(138g) Modulation of Transcriptional Kinetics Via Protein-DNA Interactions

Traditional studies of gene regulation in the Drosophila embryo centered primarily on the analysis of fixed tissues. These methods provided considerable insight into the spatial control of gene activity, such as the borders of eve stripe 2, but yielded only limited information about temporal dynamics. Here, we use a combination of live imaging and quantitative analysis in early Drosophila embryos to study the possibility that modulations in enhancer strength via changes in transcription factor (TF) binding affinity can linearly affect the transcriptional kinetics of the target genes, thereby modulating precise gradients of gene expression. We used the Rho GTPase pathway component t48 as our testbed for this hypothesis and showed that both the spatial boundary of gene expression and the total amount of mRNA production can be modulated via changes in timing of transcriptional activation. Strikingly, the timing of transcriptional activation can be significantly expedited or delayed by modulating the binding affinity of a single TF (Dorsal) binding site within the enhancer. Interestingly, once transcription is initiated, each nucleus seems to recruit Pol II at a similar rate regardless of its position along the dorsoventral axis. Furthermore, we show that optimizing the binding affinity of a pioneer factor Zelda binding site can further increase mRNA production, presumably by allowing more activators like Dorsal to bind to the target binding site and recruiting more Pol II to the promoter. We present a detailed analysis where a mechanism of how multiple TF binding sites within an enhancer regulate transcriptional dynamics is examined. Since t48 expression is linearly correlated with downstream myosin distribution, such fine-tuning of gene control via TF binding site arrangement within the t48 enhancer may play an important role in ensuring normal development during gastrulation.