Summary Transcription is episodic, consisting of a series of discontinuous bursts. Using live imaging methods and quantitative analysis, we examine transcriptional bursting in living Drosophila embryos. Different developmental enhancers positioned downstream of synthetic reporter genes produce transcriptional bursts with similar amplitudes and duration, but generate very different bursting frequencies, with strong enhancers producing more bursts than weak enhancers. Insertion of an insulator reduces the number of bursts and the corresponding level of gene expression, suggesting that enhancer regulation of bursting frequency is a key parameter of gene control in development. We also show that linked reporter genes exhibit coordinated bursting profiles when regulated by a shared enhancer, challenging conventional models of enhancer-promoter looping.

Summary Zygotic genome activation (ZGA) is a major genome programming event whereby the cells of the embryo begin to adopt specified fates. Experiments in Drosophila and zebrafish revealed that ZGA depends on transcription factors that provide large-scale control of gene expression by direct and specific binding to gene regulatory sequences [1–5]. Zelda (Zld) plays such a role in the Drosophila embryo, where it was shown to control the action of patterning signals [1, 2], however the mechanisms underlying this effect remain largely unclear. A recent model proposed that Zld binding sites act as quantitative regulators of the spatiotemporal expression of genes activated by Dorsal (Dl), the morphogen that patterns the dorsoventral (DV) axis [6]. We test this model experimentally, using enhancers of brinker (brk) and short gastrulation (sog), both of which are directly activated by Dl, but at different concentration thresholds [7–9]. In agreement with the model, we show that there is a clear positive correlation between the number of Zld binding sites and the spatial domain of enhancer activity. Likewise, the timing of expression could be advanced or delayed. We present evidence that Zld facilitates binding of Dl to regulatory DNA, and that this is associated with increased chromatin accessibility. Importantly, the change in chromatin accessibility is strongly correlated with the change in Zld binding, but not Dl. We propose that the ability of genome activators to facilitate read-out of transcriptional input is key to widespread transcriptional induction during ZGA.

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How remote enhancers interact with appropriate target genes persists as a central mystery in gene regulation. Here, we exploit the properties of transvection to explore enhancer-promoter communication between homologous chromosomes in living Drosophila embryos. We successfully visualized the activation of an MS2-tagged reporter gene by a defined developmental enhancer located in trans on the other homolog. This trans-homolog activation depends on insulator DNAs, which increase the stability-but not the frequency-of homolog pairing. A pair of heterotypic insulators failed to mediate transvection, raising the possibility that insulator specificity underlies the formation of chromosomal loop domains. Moreover, we found that a shared enhancer co-activates separate PP7 and MS2 reporter genes incis and intrans. Transvecting alleles weakly compete with one another, raising the possibility that they share a common pool of the transcription machinery. We propose that transvecting alleles form a trans-homolog "hub," which serves as a scaffold for the accumulation of transcription complexes.

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