Previous studies have described a transcriptional "memory effect," whereby transcript levels of many Abf1-regulated genes in the budding yeast Saccharomyces cerevisiae are undiminished even after Abf1 has dissociated from its regulatory sites. Here we provide additional support for this effect and investigate its molecular basis. We show that the effect is observed in a distinct abf1 ts mutant from that used in earlier studies, demonstrating that it is robust, and use chromatin immunoprecipitation to show that Abf1 association is decreased similarly from memory effect and transcriptionally responsive genes at the restrictive temperature. We also demonstrate that the association of TATA-binding protein and Pol II decreases after the loss of Abf1 binding for transcriptionally responsive genes but not for memory effect genes. Examination of genome-wide nucleosome occupancy data reveals that although transcriptionally responsive genes exhibit increased nucleosome occupancy in abf1 ts yeast, the promoter regions of memory effect targets show no change in abf1 ts mutants, maintaining an open chromatin conformation even after Abf1 eviction. This contrasting behavior reflects different inherent propensity for nucleosome formation between the two classes, driven by the presence of A/T-rich sequences upstream of the Abf1 site in memory effect gene promoters. These sequence-based differences show conservation in closely related fungi and also correlate with different gene expression noise, suggesting a physiological basis for greater access to " memory effect" promoter regions. Thus, our results establish a conserved mechanism underlying a transcriptional memory effect whereby sequences surrounding Abf1 binding sequences affect local nucleosome occupancy following loss of Abf1 binding. Furthermore, these findings demonstrate that sequence-based differences in the propensity for nucleosome occupancy can influence the transcriptional response of genes to an altered regulatory
Genome-encoded microRNAs (miRNAs) provide a posttranscriptional regulatory layer that controls the differentiation and function of various cellular systems, including hematopoietic cells. miR-142 is one of the most prevalently expressed miRNAs within the hematopoietic lineage. To address the in vivo functions of miR-142 we utilized a novel reporter and loss-of-function mouse allele that we have recently generated. Here, we show that miR-142 is broadly expressed in the adult hematopoietic system. Our data further reveal that miR-142 is critical for megakaryopoiesis. Thus, genetic miR-142 ablation caused impaired megakaryocyte maturation, inhibition of polyploidization, abnormal proplatelet formation, and thrombocytopenia. Finally, we characterize a network of miR-142-3p targets which collectively controls actin filament homeostasis, thereby ensuring proper execution of actin-dependent proplatelet formation. Our study reveals a pivotal role for miR-142 activity in megakaryocyte maturation and function, and demonstrates a critical contribution of a single miRNA in orchestrating cytoskeletal dynamics and normal haemostasis.
The bacterial community in the human gut has crucial health roles both in metabolic functions and in protection against pathogens. Phages, which are known to significantly affect microbial community composition in many ecological niches, have the potential to impact the gut microbiota, yet thorough characterization of this relationship remains elusive. We have reconstructed the content of the CRISPR bacterial immune system in the human gut microbiomes of 124 European individuals and used it to identify a catalog of 991 phages targeted by CRISPR across all individuals. Our results show that 78% of these phages are shared among two or more individuals. Moreover, a significant fraction of phages found in our study are shown to exist in fecal samples previously derived from American and Japanese individuals, identifying a common reservoir of phages frequently associated with the human gut microbiome. We further inferred the bacterial hosts for more than 130 such phages, enabling a detailed analysis of phage-bacteria interactions across the 124 individuals by correlating patterns of phage abundance with bacterial abundance and resistance. A subset of phages demonstrated preferred association with host genomes as lysogenized prophages, with highly increased abundance in specific individuals. Overall, our results imply that phage-bacterial attack-resistance interactions occur within the human gut microbiome, possibly affecting microbiota composition and human health. Our finding of global sharing of gut phages is surprising in light of the extreme genetic diversity of phages found in other ecological niches.
