Genomic Approaches to Elucidating Developmental Regulatory Networks

My laboratory investigates the genetic regulatory circuitry responsible for assigning cell fates during development, using the Drosophila embryonic mesoderm as our primary model system. Our work combines genomics and bioinformatics with the traditional molecular and genetic techniques of Drosophila research to investigate two key components of developmental regulatory networks, intercellular signaling and transcriptional regulation. This powerful combination of in silico and in vivo approaches enables us not only to make predictions but also to validate them within specific biological contexts. Our approaches have broad applicability to the study of genomes other than that of Drosophila, including the human genome. Current research in the laboratory falls into two main areas: (a) discovery and characterization of transcriptional cis-regulatory modules (CRMs), and (b) mechanisms of specificity for receptor tyrosine kinase (RTK) signaling. The combined results of our studies will provide insight into gene regulation, genome structure, intercellular signaling, and the regulatory networks that govern embryonic development.

Regulatory Networks

Gene expression is controlled by the binding of transcription factors to specific cis-regulatory elements. In the higher eukaryotes, these elements can lie 5' to, 3' to, or within introns of a gene; in some cases, they can even be found within protein coding sequences! Spatial and temporal aspects of gene expression are often controlled in a modular fashion, with individual cis-regulatory elements (termed "modules" or "enhancers") regulating expression in a particular time and place. An emerging theme is that a specific combination of transcription factors activiated as a result of intercellular signaling binds a regulatory module in conjunction with tissue-specific transcription factors ("selectors"), forming a "transcriptional code" that regulates the expression of a given gene (see Figure 1).

Together, the signaling and transcriptional events form a network of interactions in which signaling induces gene transcription, which can in turn lead to further signaling events, which then induce additional gene expression, and so on. Cascades of transcription can also occur, whereby transcription factors induce the expression of other transcription factors, which can in turn regulate still other transcription factors. These developmental regulatory networks are often complex, with multiple levels of cross-talk between different signaling pathways and both positive and negative feedback loops (see Figure 1, right). Our ultimate goal is to be able to describe all of the regulatory interactions involved in embryonic development. As a tractable step in this direction, we have begun to identify and characterize specific cis-regulatory elements and signaling pathways involved in mesoderm development.

Defining cis-regulatory elements

cis-Regulatory modules (CRMs) are critical nodes in developmental regulatory networks, as it is here that signaling pathways and transcription factors are integrated to give rise to changes in the expression of specific genes. Mutations within CRMs have been implicated in a number of diseases, underscoring the importance of being able to identify and characterize them. However, CRM identification has traditionally been difficult, relying on a trial-and-error approach using the non-coding DNA flanking the gene of interest. We are using a number of computational approaches to attempt to locate the cis-regulatory modules responsible for directing specific patterns of gene expression in a rapid and comprehensive fashion. All of our predictions are extensively tested in vivo using reporter gene assays in the fly embryo so that we can definitively assess our success rate and refine our approach to achieve better performance. Our most recent efforts, performed in collaboration with Dr. Saurabh Sinha at the University of Illinois, are proving to be highly effective.

Although our primary focus has been on CRM discovery, much can be learned from studying already-known CRMs using bioinformatics approaches. However, these studies are significantly hindered by the absence of readily available data on large numbers of CRMs. To address this shortcoming, we have constructed the REDfly database of published Drosophila CRMs. This database contains more than 600 CRMs associated with over 200 genes, along with their sequences and the expression patterns for which they are responsible. Computational analysis of this collection will allow us to discover previously unrecognized transcription factor binding sites as well as to begin to explore the "grammar" of CRMs--how differences in the order and spacing of individual binding sites affect the overall functioning of the module. We are investigating issues such as to what extent clustering of binding sites is important for enhancer activity, how modular versus nested or interrelated regulatory elements tend to be, and how subtle differences in structure and sequence affect enhancer activity.

CRMs work in concert with a gene's promoter, and we are interested in understanding how specific CRM-promoter interactions come about. The data we have collected in the REDfly database, along with promoter characterizations from a recent genome wide study we performed, are allowing us to undertake both wet-lab and computational explorations of this question.

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Downstream responses to intercellular signaling

In order to characterize developmental regulatory networks, we must also understand how upstream intercellular signaling events establish the transcriptional codes that act at the cis-regulatory elements. We have been particularly interested in receptor tyrosine kinase (RTK) signaling pathways, including the receptors Heartless (a fibroblast growth factor (FGF) receptor homolog) and Egfr (an epidermal growth factor (EGF) receptor homolog), which play important roles in establishing mesodermal cell fates. Although the FGF and EGF receptors are often believed to be acting via identical downstream signaling cascades, our data indicate that there are significant points of divergence within these pathways. We are investigating the mechanisms behind this divergent signaling with support from the American Cancer Society. These studies will contribute to our knowledge of RTK pathway regulation as well as identify additional genes important for mesodermal development.

Selected Recent Publications