The Biophysical and Molecular Mechanisms of Reliability in Development

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Project Summary Metazoan development relies on precise and reproducible cell fate decisions. However, the molecular events underlying these decisions, namely transcriptional activation of subsets of genes, are inherently stochastic. The goal of the proposed study is to bridge this gap by uncovering how precise patterns emerge from dynamic gene activation. Our past work was mostly focused on the readout of regulatory DNA sequences, such as enhancers, controlling the activation of a target gene. However, during the previous funding cycle, we identified the functional importance of the large chromosomal distances over which this regulation takes place, contributing to often rate- limiting dynamics and adding an extra source for stochasticity. Numerous studies in the past decade have pointed to the prevalence of such long-range chromosomal interactions adding another layer of complexity to the regulation of gene expression. However, investigation of temporal dynamics of chromatin is strongly limited by the prevalent use of bulk assays using fixed material, and traditional imaging methods often lack the spatiotemporal resolution to accurately capture the dynamics of gene activity. Here we propose to overcome these limitations by developing new imaging approaches and computational analyses to provide a dynamic picture of chromosomal architecture and its causal relationship to transcription. For this purpose, we will capitalize on the advantages of the early Drosophila embryo for the development of quantitative live imaging methods. In this system, changes in segmentation gene expression are position-specific determinants of cell- type identity. We will thus examine regulatory interactions at scales characteristic to flies and mammals (from tens to hundreds of kilobases) and their implications in the context of cellular specification in a developing organism. The proposed studies will help understand how robust cell type specification emerges from the stochastic gene expression that is regulated by long-range interactions and chromatin architecture. The following three complementary hypotheses are tested: 1) distinct locus-specific architectures generate different functional outputs; 2) physical order underlies long-distance chromosomal relationships; 3) a functional relationship exists between chromatin dynamics and activity. The overall goal of this project is to establish a quantitative link between chromatin architecture and transcriptional activity, which will ultimately lead us to regulate and re- engineer transcriptional programs underlying development and disease processes.