Gene expression shows a significant variation (noise) between genetically identical cells. Noise depends on the gene expression process regulated by the chromatin environment. We screened for chromatin factors that modulate noise in S. cerevisiae and analyzed the results using a theoretical model that infers regulatory mechanisms from the noise versus mean relationship. Distinct activities of the Rpd3(L) and Set3 histone deacetylase complexes were predicted. Both HDACs repressed expression. Yet, Rpd3(L)C decreased the frequency of transcriptional bursts, while Set3C decreased the burst size, as did H2B monoubiquitination (ubH2B). We mapped the acetylation of H3 lysine 9 (H3K9ac) upon deletion of multiple subunits of Set3C and Rpd3(L)C and of ubH2B effectors. ubH2B and Set3C appear to function in the same pathway to reduce the probability that an elongating PoIII produces a functional transcript (PoIII processivity), while Rpd3(L)C likely represses gene expression at a step preceding elongation.
Understanding why genes evolve at different rates is fundamental to evolutionary thinking. In species of the budding yeast, the rate at which genes diverge in expression correlates with the organization of their promoter nucleosomes: genes lacking a nucleosome-free region (denoted OPN for "Occupied Proximal Nucleosomes'') vary widely between the species, while the expression of those containing NFR (denoted DPN for "Depleted Proximal Nucleosomes'') remains largely conserved. To examine if early evolutionary dynamics contributes to this difference in divergence, we artificially selected for high expression of GFP-fused proteins. Surprisingly, selection was equally successful for OPN and DPN genes, with similar to 80% of genes in each group stably increasing in expression by a similar amount. Notably, the two groups adapted by distinct mechanisms: DPN-selected strains duplicated large genomic regions, while OPN-selected strains favored trans mutations not involving duplications. When selection was removed, DPN (but not OPN) genes reverted rapidly to wild-type expression levels, consistent with their lower diversity between species. Our results suggest that promoter organization constrains the early evolutionary dynamics and in this way biases the path of long-term evolution.
Background: Previous work showed that mRNA degradation is coordinated with transcription in yeast, and in several genes the control of mRNA degradation was linked to promoter elements through two different mechanisms. Here we show at the genomic scale that the coordination of transcription and mRNA degradation is promoter-dependent in yeast and is also observed in humans. Results: We first demonstrate that swapping upstream cis-regulatory sequences between two yeast species affects both transcription and mRNA degradation and suggest that while some cis-regulatory elements control either transcription or degradation, multiple other elements enhance both processes. Second, we show that adjacent yeast genes that share a promoter (through divergent orientation) have increased similarity in their patterns of mRNA degradation, providing independent evidence for the promoter-mediated coupling of transcription to mRNA degradation. Finally, analysis of the differences in mRNA degradation rates between mammalian cell types or mammalian species suggests a similar coordination between transcription and mRNA degradation in humans. Conclusions: Our results extend previous studies and suggest a pervasive promoter-mediated coordination between transcription and mRNA degradation in yeast. The diverse genes and regulatory elements associated with this coordination suggest that it is generated by a global mechanism of gene regulation and modulated by gene-specific mechanisms. The observation of a similar coupling in mammals raises the possibility that coupling of transcription and mRNA degradation may reflect an evolutionarily conserved phenomenon in gene regulation.
Genome-wide patterns of nucleosome occupancy and positioning have greatly impacted on studies of chromatin structure, yet these studies require extensive computational analysis which is crucial for the quality of the resulting datasets and inferred conclusions. This chapter describes the computational steps required in order to estimate genome-wide patterns of nucleosome occupancy and positioning from raw data obtained from high-throughput sequencing of mononucleosome DNA fragments. Potential pitfalls that may be encountered in such analysis and computational quality controls are further discussed in Subheading 3.
