Identification of functional transcription factor binding sites using closely related Saccharomyces species

Comparative genomics provides a rapid means of identifying functional DNA elements by their sequence conservation between species. Transcription factor binding sites (TFBSs) may constitute a significant fraction of these conserved sequences, but the annotation of specific TFBSs is complicated by the fact that these short, degenerate sequences may frequently be conserved by chance rather than functional constraint. To identify intergenic sequences that function as TFBSs, we calculated the probability of binding site conservation between Saccharomyces cerevisiae and its two closest relatives under a neutral model of evolution. We found that this probability is <5% for 134 of 163 transcription factor binding motifs, implying that we can reliably annotate binding sites for the majority of these transcription factors by conservation alone. Although our annotation relies on a number of assumptions, mutations in five of five conserved Ume6 binding sites and three of four conserved Ndt80 binding sites show Ume6- and Ndt80-dependent effects on gene expression. We also found that three of five unconserved Ndt80 binding sites show Ndt80-dependent effects on gene expression. Together these data imply that although sequence conservation can be reliably used to predict functional TFBSs, unconserved sequences might also make a significant contribution to a species' biology

Frequent Gain and Loss of Functional Transcription Factor Binding Sites

Cis-regulatory sequences are not always conserved across species. Divergence within cis-regulatory sequences may result from the evolution of species-specific patterns of gene expression or the flexible nature of the cis-regulatory code. The identification of functional divergence in cis-regulatory sequences is therefore important for both understanding the role of gene regulation in evolution and annotating regulatory elements. We have developed an evolutionary model to detect the loss of constraint on individual transcription factor binding sites (TFBSs). We find that a significant fraction of functionally constrained binding sites have been lost in a lineage-specific manner among three closely related yeast species. Binding site loss has previously been explained by turnover, where the concurrent gain and loss of a binding site maintains gene regulation. We estimate that nearly half of all loss events cannot be explained by binding site turnover. Recreating the mutations that led to binding site loss confirms that these sequence changes affect gene expression in some cases. We also estimate that there is a high rate of binding site gain, as more than half of experimentally identified S. cerevisiae binding sites are not conserved across species. The frequent gain and loss of TFBSs implies that cis-regulatory sequences are labile and, in the absence of turnover, may contribute to species-specific patterns of gene expression

MAPPFinder: using Gene Ontology and GenMAPP to create a global gene-expression profile from microarray data

MAPPFinder is a tool that creates a global gene-expression profile across all areas of biology by integrating the annotations of the Gene Ontology (GO) Project with the free software package GenMAPP . The results are displayed in a searchable browser, allowing the user to rapidly identify GO terms with over-represented numbers of gene-expression changes. Clicking on GO terms generates GenMAPP graphical files where gene relationships can be explored, annotated, and files can be freely exchanged

Identifying genetic networks underlying myometrial transition to labor

BACKGROUND: Early transition to labor remains a major cause of infant mortality, yet the causes are largely unknown. Although several marker genes have been identified, little is known about the underlying global gene expression patterns and pathways that orchestrate these striking changes. RESULTS: We performed a detailed time-course study of over 9,000 genes in mouse myometrium at defined physiological states: non-pregnant, mid-gestation, late gestation, and postpartum. This dataset allowed us to identify distinct patterns of gene expression that correspond to phases of myometrial 'quiescence', 'term activation', and 'postpartum involution'. Using recently developed functional mapping tools (HOPACH (hierarchical ordered partitioning and collapsing hybrid) and GenMAPP 2.0), we have identified new potential transcriptional regulatory gene networks mediating the transition from quiescence to term activation. CONCLUSIONS: These results implicate the myometrium as an essential regulator of endocrine hormone (cortisol and progesterone synthesis) and signaling pathways (cyclic AMP and cyclic GMP stimulation) that direct quiescence via the transcripitional upregulation of both novel and previously associated regulators. With term activation, we observe the upregulation of cytoskeletal remodeling mediators (intermediate filaments), cell junctions, transcriptional regulators, and the coordinate downregulation of negative control checkpoints of smooth muscle contractile signaling. This analysis provides new evidence of multiple parallel mechanisms of uterine contractile regulation and presents new putative targets for regulating myometrial transformation and contraction

