Large distances separate coregulated 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 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 (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 , 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 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 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 ( 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

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Fig. 1. Large distances separate coregulated genes in trans. (A) A schematic of the transvection assay. On the top allele, there is a gyspy , 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

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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. 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.) (E) Distribution of xy distances separating MS2 and PP7 foci with the distal (blue; n = 2,098) or proximal configuration in nuclear cycle 14 (red; n = 2,016). Bin widths plotted were 10 nm. These 2 distance distributions − were determined to be significantly different (P = 2.0 × 10 85, Mann–Whitney U test). (F) Boxplot showing distribution of fluorescence intensity of MS2 foci between proximal (red; n = 832) or distal configuration (blue; n = 1,066). The box represents the lower (25%) and upper (75%) quantile and the solid line represents the median. Whiskers cover up to the 10th and 90th percentile of each distribution. P value was calculated using a Mann–Whitney U test.

15064 | www.pnas.org/cgi/doi/10.1073/pnas.1908962116 Heist et al. Downloaded by guest on September 28, 2021 interleaved MS2 and PP7 stem loops (19). These measurements and MS2 target promoter since we are visualizing nascent tran- suggest a high degree of localization precision, incorporating both scripts. However, previous studies suggest that RNAs are tightly technical noise and noise arising from the MS2/PP7 system, with associated with the chromatin templates from which they are de- 32 ± 22 nm in the x axis and 26 ± 20 nm in y (SI Appendix,Fig.S1). rived (20) and, therefore, could provide a reasonable proxy for During transvection, the 2 foci of transcription do not co- visualizing enhancer-promoter interactions across homologous incide, but instead display considerable spatial separation. Rapid chromosomes. fluctuations cause some variations in the distances separating the The distances separating transvecting PP7 and MS2 alleles are PP7 and MS2 nascent transcripts within individual nuclei during consistent with molecular crowding at active promoters due to the acquisition window (e.g., Fig. 1B and Movie S1). We discuss aggregation of components of the general transcription ma- these fluctuations in more detail below. chinery (e.g., Pol II, Mediator). Further support for this view Surprisingly large distances were found to separate the 2 al- stems from measurements of coordinately expressed PP7 and leles during transvection: 282 ± 124 nm (Fig. 1C), although they MS2 alleles linked in cis in close proximity on the same chro- are considerably smaller than those seen for MS2 and PP7 alleles mosome (Fig. 2). We measured the distances separating co- that are regulated by separate enhancers (SI Appendix, Fig. S2). ordinately expressed MS2 and PP7 reporter genes organized in This underscores the fact that coordinate transcription of PP7 distal (Fig. 2A and Movie S2) or proximal (Fig. 2B and Movie and MS2 by a shared enhancer depends on pairing of the 2 al- S3) arrangements. The promoter regions of the reporter genes leles during transvection and the resultant increase in physical map ∼16.5 kb away from one another in the distal arrangement. proximity. Of course, these measurements represent only an ap- In this configuration, the distance between the transcription foci proximation of the distance separating the sna shadow enhancer is an average of 202 ± 111 nm (Fig. 2 C and E), somewhat closer

A 800

600 13.7 48.1 61.9 103.1 seconds BIOLOGY 400 DEVELOPMENTAL

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0 0 50 100 150 200 Time (seconds)

Fig. 3. Distance dynamics between coregulated genes during transvection. (A–D, Left) Stills of representative nuclei during transvection taken from max- imum 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.) (A–D, Right) xy distance trajectories between MS2 and PP7 foci over time. Time points that are indicated by blue correspond to a still image shown at Left.

