Transcription factor clusters enhance gene expression in vivo without modulating bursting dynamics.

Phase separation drives key cellular processes, yet the role transcription factor condensates play in gene regulation remains contested. Evidence comes primarily from in vitro experiments, leaving it largely unknown how these structures regulate transcription in vivo. To address this knowledge gap, we used cutting-edge microscopy to measure and manipulate transcription in live Drosophila embryos. Here, we show that Dorsal (an NF-κB homolog) forms clusters that associate with a subset of endogenous target genes. We demonstrate that binding-site abundance governs cluster-gene association, which in turn amplifies transcription. Moreover, enhancer mutations that specifically disrupt cluster-gene association cause lethal developmental defects. Through precise spatiotemporal control of Dorsal concentration, we uncovered that Dorsal clusters neither initiate nor sustain transcriptional bursts. Instead, they only augment polymerase loading during a burst. Our findings establish that Dorsal clusters have a function in vivo, while fundamentally challenging prevailing models of how clusters, hubs, or condensates regulate transcription.