Individual differences in cardioblast specification: The role of cis-regulatory variation

• Applicant: Eileen E. M. Furlong, Ph.D.
• Subject Area: Developmental Biology
• Term: from 2008 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 75711951

Changes in DNA cis-regulatory modules (CRMs) can have profound consequences for organismal phenotypes, including human disease, and play an important role in the evolution of morphological differences between species. Changes between species often arise as initial variation within species. Recent work has revealed extensive variation in CRMs within natural populations. However, little is understood about the functional impact of this variation or the role that natural selection plays in shaping its evolution. This is due, in part, to the fact that interactions between transcription factors (TFs) and CRMs are often cooperative and non-linear; the consequences of mutations affecting individual TFs binding sites cannot be understood without understanding the ‘functional rules’ by which combinatorial inputs from multiple TFs are translated into gene expression at a CRM. With support from the DFG, we previously identified an extensive set of regulatory elements at which 5 TFs cooperatively act to drive gene expression during cardiac specification in Drosophila along with thousands of sites at which these factors bind individually. This represents a unique dataset in terms of both its scale and its focus on the collective properties of TF occupancy at developmental CRMs. The goal of the current study is to leverage off this extensive collection of cardiac enhancers to 1) quantify sequence variation in developmental regulatory elements and characterize its impact on TF occupancy and gene expression and, 2) look for evidence of non-linear interactions among CRM variants that may influence evolutionary processes. Drosophila melanogaster is an ideal system to address these questions. The genetic components regulating cardiac mesoderm specification is well-characterized, highly conserved and experimentally accessible, and there is a wealth of available population genetic resources including a fully sequenced set of 162 isogenic lines derived from a single, wild population. To address these goals, we will conduct ChIP-Seq experiments for three cardiac TFs in 10 isogenic lines of Drosophila derived from a single, natural population to look for population-level variation in TF occupancy. Because these lines (and 152 others from the same source) have been fully sequenced, we can then combine these data with eQTL data and computational scans for the actions of natural selection to form a unique picture of the extent of genetic variation influencing gene expression or TF occupancy during development along with estimates of how selection acts to shape this variation. We will then follow this up with targeted, in vivo experiments on individual CRMs to characterize the role that non-linear interactions between regulatory variants may influence gene expression or TF occupancy. Taken together, this project will provide the first in-depth global view of individual variation in a developmental network and how this relates to cardioblast gene expression.

• DFG Programme: Research Grants
• Participating Person: David Garfield, Ph.D.