GenMAPP 2: New features and resources for pathway analysis

BACKGROUND: Microarray technologies have evolved rapidly, enabling biologists to quantify genome-wide levels of gene expression, alternative splicing, and sequence variations for a variety of species. Analyzing and displaying these data present a significant challenge. Pathway-based approaches for analyzing microarray data have proven useful for presenting data and for generating testable hypotheses. RESULTS: To address the growing needs of the microarray community we have released version 2 of Gene Map Annotator and Pathway Profiler (GenMAPP), a new GenMAPP database schema, and integrated resources for pathway analysis. We have redesigned the GenMAPP database to support multiple gene annotations and species as well as custom species database creation for a potentially unlimited number of species. We have expanded our pathway resources by utilizing homology information to translate pathway content between species and extending existing pathways with data derived from conserved protein interactions and coexpression. We have implemented a new mode of data visualization to support analysis of complex data, including time-course, single nucleotide polymorphism (SNP), and splicing. GenMAPP version 2 also offers innovative ways to display and share data by incorporating HTML export of analyses for entire sets of pathways as organized web pages. CONCLUSION: GenMAPP version 2 provides a means to rapidly interrogate complex experimental data for pathway-level changes in a diverse range of organisms

Event-Related Potential Effects of Object Recognition depend on Attention and Part-Whole Configuration

The effects of spatial attention and part-whole configuration on recognition of repeated objects were investigated with behavioral and event-related potential (ERP) measures. Short-term repetition effects were measured for probe objects as a function of whether a preceding prime object was shown as an intact image or coarsely scrambled (split into two halves) and whether or not it had been attended during the prime display. In line with previous behavioral experiments, priming effects were observed from both intact and split primes for attended objects, but only from intact (repeated sameview) objects when they were unattended. These behavioral results were reflected in ERP waveforms at occipital–temporal locations as more negative-going deflections for repeated items in the time window between 220 and 300 ms after probe onset (N250r).Attended intact images showed generally more enhanced repetition effects than split ones. Unattended images showed repetition effects only when presented in an intact configuration, and this finding was limited to the right-hemisphere electrodes. Repetition effects in earlier (before 200 ms) time windows were limited to attended conditions at occipito-temporal sites during the N1, a component linked to the encoding of object structure, while repetition effects at central locations during the same time window (P150) were found for attended and unattended probes but only when repeated in the same intact configuration. The data indicate that view-generalization is mediated by a combination of analytic (part-based) representations and automatic view-dependent representations

Modeling Insertional Mutagenesis Using Gene Length and Expression in Murine Embryonic Stem Cells

Background. High-throughput mutagenesis of the mammalian genome is a powerful means to facilitate analysis of gene function. Gene trapping in embryonic stem cells (ESCs) is the most widely used form of insertional mutagenesis in mammals. However, the rules governing its efficiency are not fully understood, and the effects of vector design on the likelihood of genetrapping events have not been tested on a genome-wide scale. Methodology/Principal Findings. In this study, we used public gene-trap data to model gene-trap likelihood. Using the association of gene length and gene expression with gene-trap likelihood, we constructed spline-based regression models that characterize which genes are susceptible and which genes are resistant to gene-trapping techniques. We report results for three classes of gene-trap vectors, showing that both length and expression are significant determinants of trap likelihood for all vectors. Using our models, we also quantitatively identifie

A Catalog of Neutral and Deleterious Polymorphism in Yeast

The abundance and identity of functional variation segregating in natural populations is paramount to dissecting the molecular basis of quantitative traits as well as human genetic diseases. Genome sequencing of multiple organisms of the same species provides an efficient means of cataloging rearrangements, insertion, or deletion polymorphisms (InDels) and single-nucleotide polymorphisms (SNPs). While inbreeding depression and heterosis imply that a substantial amount of polymorphism is deleterious, distinguishing deleterious from neutral polymorphism remains a significant challenge. To identify deleterious and neutral DNA sequence variation within Saccharomyces cerevisiae, we sequenced the genome of a vineyard and oak tree strain and compared them to a reference genome. Among these three strains, 6% of the genome is variable, mostly attributable to variation in genome content that results from large InDels. Out of the 88,000 polymorphisms identified, 93% are SNPs and a small but significant fraction can be attributed to recent interspecific introgression and ectopic gene conversion. In comparison to the reference genome, there is substantial evidence for functional variation in gene content and structure that results from large InDels, frame-shifts, and polymorphic start and stop codons. Comparison of polymorphism to divergence reveals scant evidence for positive selection but an abundance of evidence for deleterious SNPs. We estimate that 12% of coding and 7% of noncoding SNPs are deleterious. Based on divergence among 11 yeast species, we identified 1,666 nonsynonymous SNPs that disrupt conserved amino acids and 1,863 noncoding SNPs that disrupt conserved noncoding motifs. The deleterious coding SNPs include those known to affect quantitative traits, and a subset of the deleterious noncoding SNPs occurs in the promoters of genes that show allele-specific expression, implying that some cis-regulatory SNPs are deleterious. Our results show that the genome sequences of both closely and distantly related species provide a means of identifying deleterious polymorphisms that disrupt functionally conserved coding and noncoding sequences