Gene expression analysis in serial liver fine needle aspirates.

No method with low morbidity presently exists for obtaining serial hepatic gene expression measurements in humans. While hepatic fine needle aspiration (FNA) has lower morbidity than core needle biopsy, applicability is limited due to blood contamination, which confounds quantification of gene expression changes. The aim of this study was to validate FNA for assessment of hepatic gene expression. Liver needle biopsies and FNA procedures were simultaneously performed on 17 patients with chronic hepatitis C virus infection with an additional FNA procedure 1 week later. Nine patients had mild/moderate fibrosis and eight advanced fibrosis. Gene expression profiling was performed using Affymetrix microarrays and TaqMan qPCR; pathway analysis was performed using Ingenuity. We developed a novel strategy that applies liver-enriched normalization genes to determine the percentage of liver in the FNA sample, which enables accurate gene expression measurements overcoming biases derived from blood contamination. We obtained almost identical gene expression results (ρ = 0.99, P < 0.0001) comparing needle biopsy and FNA samples for 21 preselected genes. Gene expression results were also validated in dogs. These data suggest that liver FNA is a reliable method for serial hepatic tissue sampling with potential utility for a variety of preclinical and clinical applications.

Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins.

BACKGROUND: Signal transduction pathways with shared components must be insulated from each other to avoid the inappropriate activation of multiple pathways by a single stimulus. Scaffold proteins are thought to contribute to this specificity by binding select substrates. RESULTS: We have studied the ability of scaffold proteins to influence signaling by the yeast kinase Ste11, a MAPKKK molecule that participates in three distinct MAP kinase pathways: mating, filamentation, and HOG. We used protein fusions to force Ste11 to associate preferentially with a subset of its possible binding partners in vivo, including Ste5, Ste7, and Pbs2. Signaling became confined to a particular pathway when Ste11 was covalently attached to these scaffolds or substrates. This pathway bias was conferred upon both stimulus-activated and constitutively active forms of Ste11. We also used membrane-targeted derivatives of the mating pathway scaffold, Ste5, to show that stimulus-independent signaling initiated by this scaffold remained pathway specific. Finally, we demonstrate that loss of pathway insulation has a negative physiological consequence, as nonspecific activation of both the HOG and mating pathways interfered with proper execution of the mating pathway. CONCLUSIONS: The signaling properties of these kinase fusions support a model in which scaffold proteins dictate substrate choice and promote pathway specificity by presenting preferred substrates in high local concentration. Furthermore, insulation is inherent to scaffold-mediated signaling and does not require that signaling be initiated by pathway-specific stimuli or activator proteins. Our results give insight into the mechanisms and physiological importance of pathway insulation and provide a foundation for the design of customized signaling proteins.

Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast.

Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. In an effort to identify all genes regulated by Gcn4p during amino acid starvation, we performed cDNA microarray analysis. Data from 21 pairs of hybridization experiments using two different strains derived from S288c revealed that more than 1,000 genes were induced, and a similar number were repressed, by a factor of 2 or more in response to histidine starvation imposed by 3-aminotriazole (3AT). Profiling of a gcn4Delta strain and a constitutively induced mutant showed that Gcn4p is required for the full induction by 3AT of at least 539 genes, termed Gcn4p targets. Genes in every amino acid biosynthetic pathway except cysteine and genes encoding amino acid precursors, vitamin biosynthetic enzymes, peroxisomal components, mitochondrial carrier proteins, and autophagy proteins were all identified as Gcn4p targets. Unexpectedly, genes involved in amino acid biosynthesis represent only a quarter of the Gcn4p target genes. Gcn4p also activates genes involved in glycogen homeostasis, and mutant analysis showed that Gcn4p suppresses glycogen levels in amino acid-starved cells. Numerous genes encoding protein kinases and transcription factors were identified as targets, suggesting that Gcn4p is a master regulator of gene expression. Interestingly, expression profiles for 3AT and the alkylating agent methyl methanesulfonate (MMS) overlapped extensively, and MMS induced GCN4 translation. Thus, the broad transcriptional response evoked by Gcn4p is produced by diverse stress conditions. Finally, profiling of a gcn4Delta mutant uncovered an alternative induction pathway operating at many Gcn4p target genes in histidine-starved cells.

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.

We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

Experimental annotation of the human genome using microarray technology.

The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.

Functional discovery via a compendium of expression profiles.

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Widespread aneuploidy revealed by DNA microarray expression profiling.

