A Predictive Model of the Oxygen and Heme Regulatory Network in Yeast

Deciphering gene regulatory mechanisms through the analysis of high-throughput expression data is a challenging computational problem. Previous computational studies have used large expression datasets in order to resolve fine patterns of coexpression, producing clusters or modules of potentially coregulated genes. These methods typically examine promoter sequence information, such as DNA motifs or transcription factor occupancy data, in a separate step after clustering. We needed an alternative and more integrative approach to study the oxygen regulatory network in Saccharomyces cerevisiae using a small dataset of perturbation experiments. Mechanisms of oxygen sensing and regulation underlie many physiological and pathological processes, and only a handful of oxygen regulators have been identified in previous studies. We used a new machine learning algorithm called MEDUSA to uncover detailed information about the oxygen regulatory network using genome-wide expression changes in response to perturbations in the levels of oxygen, heme, Hap1, and Co2+. MEDUSA integrates mRNA expression, promoter sequence, and ChIP-chip occupancy data to learn a model that accurately predicts the differential expression of target genes in held-out data. We used a novel margin-based score to extract significant condition-specific regulators and assemble a global map of the oxygen sensing and regulatory network. This network includes both known oxygen and heme regulators, such as Hap1, Mga2, Hap4, and Upc2, as well as many new candidate regulators. MEDUSA also identified many DNA motifs that are consistent with previous experimentally identified transcription factor binding sites. Because MEDUSA's regulatory program associates regulators to target genes through their promoter sequences, we directly tested the predicted regulators for OLE1, a gene specifically induced under hypoxia, by experimental analysis of the activity of its promoter. In each case, deletion of the candidate regulator resulted in the predicted effect on promoter activity, confirming that several novel regulators identified by MEDUSA are indeed involved in oxygen regulation. MEDUSA can reveal important information from a small dataset and generate testable hypotheses for further experimental analysis. Supplemental data are included.Author Summary: The cell uses complex regulatory networks to modulate the expression of genes in response to changes in cellular and environmental conditions. The transcript level of a gene is directly affected by the binding of transcriptional regulators to DNA motifs in its promoter sequence. Therefore, both expression levels of transcription factors and other regulatory proteins as well as sequence information in the promoters contribute to transcriptional gene regulation. In this study, we describe a new computational strategy for learning gene regulatory programs from gene expression data based on the MEDUSA algorithm. We learn a model that predicts differential expression of target genes from the expression levels of regulators, the presence of DNA motifs in promoter sequences, and binding data for transcription factors. Unlike many previous approaches, we do not assume that genes are regulated in clusters, and we learn DNA motifs de novo from promoter sequences as an integrated part of our algorithm. We use MEDUSA to produce a global map of the yeast oxygen and heme regulatory network. To demonstrate that MEDUSA can reveal detailed information about regulatory mechanisms, we perform biochemical experiments to confirm the predicted regulators for an important hypoxia gene.