Discovering regulatory overlapping RNA transcripts.

STEREO is a novel algorithm that discovers cis-regulatory RNA interactions by assembling complete and potentially overlapping same-strand RNA transcripts from tiling expression data. STEREO first identifies coherent segments of transcription and then discovers individual transcripts that are consistent with the observed segments given intensity and shape constraints. We used STEREO to identify 1446 regions of overlapping transcription in two strains of yeast, including transcripts that comprise a new form of molecular toggle switch that controls gene variegation.

Genotype to phenotype: a complex problem.

We generated a high-resolution whole-genome sequence and individually deleted 5100 genes in Sigma1278b, a Saccharomyces cerevisiae strain closely related to reference strain S288c. Similar to the variation between human individuals, Sigma1278b and S288c average 3.2 single-nucleotide polymorphisms per kilobase. A genome-wide comparison of deletion mutant phenotypes identified a subset of genes that were conditionally essential by strain, including 44 essential genes unique to Sigma1278b and 13 unique to S288c. Genetic analysis indicates the conditional phenotype was most often governed by complex genetic interactions, depending on multiple background-specific modifiers. Our comprehensive analysis suggests that the presence of a complex set of modifiers will often underlie the phenotypic differences between individuals.

Analysis of the mouse embryonic stem cell regulatory networks obtained by ChIP-chip and ChIP-PET.

BACKGROUND: Genome-wide approaches have begun to reveal the transcriptional networks responsible for pluripotency in embryonic stem (ES) cells. Chromatin Immunoprecipitation (ChIP) followed either by hybridization to a microarray platform (ChIP-chip) or by DNA sequencing (ChIP-PET), has identified binding targets of the ES cell transcription factors OCT4 and NANOG in humans and mice, respectively. These studies have provided an outline of the transcriptional framework involved in maintaining pluripotency. Recent evidence with comparing multiple technologies suggests that expanding these datasets using different platforms would be a useful resource for examining the mechanisms underlying pluripotency regulation. RESULTS: We have now identified OCT4 and NANOG genomic targets in mouse ES cells by ChIP-chip and provided the means to compare these data with previously reported ChIP-PET results in mouse ES cells. We have mapped the sequences of OCT4 and NANOG binding events from each dataset to genomic coordinates, providing a valuable resource to facilitate a better understanding of the ES cell regulatory circuitry. Interestingly, although considerable differences are observed in OCT4 and NANOG occupancy as identified by each method, a substantial number of targets in both datasets are enriched for genes that have known roles in cell-fate specification and that are differentially expressed upon Oct4 or Nanog knockdown. CONCLUSION: This study suggests that each dataset is a partial representation of the overall ES cell regulatory circuitry, and through integrating binding data obtained by ChIP-chip and ChIP-PET, the methods presented here provide a useful means for integrating datasets obtained by different techniques in the future.

GSE: a comprehensive database system for the representation, retrieval, and analysis of microarray data.

We present GSE, the Genomic Spatial Event database, a system to store, retrieve, and analyze all types of high-throughput microarray data. GSE handles expression datasets, ChIP-chip data, genomic annotations, functional annotations, the results of our previously published Joint Binding Deconvolution algorithm for ChIP-chip, and precomputed scans for binding events. GSE can manage data associated with multiple species; it can also simultaneously handle data associated with multiple 'builds' of the genome from a single species. The GSE system is built upon a middle software layer for representing streams of biological data; we outline this layer, called GSEBricks, and show how it is used to build an interactive visualization application for ChIP-chip data. The visualizer software is written in Java and communicates with the GSE database system over the network. We also present a system to formulate and record binding hypotheses--simple descriptions of the relationships that may hold between different ChIP-chip experiments. We provide a reference software implementation for the GSE system.

Tissue-specific transcriptional regulation has diverged significantly between human and mouse.

We demonstrate that the binding sites for highly conserved transcription factors vary extensively between human and mouse. We mapped the binding of four tissue-specific transcription factors (FOXA2, HNF1A, HNF4A and HNF6) to 4,000 orthologous gene pairs in hepatocytes purified from human and mouse livers. Despite the conserved function of these factors, from 41% to 89% of their binding events seem to be species specific. When the same protein binds the promoters of orthologous genes, approximately two-thirds of the binding sites do not align.

High-resolution computational models of genome binding events.

Direct physical information that describes where transcription factors, nucleosomes, modified histones, RNA polymerase II and other key proteins interact with the genome provides an invaluable mechanistic foundation for understanding complex programs of gene regulation. We present a method, joint binding deconvolution (JBD), which uses additional easily obtainable experimental data about chromatin immunoprecipitation (ChIP) to improve the spatial resolution of the transcription factor binding locations inferred from ChIP followed by DNA microarray hybridization (ChIP-Chip) data. Based on this probabilistic model of binding data, we further pursue improved spatial resolution by using sequence information. We produce positional priors that link ChIP-Chip data to sequence data by guiding motif discovery to inferred protein-DNA binding sites. We present results on the yeast transcription factors Gcn4 and Mig2 to demonstrate JBD's spatial resolution capabilities and show that positional priors allow computational discovery of the Mig2 motif when a standard approach fails.

Zebrafish promoter microarrays identify actively transcribed embryonic genes.

We have designed a zebrafish genomic microarray to identify DNA-protein interactions in the proximal promoter regions of over 11,000 zebrafish genes. Using these microarrays, together with chromatin immunoprecipitation with an antibody directed against tri-methylated lysine 4 of Histone H3, we demonstrate the feasibility of this method in zebrafish. This approach will allow investigators to determine the genomic binding locations of DNA interacting proteins during development and expedite the assembly of the genetic networks that regulate embryogenesis.

Core transcriptional regulatory circuitry in human hepatocytes.

We mapped the transcriptional regulatory circuitry for six master regulators in human hepatocytes using chromatin immunoprecipitation and high-resolution promoter microarrays. The results show that these regulators form a highly interconnected core circuitry, and reveal the local regulatory network motifs created by regulator-gene interactions. Autoregulation was a prominent theme among these regulators. We found that hepatocyte master regulators tend to bind promoter regions combinatorially and that the number of transcription factors bound to a promoter corresponds with observed gene expression. Our studies reveal portions of the core circuitry of human hepatocytes.

Transcriptional regulatory code of a eukaryotic genome.

DNA-binding transcriptional regulators interpret the genome's regulatory code by binding to specific sequences to induce or repress gene expression. Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites. We have constructed an initial map of yeast's transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species. The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed. We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeast's transcriptional regulators.