Highly parallel assays of tissue-specific enhancers in whole <i>Drosophila</i> embryos.

Highly parallel assays of tissue-specific enhancers in whole Drosophila embryos. Stephen S. Gisselbrecht¹, Luis Barrera^1,2, Martin Porsch^1,3, Preston W. Estep⁴, Anastasia Vedenko¹, Anton Aboukhalil^1,5, Alexandre Palagi^1,6, Yongsok Kim⁷, Xianmin Zhu⁷, Brian Busser⁷, Alan M. Michelson⁷, Martha L. Bulyk^1,2,8,9. 1) Division of Genetics, Brigham & Women's Hospital, Boston, MA 02115; 2) Committee on Higher Degrees in Biophysics, Harvard University, Cambridge, MA 02138; 3) Institute of Computer Science, Martin Luther University of Halle-Wittenberg, 06099 Halle, Germany; 4) TeloMe, Inc., Waltham, MA 02451; 5) Department of Aeronautics and Astronautics, Massachusetts Institute of Technology, Cambridge, MA 02139; 6) Bioengineering Department, Polytech Nice Sophia, University of Nice Sophia Antipolis, 06903, France; 7) Laboratory of Developmental Systems Biology, Genetics and Developmental Biology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892; 8) Dept. of Pathology, Brigham & Women's Hospital, Boston, MA 02115; 9) Harvard-MIT Division of Health Sciences and Technology (HST); Harvard Medical School, Boston, MA 02115.

Understanding transcriptional regulatory networks requires the identification and characterization of cis-regulatory modules (CRMs), DNA sequences which can direct expression of associated genes to a specific cell type and/or developmental stage. Reporter assays for the capacity of a candidate CRM to activate a heterologous promoter have been productive but suffer limited throughput or fail to convey information on cell type specificity. We have developed an assay in which reporter constructs containing a pool of candidate CRMs are introduced in parallel to Drosophila embryos along with a common cell-type-specific second marker; candidate CRMs isolated by PCR from FACS-purified double-positive cells can by quantitated by high-throughput sequencing, and their relative abundance compared to those in the input cell population to detect activity in the cell type (or types) of interest.