CBE Doctoral Dissertation Defense: “Configuration Matters: Unveiling Cis-Regulatory Arrangement as a Mechanism of Transcriptional Fine-Tuning” (Emilia Leyes Porello)

The precise regulation of gene expression depends on coordinated enhancer-promoter (E-P) interactions, yet the principles governing how enhancer configuration influences transcription remain unclear. Classical models describe enhancers as modular and independent of orientation or relative positioning, but accumulating evidence suggests these assumptions may not hold true. To investigate this problem, systematic manipulations of enhancer configuration were performed in early Drosophila melanogaster embryos using reporter constructs designed to vary E-P distance, enhancer placement upstream or downstream of the promoter, and orientation (sense versus antisense). Site-specific insertion of these transgenic reporters coupled with quantitative single-nucleus live imaging of transcription via the MS2/MCP system enabled direct measurement of transcriptional activity and bursting dynamics in real time. These experiments revealed two distinct regulatory effects: linear E-P distance primarily determined transcriptional onset time, consistent with a diffusion-controlled search process, while relative enhancer positioning dictated the stability of the transcriptionally active state. Downstream enhancers exhibited reduced amplitude and stability compared to upstream counterparts, and inversion of the enhancer into the antisense orientation further decreased transcriptional output. Mechanistic analysis identified the GAGA factor (GAF) as a key contributor to orientation-dependent repression at the snail proximal enhancer. Deletion of GAF-binding sites restored transcriptional activity, supporting a model in which GAF acts as an insulator when positioned between enhancer and promoter. To extend these findings to larger genomic scales, a live-imaging framework was developed in murine erythroblasts to quantify long-range E-P interactions mediated by the architectural protein YY1. Collectively, these results demonstrate that enhancer function is not universally independent of configuration but instead emerges from an interplay between genomic arrangement, local factor occupancy, and higher-order chromatin organization, refining models of transcriptional regulation across both short- and long-range contexts. By challenging long-standing assumptions of enhancer independence and revealing new principles of regulatory fine-tuning, this work provides a framework for understanding how cis-regulatory architecture encodes developmental precision, with implications for interpreting noncoding variation in human disease and for advancing targeted gene engineering approaches.