Background: GC-skews have previously been linked to transcription in some eukaryotes. They have been associated with transcription start sites, with the coding strand G-biased in mammals and C-biased in fungi and invertebrates. Results: We show a consistent and highly significant pattern of GC-skew within genes of almost all unicellular fungi. The pattern of GC-skew is asymmetrical: the coding strand of genes is typically C-biased at the 5' ends but G-biased at the 3' ends, with intermediate skews at the middle of genes. Thus, the initiation, elongation, and termination phases of transcription are associated with different skews. This pattern influences the encoded proteins by generating differential usage of amino acids at the 5' and 3' ends of genes. These biases also affect fourfold-degenerate positions and extend into promoters and 3' UTRs, indicating that skews cannot be accounted by selection for protein function or translation. Conclusions: We propose two explanations, the mutational pressure hypothesis, and the adaptive hypothesis. The mutational pressure hypothesis is that different co-factors bind to RNA pol II at different phases of transcription, producing different mutational regimes. The adaptive hypothesis is that cytidine triphosphate deficiency may lead to C-avoidance at the 3' ends of transcripts to control the flow of RNA pol II molecules and reduce their frequency
To examine the role of nucleosome occupancy in the evolution of gene expression, we measured the genome-wide nucleosome profiles of four yeast species, three belonging to the Saccharomyces sensu stricto lineage and the more distantly related Candida glabrata. Nucleosomes and associated promoter elements at C. glabrata genes are typically shifted upstream by similar to 20 bp, compared to their orthologs from sensu stricto species. Nonetheless, all species display the same global organization features first described for Saccharomyces cerevisiae: a stereotypical nucleosome organization along genes and a division of promoters into those that contain or lack a pronounced nucleosome-depleted region (NDR), with the latter displaying a more dynamic pattern of gene expression. Despite this global similarity, however, nucleosome occupancy at specific genes diverged extensively between sensu stricto and C. glabrata orthologs (similar to 50 million years). Orthologs with dynamic expression patterns tend to maintain their lack of NDR, but apart from that, sensu stricto and C. glabrata orthologs are nearly as similar in nucleosome occupancy patterns as nonorthologous genes. This extensive divergence in nucleosome occupancy contrasts with a conserved pattern of gene expression. Thus, while some evolutionary changes in nucleosome occupancy contribute to gene expression divergence, nucleosome occupancy often diverges extensively with apparently little impact on gene expression.
Closely related species show a high degree of differences in gene expression, but the functional significance of these differences remains unclear. Similarly, stress responses in yeast typically involve differential expression of numerous genes, and it is unclear how many of these are functionally significant. To address these issues, we compared the expression programs of four yeast species under different growth conditions, and found that the response of these species to stress has diverged extensively. On an individual gene basis, most transcriptional responses are not conserved in any pair of species, and there are very limited common responses among all four species. We present evidence that many evolutionary changes in stress responses are compensated either (i) by the response of related genes or (ii) by changes in the basal expression levels of the genes whose responses have diverged. Thus, stress-related genes are often induced upon stress in some species but maintain high levels even in the absence of stress at other species, indicating a transition between induced and constitutive activation. In addition, similar to 15% of the stress responses are specific to only one of the four species, with no evidence for compensating effects or stress-related annotations, and these may reflect fortuitous regulation that is unimportant for the stress response (i. e., biological noise). Frequent compensatory changes and biological noise may explain how diverged expression responses support similar physiological responses.
The number of sequenced species is increasing at a staggering rate, calling for new approaches for incorporating evolutionary information in the study of biological mechanisms. Evolutionary conservation is widely used for assigning a function to new proteins and for predicting functional coding or non-coding sequences. Here, we argue for a complementary approach that focuses on the divergence of regulatory programs. Regulatory mechanisms can be learned from patterns of evolutionary divergence in regulatory properties such as gene expression, transcription factor binding or nucleosome positioning. We review examples of this concept using yeast as a model system, and highlight a hybrid-based approach that is highly instrumental in this analysis. Molecular Systems Biology 7: 530; published online 13 September 2011; doi:10.1038/msb.2011.60
mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast.
