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TRANSCRIPTIONAL REGULATION Visualizing long-range enhancer–promoter interaction A new study uses multicolor live imaging to simultaneously visualize enhancer–promoter interaction and transcription in Drosophila embryos. Albert Tsai and Justin Crocker

ranscriptional enhancers are short DNA fragments that remotely Distal OFF Proximal ON control gene expression, being able Insulator T eve Enhancer to drive transcription from promoters ✓ located tens of thousands of base pairs away. Several decades have passed since their initial discovery, and we are just beginning to unravel how enhancers and promoters interact in vivo. A long-standing ✓ question in the field remains: how does the Distal ParB Distal ParB physical interaction between enhancers promoter promoter and promoters impact gene expression? In 1 this issue, Chen et al. describe an imaging Fig. 1 | Models of enhancer–promoter interaction. Left, MS2-tagged mRNA (magenta) provides a approach to directly monitor long-range direct readout of native eve transcription by nearby enhancers. The distal eve promoter, marked with interactions between promoters and distal ParB (green), is inactive when it is physically far from enhancers in the ‘distal off’ state. Right, insulator enhancers and their consequences on elements facilitate interactions that bring distal elements into proximity. Additional interactions between transcriptional output. the enhancer and promoter are needed for the reporter to enter the transcriptionally active ‘proximal Transcriptional enhancers in on’ state. PP7-tagged mRNA (orange) provides a direct readout of the distal promoter. The promoter on multicellular eukaryotes greatly outnumber the reporter construct can compete with the native eve promoter, leading to reduced eve expression and genes. In the case of humans, estimates developmental defects. for the total number of enhancers exceed a million elements2,3. This number stands in stark contrast to the estimated that transcription is dynamic and even promoter. Collectively, this three-color setup ~20,000 genes. Thus, each promoter is, on stochastic, live imaging studies will allowed the authors to measure the physical average, under the control of more than necessarily have a pivotal role. distance between the native eve enhancers ten regulatory elements. Furthermore, and the distal reporter construct, providing a the distances between enhancers and Enhancer–promoter interaction goes live direct readout of long-range transcriptional promoters vary over a wide range—with Chen et al.1 imaged the positions of regulation (Fig. 1). some separated by over a million base enhancers relative to eve promoters within A hallmark of early Drosophila embryos pairs4. However, the mechanisms and live Drosophila embryos. eve is a well- is their rapid nuclear divisions, occurring functional significance of enhancer– characterized developmental locus expressed with timing on the order of 30 min or less. promoter interaction have remained in seven stripes along the anterior-to- These rapid cell cycles potentially leave enduring questions. posterior axis of early Drosophila embryos, insufficient time for long-range interactions One shortcoming in the field has been patterning corresponding body segments. to form. To facilitate the formation of stable that most measurements concerning The authors first tagged the native eve looping interactions, the authors took enhancer–promoter communication are mRNA with binding sites for fluorescent advantage of a homie insulator element based on imaging or sequencing studies viral coat proteins (from MS2)5,6, providing present in the native eve locus9 and added from fixed samples. Such assays do not a direct readout of transcription from the a corresponding insulator to the distal often capture the temporal dynamics of endogenous locus. Enhancers driving stripes reporter construct. With pairing insulator transcriptional interactions, leading to of eve expression are located proximal to the elements, the distal reporter became many outstanding questions. What kind native promoter, which allowed the authors transcriptionally active, even with the rapid of physical interactions are required to to monitor the location of eve enhancers nuclear divisions. drive transcription? How close must the using MS2 coat protein fluorescence as a The authors observed that the enhancer be to the promoter to drive proxy. The authors additionally inserted a distances between enhancers and transcription? How frequent or stable remote reporter construct 142 kb away. It promoters were shorter when the are enhancer–promoter interactions? contained ParB binding sites to mark its reporter was transcriptionally active. In How do these interactions shape animal location7,8 and a reporter mRNA tagged with fact, immediately before transcriptional development? With recent reports hinting PP7 stem loops under the control of an eve activation, the distance between the

