Project Summary Abstract How remote transcriptional enhancers communicate with their target promoters to regulate gene activity persists as one of the central mysteries in cell and developmental biology. Classical models envisioned the formation of chromosomal loops that result in direct physical association of enhancers and promoters. However, there is emerging evidence that large distances (~200 nm) separate enhancers and promoters during transcription activation. Moreover, shared enhancers can co-activate linked genes, both in cis and in trans across homologous chromosomes. These observations are not obviously compatible with classical models and have prompted an alternate view, “transcription hubs”, whereby enhancers and promoters recruit large clusters or condensates of transcriptional activators. A major goal of the proposed study is to determine the role of insulator DNAs and tethering elements in the formation and function of transcription hubs. Insulators function as boundary elements when interposed between a distal enhancer and target promoter. They also foster the formation of topological associating domains (TADs) and paring of alleles located on different homologous chromosomes. In Drosophila, tethering elements are GAGA-rich sequences that facilitate long-range enhancer-promoter interactions. We will employ a combination of genome editing, Micro-C XL assays, and quantitative live imaging methods in living Drosophila embryos to visualize even subtle changes in the timing and levels of gene expression arising from mutations, inversions, and deletions of defined insulators and tethering elements. Particular efforts will focus on long-range enhancer-promoter communication within complex genetic loci. For example, we will examine the role of tethering elements in the activation of Scr transcription by a remote enhancer that must bypass an intervening TAD within the Antennapedia Hox gene complex. We will also explore the contributions of individual insulators and tethering elements for the co-regulation of linked genes by shared enhancers over distances of 75 kb and 235 kb in the knirps and Scylla loci, respectively. Finally, we will examine the possibility that low complexity sequences in tethering factors (e.g., Trithorax-like) contribute to the stability of enhancer-promoter loops by nucleating the formation of activation condensates. These studies have the potential to unravel current controversies regarding the importance of insulators, TADs, and tethering elements in gene regulation during development. They might also reveal new connections between chromosomal loops and the formation of transcriptional hubs.