Gene expression varies widely between closely related species and strains, yet the genetic basis of most differences is still unknown. Several studies suggested that chromatin regulators have a key role in generating expression diversity, predicting a reduction in the interspecies differences on deletion of genes that influence chromatin structure or modifications. To examine this, we compared the genome-wide expression profiles of two closely related yeast species following the individual deletions of eight chromatin regulators and one transcription factor. In all cases, regulator deletions increased, rather than decreased, the expression differences between the species, revealing hidden genetic variability that was masked in the wild-type backgrounds. This effect was not observed for individual deletions of 11 enzymes involved in central metabolic pathways. The buffered variations were associated with trans differences, as revealed by allele-specific profiling of the interspecific hybrids. Our results support the idea that regulatory proteins serve as capacitors that buffer gene expression against hidden genetic variability. Molecular Systems Biology 6: 435; published online 30 November 2010; doi:10.1038/msb.2010.84
Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.
Gene regulation differs greatly between related species, constituting a major source of phenotypic diversity. Recent studies characterized extensive differences in the gene expression programs of closely related species. In contrast, virtually nothing is known about the evolution of chromatin structure and how it influences the divergence of gene expression. Here, we compare the genome-wide nucleosome positioning of two closely related yeast species and, by profiling their interspecific hybrid, trace the genetic basis of the observed differences into mutations affecting the local DNA sequences (cis effects) or the upstream regulators (trans effects). The majority (similar to 70%) of inter-species differences is due to cis effects, leaving a significant contribution (30%) for trans factors. We show that cis effects are well explained by mutations in nucleosome-disfavoring AT-rich sequences, but are not associated with divergence of nucleosome-favoring sequences. Differences in nucleosome positioning propagate to multiple adjacent nucleosomes, supporting the statistical positioning hypothesis, and we provide evidence that nucleosome-free regions, but not the +1 nucleosome, serve as stable border elements. Surprisingly, although we find that differential nucleosome positioning among cell types is strongly correlated with differential expression, this does not seem to be the case for evolutionary changes: divergence of nucleosome positioning is excluded from regulatory elements and is not correlated with gene expression divergence, suggesting a primarily neutral mode of evolution. Our results provide evolutionary insights to the genetic determinants and regulatory function of nucleosome positioning. Molecular Systems Biology 6: 365; published online 11 May 2010; doi: 10.1038/msb.2010.20
Background: The positions of nucleosomes along eukaryotic DNA are defined by the local DNA sequence and are further tuned by the activity of chromatin remodelers. While the genome-wide effect of most remodelers has not been described, recent studies in Saccharomyces cerevisiae have shown that Isw2 prevents ectopic expression of anti-sense and suppressed transcripts at gene ends. Results: We examined the genome-wide function of the Isw2 homologue, Isw1, by mapping nucleosome positioning in S. cerevisiae and Saccharomyces paradoxus strains deleted of ISW1. We found that Isw1 functions primarily within coding regions of genes, consistent with its putative role in transcription elongation. Upon deletion of ISW1, mid-coding nucleosomes were shifted upstream (towards the 5' ends) in about half of the genes. Isw1-dependent shifts were correlated with trimethylation of H3K79 and were enriched at genes with internal cryptic initiation sites. Conclusions: Our results suggest a division of labor between Isw1 and Isw2, whereby Isw2 maintains repressive chromatin structure at gene ends while Isw1 has a similar function at mid-coding regions. The differential specificity of the two remodelers may be specified through interactions with particular histone marks.
Keywords: Biochemistry & Molecular Biology
During evolution, novel phenotypes emerge through changes in gene expression, but the genetic basis is poorly understood. We compared the allele-specific expression of two yeast species and their hybrid, which allowed us to distinguish changes in regulatory sequences of the gene itself (cis) from changes in upstream regulatory factors (trans). Expression divergence between species was generally due to changes in cis. Divergence in trans reflected a differential response to the environment and explained the tendency of certain genes to diverge rapidly. Hybrid-specific expression, deviating from the parental range, occurred through novel cis-trans interactions or, more often, through modified trans regulation associated with environmental sensing. These results provide insights on the regulatory changes in cis and trans during the divergence of species and upon hybridization.
