Direct Quantification of Transcriptional Regulation at an Endogenous Gene Locus. Heng Xu¹, Anna Sokac¹, Ido Golding^1,2. 1) Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX; 2) Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL.

   Gene regulation by sequence-specific transcription factors underlies the diversity of cell fates and behavior observed during development and homeostasis. Our quantitative understanding of this process is limited by the fact that most experimental methods involve averaging over many individual cells, while single-cell studies typically require the use of reporter fusions which perturb the function of the system under study. Here we present a direct quantification of gene regulation by a transcription factor at the level of a single, genetically unmodified gene locus, at single-molecule resolution. By combining single-molecule fluorescence in situ hybridization (smFISH) with quantitative immunofluorescence, and introducing novel image analysis algorithms, we are able to simultaneously measure the numbers of endogenous Bicoid (Bcd) transcription factors and the nascent mRNAs from the hunchback (hb) gene they regulate, in individual nuclei of the early Drosophila embryo. We measure the binding of Bcd at the gene locus and correlate this binding with the transcriptional activity of the hb gene. Our measurements allow us to derive a detailed stochastic model of hb regulation by Bcd and extract the in vivo biochemical parameters governing the process. The experimental and numerical procedures described here can be directly applied to the study of transcriptional regulation in other genes gene networks and organisms.