Nature Genetics | www.nature.com/naturegenetics news & views enhancer and promoter decreased by how many of these regions are functional or transcriptional environments may be the around twofold. Conversely, when the how many elements have ‘homing’ activity. key to understanding the mechanisms distance between the enhancer and Future work could address the generalities of transcriptional regulation during promoter increased, transcription stopped. of such insulator function and its dynamics, development. It is intriguing that many These findings suggest that sustained leading to a deeper understanding of of these results are converging on similarly physical proximity of regulatory elements is transcriptional regulation. sized transcriptional ‘hubs’, which may required for transcription. These findings are consistent with the form transiently on the stable chromatin Further characterizing the dynamics suggestion that chromosomal boundaries scaffolds formed by insulator proteins. of long-range gene regulation, the authors are rather stable, based on the similarity of Ultimately, continued work to found that a model with three states fit the boundaries across cell types from genomic connect genomics to these in vivo distribution of distances between regulatory data11. However, the dynamics of the measurements promises to lead to a elements. These states represented three loop structures that form such insulated better understanding of how the molecular functional conformations, concerning neighborhoods10 and the enhancer– interactions within the nucleus lead to a physical distance (distal versus proximal) promoter interactions within these regions fully functional organism. ❐ and transcriptional state (on versus off): are not yet fully understood. Furthermore, distal off, proximal off and proximal on. in the Drosophila genome, there is evidence Albert Tsai and Justin Crocker* Deletion of individual components in for stable, long-range interactions between Developmental Biology Unit, European Molecular the reporter construct supported this loci12, consistent with the findings of Chen Biology Laboratory, Heidelberg, Germany. interpretation. Tracking the populations of et al.1. In fact, the majority of enhancer *e-mail: [email protected] each state over time highlighted the striking interactions show no evidence of dynamic observation that sustained proximity is changes across development12. The Published: xx xx xxxx required to transition into the on state. experimental approaches used by Chen et al.1 https://doi.org/10.1038/s41588-018-0198-5 could be applied to study the dynamics of The future of live-imaging transcription such neighborhoods of gene expression and References Chen et al.1 demonstrated the power of enhancer–promoter interactions. 1. Chen, H. et al. Nat. Genet. https://doi.org/10.1038/s41588-018- 0175-z (2018). using in vivo imaging to confirm that Intriguingly, the finding that stable 2. Lim, B., Heist, T., Levine, M. & Fukaya, T. Mol. Cell 70, enhancer–promoter proximity is required physical proximity of regulatory elements 287–296 (2018). for sustained transcription. These results is required for transcription seems to be 3. ENCODE Project Consortium. Nature 489, 57–74 (2012). 4. Lettice, L. A. et al. Hum. Mol. Genet. 12, 1725–1735 (2003). highlight the relationship between DNA at odds with recent work demonstrating 5. Bertrand, E. et al. Mol. Cell 2, 437–445 (1998). topological association and physical that active transcription sites are highly 6. Garcia, H. G. G., Tikhonov, M., Lin, A. & Gregor, T. Curr. Biol. 23, proximity. That is, elements that strongly dynamic13–15 and can diffuse more 2140–2145 (2013). 16 7. Saad, H. et al. PLoS Genet. 10, e1004187 (2014). influence the conformation of chromosomes freely . These differences may be 8. Dubarry, N., Pasta, F. & Lane, D. J. Bacteriol. 188, directly alter gene regulation. The homie the result of the insulator elements 1489–1496 (2006). insulator element chosen by the authors inducing a very stable chromatin 9. Fujioka, M., Wu, X. & Jaynes, J. B. Development 136, is a particularly strong example of this conformation, working with timing 3077–3087 (2009). 10. Hnisz, D., Day, D. S. & Young, R. A. Cell 167, 1188–1200 (2016). 9 kind of element , as it was able to hijack on the order of tens of minutes. In 11. Dixon, J. R. et al. Nature 518, 331–336 (2015). transcription from the native eve promoter contrast, recent studies focused on 12. Ghavi-Helm, Y. et al. Nature 512, 96–100 (2014). to the distal reporter construct, leading to transcriptional dynamics occurring 13. Mir, M. et al. Genes Dev. 31, 1784–1794 (2017). 14. Cisse, I. I. et al. Science 341, 664–667 (2013). competition between promoters resulting in in time frames from milliseconds 15. Tsai, A. et al. eLife 6, e28975 (2017). developmental defects. Furthermore, while to minutes. Resolving the apparent 16. Gu, B. et al. Science 359, 1050–1055 (2018). many regions in the genome are bound by inconsistencies between the stable, similar insulator or architectural proteins long-range chromatin topologies found Competing interests (i.e., CTCF and BEAF32)10, it is not clear by Chen et al.1 and the dynamic local The authors declare no competing interests.

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