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Large Distances Separate Coregulated Genes in Living Drosophila Embryos Large distances separate coregulated genes in living Drosophila embryos Tyler Heista, Takashi Fukayab,c, and Michael Levinea,d,1 aLewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544; bInstitute for Quantitative Biosciences, The University of Tokyo, 113-0032 Tokyo, Japan; cDepartment of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 113-0032 Tokyo, Japan; and dDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544 Contributed by Michael Levine, June 11, 2019 (sent for review May 24, 2019; reviewed by Cornelis Murre and Christine Rushlow) Transcriptional enhancers are short segments of DNA that switch apparent distances of 300–400 nm (14). Similar distances (∼300– genes on and off in response to a variety of cellular signals. Many 350 nm) were observed between the Sox2 control region (SCR) enhancers map quite far from their target genes, on the order of enhancer and Sox2 promoter in mouse embryonic stem cells (15). tens or even hundreds of kilobases. There is extensive evidence High-resolution fixed tissue methods may be consistent with such that remote enhancers are brought into proximity with their distances in the Bithorax complex in Drosophila embryos (16). target promoters via long-range looping interactions. However, Here, we use a recently reported transvection assay and high- the exact physical distances of these enhancer–promoter interac- resolution imaging of living Drosophila embryos to measure the tions remain uncertain. Here, we employ high-resolution imaging distances separating coexpressed reporter genes that are regu- of living Drosophila embryos to visualize the distances separating lated by a single enhancer across homologous chromosomes (9). linked genes that are coregulated by a shared enhancer. Cotransvection These studies reveal large distances of nascent transcripts asso- assays (linked genes on separate homologs) suggest a surpris- ciated with active alleles, on the order of 250–300 nm. These ingly large distance during transcriptional activity: at least distances include a relatively large standard deviation (SD) 100–200 nm. Similar distances were observed when a shared en- (∼120 nm). We present evidence that this variance is not strictly hancer was placed into close proximity with linked reporter genes due to technical or biological noise in our measurements, but in cis. These observations are consistent with the occurrence of instead might arise from rapid fluctuations in the distances “ ” transcription hubs, whereby clusters (or condensates) of multi- separating coordinately expressed alleles. We discuss the impli- ple RNA polymerase II complexes and associated cofactors are pe- cations of these measurements with the hub hypothesis and the riodically recruited to active promoters. The dynamics of this dynamics of Pol II aggregation/condensation at active promoters. process might be responsible for rapid fluctuations in the distances separating the transcription of coregulated reporter genes during Results and Discussion transvection. We propose that enhancer-promoter communication As a first step toward estimating the distance between a distal depends on a combination of classical looping and linking models. enhancer and its target promoter during gene activation, we performed high-resolution measurements of coordinately active transcription | enhancers | transvection | Drosophila embryos | alleles during transvection (summarized in Fig. 1A). Briefly, this live-imaging assay employs a PP7 reporter gene with a closely linked upstream snail (sna) shadow enhancer, which mediates expression in the here is emerging evidence for transcription hubs, whereby presumptive mesoderm (17, 18). The other allele contains an Tgene activation is mediated by the recruitment of clusters or MS2 reporter gene that lacks its own enhancer. Consequently, condensates of multiple RNA polymerase II (Pol II) complexes MS2 expression depends on pairing of the alleles to allow the sna (reviewed in ref. 1). Transient clusters of Pol II were detected in living mammalian cells using high-resolution live imaging Significance methods (2). At least some of these clusters appear to be asso- ciated with foci of active transcription, visualized by the insertion The prevailing model of gene activation is the looping of a of MS2 RNA stem loops into defined genes (3, 4). In vitro and distal enhancer to its target promoter, although it remains in vivo assays suggest that the low complexity domains contained uncertain whether enhancers must come into direct contact. in many components of the preinitiation complex (PIC) mediate Recent studies suggest that clusters of RNA polymerase II are liquid-liquid condensation, including Pol II, TAF, and different – recruited to active genes, raising the possibility that enhancers subunits of the Mediator complex (5 7). Coordinate activation cannot get too