Expression profiling using DNA microarrays holds great promise for a variety of research applications, including the systematic characterization of genes discovered by sequencing projects. To demonstrate the general usefulness of this approach, we recently obtained expression profiles for nearly 300 Saccharomyces cerevisiae deletion mutants. Approximately 8% of the mutants profiled exhibited chromosome-wide expression biases, leading to spurious correlations among profiles. Competitive hybridization of genomic DNA from the mutant strains and their isogenic parental wild-type strains showed they were aneuploid for whole chromosomes or chromosomal segments. Expression profile data published by several other laboratories also suggest the use of aneuploid strains. In five separate cases, the extra chromosome harboured a close homologue of the deleted gene; in two cases, a clear growth advantage for cells acquiring the extra chromosome was demonstrated. Our results have implications for interpreting whole-genome expression data, particularly from cells known to suffer genomic instability, such as malignant or immortalized cells.

Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles.

Genome-wide transcript profiling was used to monitor signal transduction during yeast pheromone response. Genetic manipulations allowed analysis of changes in gene expression underlying pheromone signaling, cell cycle control, and polarized morphogenesis. A two-dimensional hierarchical clustered matrix, covering 383 of the most highly regulated genes, was constructed from 46 diverse experimental conditions. Diagnostic subsets of coexpressed genes reflected signaling activity, cross talk, and overlap of multiple mitogen-activated protein kinase (MAPK) pathways. Analysis of the profiles specified by two different MAPKs-Fus3p and Kss1p-revealed functional overlap of the filamentous growth and mating responses. Global transcript analysis reflects biological responses associated with the activation and perturbation of signal transduction pathways.

Drug target validation and identification of secondary drug target effects using DNA microarrays.

We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.

Interaction between the retinoid X receptor and transcription factor IIB is ligand-dependent in vivo.

The retinoid X receptor (RXR) influences gene activation through heterodimeric and homodimeric association with DNA and associates with TATA binding protein, TAF110, and cAMP response element-binding protein-binding protein; yet the molecular mechanisms responsible for gene activation by RXRs remain incompletely defined. Since the general transcription factor IIB (TFIIB) is a common target of sequence-specific transcriptional activators, we suspected that RXR might regulate target genes via an interaction with TFIIB. Coimmunoprecipitation, far Western analysis, and glutathione S-transferase binding studies indicated that murine RXR beta (mRXR beta) was capable of binding to human TFIIB in vitro. Functional analysis with a dual-hybrid yeast system and cotransfection assays revealed the interaction of mRXR beta with TFIIB to be ligand-dependent in vivo. Truncation experiments mapped the essential binding regions to the carboxyl region of mRXR beta (amino acids (aa) 254-389) and two regions in the carboxyl region of TFIIB (aa 178-201 and aa 238-271). Furthermore, the delta 390-410 mRXR beta mutant bound to TFIIB in vitro but was not active in the dual-hybrid yeast system, suggesting that the extreme carboxyl region of RXR was required for in vivo interaction with TFIIB. These data indicate that interaction of mRXR beta with TFIIB is specific, direct, and ligand-dependent in vivo and suggest that gene activation by RXR involves TFIIB.

Evidence that GCN1 and GCN20, translational regulators of GCN4, function on elongating ribosomes in activation of eIF2alpha kinase GCN2.

In the yeast Saccharomyces cerevisiae, phosphorylation of translation initiation factor eIF2 by protein kinase GCN2 leads to increased translation of the transcriptional activator GCN4 in amino acid-starved cells. The GCN1 and GCN20 proteins are components of a protein complex required for the stimulation of GCN2 kinase activity under starvation conditions. GCN20 is a member of the ATP-binding cassette (ABC) family, most of the members of which function as membrane-bound transporters, raising the possibility that the GCN1/GCN20 complex regulates GCN2 indirectly as an amino acid transporter. At odds with this idea, indirect immunofluorescence revealed cytoplasmic localization of GCN1 and no obvious association with plasma or vacuolar membranes. In addition, a fraction of GCN1 and GCN20 cosedimented with polysomes and 80S ribosomes, and the ribosome association of GCN20 was largely dependent on GCN1. The C-terminal 84% of GCN20 containing the ABCs was found to be dispensable for complex formation with GCN1 and for the stimulation of GCN2 kinase function. Because ABCs provide the energy-coupling mechanism for ABC transporters, these results also contradict the idea that GCN20 regulates GCN2 as an amino acid transporter. The N-terminal 15 to 25% of GCN20, which is critically required for its regulatory function, was found to interact with an internal segment of GCN1 similar in sequence to translation elongation factor 3 (EF3). Based on these findings, we propose that GCN1 performs an EF3-related function in facilitating the activation of GCN2 by uncharged tRNA on translating ribosomes. The physical interaction between GCN20 and the EF3-like domain in GCN1 could allow for modulation of GCN1 activity, and the ABC domains in GCN20 may be involved in this regulatory function. A human homolog of GCN1 has been identified, and the portion of this protein most highly conserved with yeast GCN1 has sequence similarity to EF3. Thus, similar mechanisms for the detection of uncharged tRNA on translating ribosomes may operate in yeast and human cells.

