Direct Quantification of Gene Regulation by Transcription-Factor Binding at an Endogenous Gene Locus

Gene activity is controlled by the binding of transcription factors (TFs) to the regulatory sequence of the gene, yet a direct in situ mapping from TF binding to mRNA production of a single gene remains elusive, due to the difficulty of capturing individual TF binding events of a specific gene from the ocean of TF signal in cell nucleus. Here we combine single-molecule fluorescent imaging of protein, mRNA, and gene loci to detect and quantify distinct binding configurations of multiple TFs and epigenetically modified histones, as well as the resulting nascent mRNAs at endogenous hunchback (hb) gene loci in early Drosophila embryos. Using stochastic theoretical analysis, we show that TF binding follows nonequilibrium multi-state kinetics, breaking the law of mass action. hb transcription activation is a multi-step process initiated by transient binding of the Bicoid transcription factor. Unlike TF binding at hb, the histone signal is deprived at active gene loci, indicating that nucleosome unwinding is necessary for gene activation. Our method provides a general framework to decipher the dynamics of complex gene regulatory networks in situ.