Heist et al. PNAS | July 23, 2019 | vol. 116 | no. 30 | 15065 Downloaded by guest on September 28, 2021 than that seen in the transvection assay. Surprisingly, the dis- minimum distances of ∼100 nm separate the enhancer from its tance separating the transcription foci is larger for the proximal target promoter at transient periods during transcription, al- arrangement, whereby the PP7 and MS2 promoters are sepa- though distances of 200–300 nm (or more) are equally plausible. rated by just ∼3.5 kb. The average distance that was measured is We suggest that even a distance of ∼100 nm is larger than what is 268 ± 119 nm, more similar to the distance seen in the trans- expected by traditional looping models. vection assay (Fig. 2 D and E). In summary, we have presented evidence that large distances Why would the proximal arrangement exhibit a larger distance separate coordinately expressed reporter genes regulated by a than the distal arrangement? These distances correlate with the shared enhancer. These distances are consistent with the hub level of transcriptional activity (Fig. 2F). In the proximal orien- model,wherebyclustersorcondensatesofPolIIorassociated tation, the sna shadow enhancer is located immediately upstream factors are recruited to active foci (2–4, 21). Further support for this of both the MS2 and PP7 reporter genes, and the total fluores- view is the correlation between the distances of separation and the ∼ cence intensity (burst size) is 33% higher than that observed size of transcriptional bursts (summarizedinFig.4).Whetheritis based on MS2 signal when the enhancer is positioned in the Pol II or associated factors that are the major contributors of distal orientation (8). We suggest that this difference in burst size molecular crowding at active promoters is currently an area of ac- is due to the different numbers of Pol II or associated factors tive study (2–4, 21). Nonetheless, the magnitude of the distances (e.g., Mediator) that are recruited. More factors are recruited documented in this study is consistent with several recent studies when the enhancer is proximal, leading to a larger separation of that have employed various measurement methods, in both fixed the 2 reporter genes. In contrast, fewer factors might be and living tissues (14–16). The picture that is emerging from these recruited when the shared enhancer is located in the distal ori- entation. It is possible that this same phenomenon might occur studies is that enhancer-promoter communication is mediated by a between transgenic constructs inserted at different genomic lo- combination of the classical looping and linking models. Once en- cations (SI Appendix, Fig. S3). An alternative possibility is that hancers come into proximity, they influence transcription via many – the greater distances separating the proximal vs. distal orienta- protein protein interactions within a hub across the distance of the tions of the PP7 and MS2 reporters are due to divergent elon- enhancer to its promoter. gation of Pol II along reporter genes positioned in opposing Materials and Methods directions. As discussed earlier, there is a relatively large SD in our Fly Strains. The fly strains gypsy-sna shadow enhancer-evePr-PP7-lacZ (9), measurements, ∼110–120 nm. This does not appear to be strictly gypsy-sna shadow enhancer-evePr-MS2-lacZ (9), gypsy-evePr-MS2-lacZ (9), due to technical and biological noise (SI Appendix, Fig. S1), but rather, arises (at least in part) from rapid fluctuations in the physical separation of the PP7 and MS2 reporter genes during A their coordinate expression (Fig. 3). We examined these fluctu- ations in >100 living nuclei and several examples are presented PP7 in Fig. 3, e.g., the nucleus shown in Fig. 3D exhibits a full fluc- enhancer tuation cycle over an interval of ∼40 s. The timing and distances ~280nm of these fluctuations are variable, with distances of 50–200 nm and time intervals of ∼10–40 s (SI Appendix, Fig. S4). MS2 It is possible that these fluctuations are caused by the dynamic recruitment of Pol II and associated cofactors. Perhaps Pol II clusters of variable sizes are recruited to the hub. Large clusters B – (e.g., 10 20 Pol II complexes) lead to a larger separation (e.g., PP7 ∼200 nm) of the active reporter genes compared with small clusters (5–6 Pol II complexes; ∼100 nm). As Pol II is released

hne

and undergoes elongation, the reporter genes may come into a

n ~200nm closer proximity with one another. An alternate possibility is that

ec

these fluctuations might be a consequence of dynamic “kiss and r run” interactions, whereby an enhancer comes into sporadic and MS2 transient contact with its target promoter to trigger transcription (4). However, we do not favor this view as we observed several instances of coregulated transcription foci that are separated by substantial distances (>200 nm) during the entirety of a fluctu- C ation cycle (SI Appendix, Fig. S4). PP7

We believe that the transvection assay (Fig. 1) provides the e

n

best opportunity for estimating the distance separating an en- h hancer from its target promoter due to the tight linkage of the a ~270nm

sna shadow enhancer with the PP7 reporter gene and the ecn

r uncoupling of the MS2 target gene on the other . As a result, the PP7 signals provide a reasonable MS2 estimate for the position of the enhancer. As discussed above, the mean distance separating PP7 and MS2 signals in the transvection assay is ∼250–300 nm. However, this might over- Fig. 4. Molecular crowding as the source for the distances separating estimate the actual distance between the PP7-linked enhancer coregulated genes in different configurations. (A–C)Ineachassay,wepropose from the MS2 target promoter, as we are measuring nascent that clustering and aggregation of Pol II (purple), transcription factors (orange), and other components of the general transcription machinery (e.g., transcripts that are located downstream of the transcription start PIC components, Mediator; yellow) leads to the separation seen between state. If we conservatively apply our error in localization (SI coregulated reporter genes. With the cis-linked assays, recruitment of fewer Appendix, Fig. S1) to our measurements, the minimal distance such molecules (e.g., Pol II, Mediator) might explain the smaller distance separating the 2 alleles is then on the order of ∼200 nm. Further, observed between reporter genes in the distal configuration (B) compared with the fluctuations as described above, it is possible that with the proximal configuration (C).

