) tumor suppressor gene regulates the extracellular matrix by controlling fibronectin deposition. To identify novel VHL target genes, we subjected mRNA from VHL-deficient RCC cells (786-0-pRC) and a transfectant re-expressing wildtype VHL (786-0-VHL) to differential expression profiling. Among the differentially expressed genes, we detected that fibronectin is upregulated in the presence of VHL, while it is not affected by hypoxia. Thus regulation of fibronectin deposition by VHL occurs at the transcriptional level, irrespective of oxygen levels.

Carcinoma, Renal Cell↗