Histone monoubiquitylation is implicated in critical regulatory processes. We explored the roles of histone H2B ubiquitylation in human cells by reducing the expression of hBRE1/RNF20, the major H2B-specific E3 ubiquitin ligase. While H2B ubiquitylation is broadly associated with transcribed genes, only a subset of genes was transcriptionally affected by RNF20 depletion and abrogation of H2B ubiquitylation. Gene expression dependent on RNF20 includes histones H2A and H2B and the p53 tumor suppressor. In contrast, RNF20 suppresses the expression of several proto-oncogenes, which reside preferentially in closed chromatin and are modestly transcribed despite bearing marks usually associated with high transcription rates. Remarkably, RNF20 depletion augmented the transcriptional effects of epidermal growth factor (EGF), increased cell migration, and elicited transformation and tumorigenesis. Furthermore, frequent RNF20 promoter hypermethylation was observed in tumors. RNF20 may thus be a putative tumor suppressor, acting through selective regulation of a distinct subset of genes.
Chromatin structure is central for the regulation of gene expression, but its genome-wide organization is only beginning to be understood. Here, we examine the connection between patterns of nucleosome occupancy and the capacity to modulate gene expression upon changing conditions, i.e., transcriptional plasticity. By analyzing genome-wide data of nucleosome positioning in yeast, we find that the presence of nucleosomes close to the transcription start site is associated with high transcriptional plasticity, while nucleosomes at more distant upstream positions are negatively correlated with transcriptional plasticity. Based on this, we identify two typical promoter structures associated with low or high plasticity, respectively. The first class is characterized by a relatively large nucleosome-free region close to the start site coupled with well-positioned nucleosomes further upstream, whereas the second class displays a more evenly distributed and dynamic nucleosome positioning, with high occupancy close to the start site. The two classes are further distinguished by multiple promoter features, including histone turnover, binding site locations, H2A.Z occupancy, expression noise, and expression diversity. Analysis of nucleosome positioning in human promoters reproduces the main observations. Our results suggest two distinct strategies for gene regulation by chromatin, which are selectively employed by different genes.
We show that, in yeast, the divergence rate of gene expression is not correlated with that of its associated coding sequence. Gene essentiality influences both modes of evolution, but other properties related to protein structure or promoter composition are only correlated with coding-sequence divergence or gene expression divergence, respectively. Based on these findings, we discuss the possibilities of neutral evolution of gene expression and of different modes of evolution in unicellular versus multicellular organisms.
Recent studies have characterized significant differences in the cis-regulatory sequences of related organisms, but the impact of these differences on gene expression remains largely unexplored. Here, we show that most previously identified differences in transcription factor (TF)-binding sequences of yeasts and mammals have no detectable effect on gene expression, suggesting that compensatory mechanisms allow promoters to rapidly evolve while maintaining a stabilized expression pattern. To examine the impact of changes in cis-regulatory elements in a more controlled setting, we compared the genes induced during mating of three yeast species. This response is governed by a single TF (STE12), and variations in its predicted binding sites can indeed account for about half of the observed expression differences. The remaining unexplained differences are correlated with the increased divergence of the sequences that flank the binding sites and an apparent modulation of chromatin structure. Our analysis emphasizes the flexibility of promoter structure, and highlights the interplay between specific binding sites and general chromatin structure in the control of gene expression.
Comparative analysis is a fundamental tool in biology. Conservation among species greatly assists the detection and characterization of functional elements, whereas inter-species differences are probably the best indicators of biological adaptation. Traditionally, comparative approaches were applied to the analysis of genomic sequences. With the growing availability of functional genomic data, comparative paradigms are now being extended also to the study of other functional attributes, most notably the gene expression. Here we review recent works applying comparative analysis to large-scale gene expression datasets and discuss the central principles and challenges of such approaches.
In Saccharomyces cerevisiae, transcription factor binding sites are found preferentially similar to 100-200 bp upstream of the start codon. Here, we show that this region is associated with rigid DNA in promoters lacking a TATA box, but not in TATA-containing promoters. The association of rigid DNA with transcription factor binding sites is conserved in TATA-less promoters from 11 yeast species, whereas the position of the rigid DNA varies substantially among species. Rigid DNA could influence nucleosome positioning and assist in the assembly of the transcriptional machinery at TATA-less promoters.
