Computational and Biological Approaches for Identification of Hedgehog Signaling Targets and Their Application to Intestinal Visceral Smooth Muscle Development in the Mouse.

Abstract

The Hedgehog (Hh) pathway is an evolutionarily conserved cell-cell signaling pathway that controls organ development and homeostasis in embryos and adults. Hh signaling functions in cell fate choice, patterning, cell survival, proliferation and/or differentiation. Several birth defects are known to result from altered Hh signaling and aberrant Hh signaling is also responsible for several cancers. Despite its central role in development and disease, very little is known about the precise genetic targets of Hh signaling or the genomic enhancers that activate those genes. These target genes and associated Hh-responsive enhancers are themselves responsible for disease initiation and progression. A comprehensive effort to identify these signaling targets and to dissect the context specificity that underlies their expression is therefore a high priority. This work was driven by two Aims: 1) to explore novel computational approaches for the identification of Hh-responsive enhancers; and 2) to understand the contribution of Hh-driven gene expression in the context of a single Hh-responsive cell type, intestinal visceral smooth muscle (ISM). This work comprised a multi-pronged approach, integrating both computational and biological methods in parallel, to achieve these Aims. First, we explored the degree to which clustered binding sites for the Hh transcription factor, ci/GLI, would predict functional enhancers. While this method was somewhat successful in the fly, it could not be applied to the mouse, where Hh enhancers tend not to be homotypically clustered. Therefore, a machine learning strategy was explored with substantial success, resulting in the identification of seven new enhancers in genes encoding Hh pathway components. Finally, RNA-seq and ChIP-seq data were collected to generate a catalog of smooth muscle genes that are expressed in a specific layer of developing intestinal smooth muscle. Analysis of this data implicated cJUN as a regulatory component in ISM formation and established Hh as an upstream regulator of cJun expression in that tissue. Though this work has focused on Hh signaling, a similar approach could be applied to any transcription factor or signaling pathway to comprehensively analyze the gene regulatory networks governing many normal and disease-related cell states.