Periodic Reporting for period 3 - TF-Dynamics (Illuminating the role of transcription factor dynamics in development)
Période du rapport: 2024-01-01 au 2025-06-30
Many believe that civilization is about to enter an exciting new age as a result of our newfound ability to control DNA. For the first time in history, we can easily sequence and edit complex genomes, yielding the potential to revolutionize the way that we treat and cure human diseases. While tantalizing, this possibility remains largely unrealized because we lack a predictive understanding of how genomic sequence encodes biological form and function in multicellular organisms. This gap is clearly illustrated by a trait like human height: association studies have identified the vast majority of the sequence variants that control it, and yet we have no idea what most of them do at the molecular level. The broad goal of this proposal is to achieve a predictive understanding of how genomic sequence encodes biological form and function, in order for mankind to begin to realize the full transformative potential of the age of genomics. To do this we need to first understand how enhancer sequence regulates transcription in model organisms using new tools and approaches that yield access to dynamic information. Achieving this goal necessitates doing two things. 1) We need to understand the mechanistic details of how transcription factors (TFs) regulate transcription, in real time, in vivo. 2) We need to understand how enhancer sequence modulates how TFs behave in order to regulate gene expression.
To understand how TF concentration dynamics regulate gene expression, we used recently developed single-cell live imaging tools to dissect the regulation of Fushi tarazu (Ftz) in Drosophila melanogaster embryos. Ftz is a transcription factor that is expressed in asymmetric stripes by two distinct enhancers: autoregulatory and zebra. The anterior edge of each stripe needs to be sharply defined to specify essential line- age boundaries. Here, we tracked how boundary cells commit to either a high-Ftz or low-Ftz fate by measuring Ftz protein traces in real time and simultaneously quantifying transcription from the endogenous locus and individual enhancers. This revealed that the autoregulatory enhancer does not establish this fate choice. Instead, it perpetuates the decision defined by zebra. This is contrary to the prevailing view that autoregulation drives the fate decision by causing bi-stable Ftz expression. Furthermore, we showed that the autoregulatory enhancer is not activated based on a Ftz-concentration threshold but through a timing-based mechanism. We hypothesize that this is regulated by several ubiquitously expressed pioneer-like transcription factors, which have recently been shown to act as timers in the embryo. Our work provides new insight into how precisely timed enhancer activity can directly regulate the dynamics of gene regulatory networks, which may be a general mechanism for ensuring that embryogenesis runs like clockwork.
This ambitious work spans multiple length scales, from examining changes in TF concentration at the single-nucleus level, to measuring the formation of local clusters of TFs at the locus level, to watching individual protein-protein interactions as they drive transcription at the molecular level. First, we will understand how TF concentration dynamics regulate gene expression. Second, we will uncover how recently discovered dynamic TF clusters or condensates regulate transcription and how their properties are shaped by enhancer sequence. Finally, we will overcome one of the most daunting technical challenges facing the field of transcription by developing a method to directly visualize the transient protein-protein interactions that drive transcription in vivo. Simultaneously visualizing protein-protein interactions and tracking transcription will make it possible to address the most pressing questions about transcription mechanisms in vivo, by revealing precisely when they occur and how they modulate transcription.