Abstract
High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain–containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades.
Highlights
High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification
To identify the genes directly regulated by Gcm, we mapped its binding sites using a genome-wide DNA adenine methyltransferase identification (DamID) screen
Because Gcm is expressed in several cell types: glia, hemocytes, and tendon cells (Soustelle et al 2004), as well as neuronal (Chotard et al 2005; Soustelle and Giangrande 2007) and peritracheal cell subsets (Laneve et al 2013), we decided to search for all its direct targets and did not restrict expression of the DNA adenine methyltransferase (Dam)-Gcm fusion to a specific cell type
Summary
High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. We found a significantly high number of effector genes, including numerous members of the Ig domain–containing protein family These are molecules that affect cell function or late developmental events, including cell migration, a key feature of glia and hemocytes (Schmucker et al 2000; Watson et al 2005; Kumar et al 2015) [reviewed in Schwabe et al 2009)]. This suggests that early genes such as gcm may have a much broader impact than expected in cell specification/physiology. To the best of our knowledge, this is the first evidence of functional conservation and sets the basis to further understand the Gcm network in mammals
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