GCN20, a novel ATP binding cassette protein, and GCN1 reside in a complex that mediates activation of the eIF-2 alpha kinase GCN2 in amino acid-starved cells.

GCN2 is a protein kinase that phosphorylates the alpha-subunit of translation initiation factor 2 (eIF-2) and thereby stimulates translation of GCN4 mRNA in amino acid-starved cells. We isolated a null mutation in a previously unidentified gene, GCN20, that suppresses the growth-inhibitory effect of eIF-2 alpha hyperphosphorylation catalyzed by mutationally activated forms of GCN2. The deletion of GCN20 in otherwise wild-type strains impairs derepression of GCN4 translation and reduces the level of eIF-2 alpha phosphorylation in vivo, showing that GCN20 is a positive effector of GCN2 kinase function. In accordance with this conclusion, GCN20 was co-immunoprecipitated from cell extracts with GCN1, another factor required to activate GCN2, and the two proteins interacted in the yeast two-hybrid system. We conclude that GCN1 and GCN20 are components of a protein complex that couples the kinase activity of GCN2 to the availability of amino acids. GCN20 is a member of the ATP binding cassette (ABC) family of proteins and is closely related to ABC proteins identified in Caenorhabditis elegans, rice and humans, suggesting that the function of GCN20 may be conserved among diverse eukaryotic organisms.

GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2.

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) by the protein kinase GCN2 mediates increased translation of the transcriptional activator GCN4 in amino acid-starved yeast cells. We show that this key phosphorylation event and the attendant translational induction of GCN4 are dependent on the product of a previously uncharacterized gene, GCN1. Inactivation of GCN1 did not affect the level of eIF-2 alpha phosphorylation when mammalian eIF-2 alpha kinases were expressed in yeast cells in place of GCN2, arguing against an involvement of GCN1 in dephosphorylation of eIF-2 alpha. In addition, while GCN1 is required in vivo for phosphorylation of eIF-2 alpha by GCN2, cell extracts from gcn1 delta strains contained wild-type levels of GCN2 eIF-2 alpha-kinase activity. On the basis of these results, we propose that GCN1 is not needed for GCN2 kinase activity per se but is required for in vivo activation of GCN2 in response to the starvation signal, uncharged tRNA. GCN1 encodes a protein of 297 kDa with an 88-kDa region that is highly similar in sequence to translation elongation factor 3 identified in several fungal species. This sequence similarity raises the possibility that GCN1 interacts with ribosomes or tRNA molecules and functions in conjunction with GCN2 in monitoring uncharged tRNA levels during the process of translation elongation.

The adenovirus E4 17-kilodalton protein complexes with the cellular transcription factor E2F, altering its DNA-binding properties and stimulating E1A-independent accumulation of E2 mRNA.

E2F is a cellular DNA-binding factor. Its binding activity is changed within adenovirus-infected cells so that it binds cooperatively to pairs of properly spaced and oriented E2F recognition sites. In the work described in this report, the conversion to cooperative binding was shown to require the adenovirus E4 17-kilodalton (kDa) polypeptide. Mutant viruses carrying alterations within the E4 17-kDa coding region failed to generate the infection-specific, cooperatively binding form of E2F. It was possible to alter E2F from uninfected cells so that it bound cooperatively by incubation with a partially purified fraction obtained from infected cells. The E4 17-kDa protein copurified with this activity and was also found to be present in a complex containing E2F. Consistent with its ability to alter the binding of E2F to its recognition sites within the E2 promoter, the E4 17-kDa polypeptide contributed to maximal expression of E2 mRNAs in some cell types. Its ability to enhance E2 transcription did not require expression of the E1A transactivator protein. These results are consistent with a model which proposes that the E4 17-kDa polypeptide binds to the cellular E2F factor, altering its binding behavior and thereby enhancing its ability to stimulate transcription.