15066 | www.pnas.org/cgi/doi/10.1073/pnas.1908962116 Heist et al. Downloaded by guest on September 28, 2021 evePr-MS2-yellow-sna shadow enhancer-yellow-PP7-evePr (8), snaPr-MS2- Image Analysis. All images were first deconvolved and processed in Zen Black, yellow-sna primary enhancer-yellow-PP7-snaPr (8), and hb proximal version 2.3 (Zeiss). Processed images were then moved to Imaris, version 9.2.1 enhancer-hb P2 promoter-12×(PP7-MS2)-lacZ (19) used in this study were (Bitplane). Objects were fit to MS2 and PP7 foci using the Surfaces tool and previously published and have all been integrated into the third chromo- tracked across time in the processed movie. For each time point in each some (VK00033). The pbphi-snaPr-MS2-yellow-sna primary enhancer-yellow- nucleus, the centers of both foci were recorded. Time points in which a single PP7-snaPr plasmid (8) was also injected and integrated into the second MS2 or PP7 focus was split into 2 distinct foci, likely due to sister chromatid chromosome (VK00002) by BestGene. separation, were excluded from further analysis by manual curation. Four- color 200-nm diameter Tetraspeck beads (Thermo Fisher) were imaged as Cloning and Transgenesis. The pbphi-yellow-MS2-snaPr-linker-sna shadow- described above, and the centers of each bead signal upon 488-nm and 561- linker-snaPr-PP7-yellow plasmid was constructed as previously described (8), nm excitation were calculated. The optical offsets in these channels were used except the insertion of the DNA fragment containing linker-snaPr-MS2- to fit a second-order polynomial transform in MATLAB R2016b (Mathworks) yellow in the opposite orientation was selected after sequencing. Injection was using the cp2tform function (23). This mapping was used to correct the offset done as previously described (22) into the third chromosome (VK00033). between the 2 color channels in the experimental images. For Fig. 2F, fluorescence intensity was determined as the highest intensity pixel found in Live Imaging. For the assays containing both MS2 and PP7 transgenes on the each MS2 surface per time point and nucleus (this is due to the higher same allele, MCP-GFP, mCherry-PCP, His2Av-eBFP2 homozygous virgins (9) photostability of GFP vs. mCherry). were mated with homozygous males containing the transgenic construct. For the assays containing MS2 and PP7 transgenes on separate alleles, MCP- Statistical Analysis. All plots and statistical tests were done in R, version 3.5.1. GFP, mCherry-PCP, His2Av-eBFP2 homozygous virgins were first mated with A 2-sided Mann–Whitney U test was used to calculate statistical significance homozygous males carrying the PP7-containing transgenic construct. Re- of the difference between 2 distributions of fluorescence intensity (Fig. 2F) sultant trans-heterozygote virgins were obtained and mated with homozy- gous males carrying the MS2-containing transgenic construct. For all crosses, and between 2 distributions of distance (Fig. 2E and SI Appendix, Fig. S3D). resulting embryos were treated in preparation for imaging as previously described (9). All imaging was done at room temperature using a Zeiss LSM ACKNOWLEDGMENTS. We thank Evangelos Gatzogiannis for instruction 880 with the Fast Airyscan module and using a Plan-Apochromat 63×/1.4 and assistance with imaging, the Shvartsman laboratory for assistance with N.A. oil immersion objective. The 405-nm, 488-nm, and 561-nm lasers were Imaris, as well as Paul Schedl, Stas Shvartsman, Benjamin Bratton, and members of the M.L. laboratory for helpful discussions. T.F. was supported used across all imaging. For the transvection assay, 30 z-slices of 0.17 um by the Grants-in-Aid for Leading Initiative for Excellent Young Researchers were acquired per time point. For all other experiments, either 45 or 60 z-slices from the Ministry of Education, Culture, Sports, Science and Technology in of 0.17 um were acquired per time point. For the experiments examining Japan, and the Grants-in-Aid for Research Activity Start-up JP18H06040 and the 2 cis-linked reporter genes at different integration sites (SI Appendix, the Grants-in-Aid for Scientific Research B JP19H03154 from the Japan BIOLOGY × × Fig. S3), 0.7 Nyquist xy sampling was used. For all other experiments, 1.8 Society for the Promotion of Science, and research grants from the Molecular DEVELOPMENTAL Nyquist xy sampling was used. For assays in which we compared fluores- Biology Society Japan, the Mochida Memorial Foundation for Medical and cence intensities across constructs (Fig.2),laserpowerwaskeptconstant. Pharmaceutical Research, the Inamori Foundation, and the Nakajima Founda- Images were captured in 8 bits. tion. This study was funded by NIH Grant GM118147 (to M.L.).

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