close to their target promoters due to molecular of linked reporter genes by a shared enhancer lends further crowding. Here, we use live imaging methods to visualize support for the occurrence of transcription hubs (8, 9). genes that are simultaneously regulated by a single enhancer This hub model contrasts with the classical view of gene ex- bothincisandintrans.Wepresentevidencethatthese pression, which suggests the formation of a single PIC and re- coregulated genes are separated by a substantial distance—at cruitment of individual Pol II complexes at active promoters least 100–200 nm—during transcription. These observations (10). An important implication of the hub hypothesis is that challenge the textbook view of enhancer function and raise the distal enhancers need not, and indeed cannot, come into close possibility of an action-at-a-distance model for gene expression. contact with their target promoters due to molecular crowding caused by the collective size of multiple Pol II complexes and Author contributions: T.H. and M.L. designed research; T.H. and T.F. performed research; other components of the PIC (11, 12). T.H. analyzed data; and T.H. and M.L. wrote the paper. Hi-C (chromosome conformation capture) contact maps have Reviewers: C.M., University of California San Diego; and C.R., New York University. been interpreted to suggest close contact of distal enhancers with The authors declare no conflict of interest. their target promoters (13). However, recent high-resolution Published under the PNAS license. imaging methods raise the possibility that distal enhancers can 1To whom correspondence may be addressed. Email: [email protected]. map quite far during transcriptional activation. For example, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. activation of a synthetic lacZ reporter gene by endogenous even- 1073/pnas.1908962116/-/DCSupplemental. skipped (eve) stripe enhancers is detected in living embryos over Published online July 8, 2019. 15062–15067 | PNAS | July 23, 2019 | vol. 116 | no. 30 www.pnas.org/cgi/doi/10.1073/pnas.1908962116 Downloaded by guest on September 28, 2021 A gypsy eve Pr sna shadow enhancer 24xPP7 lacZ 24xMS2 lacZ eve Pr B 0 6.6 13.2 19.8 26.4 33.1 39.7 46.3 seconds nucleus 1 0 6.6 13.2 19.8 26.4 33.1 39.7 46.3 seconds nucleus 2 BIOLOGY MS2 PP7 His2Av DEVELOPMENTAL C 1.00 0.75 0.50 Density 0.25 0.00 0 250 500 750 1000 XY distance (nm) Fig. 1. Large distances separate coregulated genes in trans. (A) A schematic of the transvection assay. On the top allele, there is a gyspy insulator, the sna shadow enhancer, and a PP7-lacZ reporter gene. On the bottom allele, there is a gypsy insulator and an MS2-lacZ reporter gene. (B) Stills of 2 representative nuclei taken from maximum projected Airyscan-processed images. MS2 foci were visualized by MCP-GFP (green), PP7 foci were visualized by mCherry-PCP (red), and nuclei were visualized using His2Av-eBFP2 (blue). (Scale bar, 500 nm.) (C) Distribution of xy distances separating MS2 and PP7 foci during transvection in nuclear cycle 14 (n = 2,441). Bin widths plotted were 10 nm. shadow enhancer on the PP7 allele to act in trans (9). To improve shown in Fig. 1B). We focused on the xy axes due to technical the frequency of pairing and transvection, gypsy insulator DNAs difficulties in achieving similar resolution in the axial (z) di- were placed upstream of the 2 reporter genes. mension. To compensate for this emphasis on xy resolution, we To address this question of distances separating enhancers and observed a large number of transcription foci to ensure reliable target genes, we measured the center-to-center distances of ac- capture of the distances separating dynamically expressed al- tive MS2 and PP7 foci in xy dimensions (representative nuclei are leles. As a control, we analyzed a reporter gene that contains Heist et al. PNAS | July 23, 2019 | vol. 116 | no. 30 | 15063 Downloaded by guest on September 28, 2021 A eve Pr eve Pr sna shadow 24xMS2 yellow enhancer yellow 24xPP7 B sna Pr sna Pr sna shadow yellow 24xMS2 enhancer 24xPP7 yellow C 0 9.9 19.8 29.7 39.6 49.5 59.4 69.3 seconds distal D 0 10.3 20.5 30.8 41.2 51.5 61.7 72.0 seconds proximal MS2 PP7 His2Av EF 1.00 distal promoters 2000 p = 1.2 x 10-46 proximal promoters 0.75 1500 0.50 1000 Density 500 0.25 Fluorescence Intensity (AU) 0 0.00 Distal Proximal 0 250 500 750 1000 XY distance (nm) Fig. 2. Distal and proximal configurations of 2 cis-linked reporter genes yield unique distance distributions. (A and B) Schematics for 2 configurations with 2 cis-linked reporter genes. The sna shadow enhancer is regulating MS2-yellow and PP7-yellow reporter genes with either distal eve promoters (A) or proximal sna promoters (B). The promoters are separated by either ∼16.5 kb (A)or∼3.5 kb (B). (C and D) Stills of representative nuclei with either the distal (C)or proximal (D) configuration taken from maximum projected Airyscan-processed images.
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