Background: DNA microarrays provide the ability to interrogate multiple genes in a single experiment and have revolutionized genomic research. However, the microarray technology suffers from various forms of biases and relatively low reproducibility. A particular source of false data has been described, in which non- random placement of gene probes on the microarray surface is associated with spurious correlations between genes. Results: In order to assess the prevalence of this effect and better understand its origins, we applied an autocorrelation analysis of the relationship between chromosomal position and expression level to a database of over 2000 individual yeast microarray experiments. We show that at least 60% of these experiments exhibit spurious chromosomal position- dependent gene correlations, which nonetheless appear in a stochastic manner within each experimental dataset. Using computer simulations, we show that large spatial biases caused in the microarray hybridization step and independently of printing procedures can exclusively account for the observed spurious correlations, in contrast to previous suggestions. Our data suggest that such biases may generate more than 15% false data per experiment. Importantly, spatial biases are expected to occur regardless of microarray design and over a wide range of microarray platforms, organisms and experimental procedures. Conclusions: Spatial biases comprise a major source of noise in microarray studies; revision of routine experimental practices and normalizations to account for these biases may significantly and comprehensively improve the quality of new as well as existing DNA microarray data.
Background: Gene duplication provides raw material for the generation of new functions, but most duplicates are rapidly lost due to the initial redundancy in gene function. How gene function diversifies following duplication is largely unclear. Previous studies analyzed the diversification of duplicates by characterizing their coding sequence divergence. However, functional divergence can also be attributed to changes in regulatory properties, such as protein localization or expression, which require only minor changes in gene sequence. Results: We developed a novel method to compare expression profiles from different organisms and applied it to analyze the expression divergence of yeast duplicated genes. The expression profiles of Saccharomyces cerevisiae duplicate pairs were compared with those of their pre-duplication orthologs in Candida albicans. Duplicate pairs were classified into two classes, corresponding to symmetric versus asymmetric rates of expression divergence. The latter class includes 43 duplicate pairs in which only one copy has a significant expression similarity to the C. albicans ortholog. These may present cases of regulatory neofunctionalization, as supported also by their dispensability and variability. Conclusion: Duplicated genes may diversify through regulatory neofunctionalization. Notably, the asymmetry of gene sequence evolution and the asymmetry of gene expression evolution are only weakly correlated, underscoring the importance of expression analysis to elucidate the evolution of novel functions.
Phenotypic diversity is generated through changes in gene structure or gene regulation. The availability of full genomic sequences allows for the analysis of gene sequence evolution. In contrast, little is known about the principles driving the evolution of gene expression. Here we describe the differential transcriptional response of four closely related yeast species to a variety of environmental stresses. Genes containing a TATA box in their promoters show an increased interspecies variability in expression, independent of their functional association. Examining additional data sets, we find that this enhanced expression divergence of TATA-containing genes is consistent across all eukaryotes studied to date, including nematodes, fruit flies, plants and mammals. TATA-dependent regulation may enhance the sensitivity of gene expression to genetic perturbations, thus facilitating expression divergence at particular genetic loci.
Background: High-throughput methods identify an overwhelming number of protein-protein interactions. However, the limited accuracy of these methods results in the false identification of many spurious interactions. Accordingly, the resulting interactions are regarded as hypothetical and computational methods are needed to increase their confidence. Several methods have recently been suggested for this purpose including co-expression as a confidence measure for interacting proteins, but their performance is still quite poor. Results: We introduce a novel computational method for verification of protein-protein interactions based on the co-expression of orthologs of interacting partners. The performance of our method is analysed using known S. cerevisiae interactions, and is shown to overcome limitations of previous methods. We present specific examples of known and putative interactions that are detected by our method and not by previous methods, and suggest that they represent transient interactions that might have been conserved and stabilized in other species. Conclusion: Co-expression of orthologous protein-pairs can be used to increase the confidence of hypothetical protein-protein interactions in S. cerevisiae as well as in other species. This approach may be especially useful for species with no available expression profiles and for transient interactions.