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Dynamic Interplay Between Enhancer–Promoter Topology and Gene Activity

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Dynamic Interplay Between Enhancer–Promoter Topology and Gene Activity ARTICLES https://doi.org/10.1038/s41588-018-0175-z Dynamic interplay between enhancer–promoter topology and gene activity Hongtao Chen1, Michal Levo1, Lev Barinov2, Miki Fujioka3, James B. Jaynes! !3 and Thomas Gregor! !1,4,5* A long-standing question in gene regulation is how remote enhancers communicate with their target promoters, and specifically how chromatin topology dynamically relates to gene activation. Here, we combine genome editing and multi-color live imag- ing to simultaneously visualize physical enhancer–promoter interaction and transcription at the single-cell level in Drosophila embryos. By examining transcriptional activation of a reporter by the endogenous even-skipped enhancers, which are located 150!kb away, we identify three distinct topological conformation states and measure their transition kinetics. We show that sustained proximity of the enhancer to its target is required for activation. Transcription in turn affects the three-dimensional topology as it enhances the temporal stability of the proximal conformation and is associated with further spatial compaction. Furthermore, the facilitated long-range activation results in transcriptional competition at the locus, causing corresponding developmental defects. Our approach offers quantitative insight into the spatial and temporal determinants of long-range gene regulation and their implications for cellular fates. nhancers play a key role in the control of gene expression which contains a set of five enhancers that drive a seven-striped that is essential for development1–3. These 50–1,500 base pair expression pattern in the cellular blastoderm (Supplementary E(bp) cis-regulatory elements stimulate transcription from Fig. 1). While this chosen distance is generally larger than that core promoters in a time- and tissue-specific manner by recruit- observed for enhancer–promoter interactions in the early fly ing context-dependent transcriptional activators and repressors4–6. embryo, it is comparable to and even smaller than the distances Whole-genome methods have shown that the human genome is rid- over which many enhancers function in higher eukaryotes8–10,20. dled with enhancers, with estimates ranging from 200,000 to over a Notably, at such distance the chromatin fiber can display fast ran- million7. Importantly, a significant fraction of enhancers are located dom movements, which creates an entropic hurdle for specific at large genomic distances from the promoters they regulate8–10. long-range chromatin interactions and thus a kinetic barrier for Even for a compact genome such as Drosophila melanogaster, at the establishment of a productive pre-initiation complex. We there- least 30% of enhancer–promoter interactions occur over 20 kb, and fore included in our reporter cassette the 368 bp insulator element in many cases over intervening genes11–13. homie (Supplementary Fig. 1a)21,22, which facilitates the formation Despite extensive studies over more than three decades, many of a stable loop by self-pairing with the endogenous homie element23 questions still remain as to how enhancers communicate with their located at the 3’ end of the eve locus21,22. In fixed embryos containing target promoters over large genomic distances14. Static measure- our reporter cassette, we observe sporadic expression (~15%) of the ments, employing, for example, fluorescence in situ hybridization reporter gene, solely within the limits of the endogenous eve stripes (FISH) and chromosome conformation capture based genomic (Supplementary Fig. 1b), which suggests that the reporter is specifi- experiments, provided evidence supporting physical interactions cally activated by the eve enhancers 142 kb away21. between a distal enhancer and a target promoter15–19. Yet we still To simultaneously visualize the location of the endogenous eve lack a dynamic characterization that could distinguish transient enhancers, the location of the promoter of the reporter, and its tran- contact from the formation of stable topological structures and scriptional activity in living embryos, we designed a three-color disentangle cause from consequence in the relationship between imaging system. First, we used two orthogonal stem-loop-based such topological structures and transcription. To address these fun- labeling cassettes24–26; MS2 stem loops were introduced via CRISPR damental questions, we have developed a live imaging approach to genome editing to the endogenous eve gene, and PP7 stem loops track the spatial positions of an enhancer and its target promoter were added to the reporter gene (Fig. 1a, Supplementary Fig. 2a,b, and to simultaneously monitor transcriptional activity in develop- Supplementary Video 1). Maternally expressed fluorescent coat pro- ing fly embryos. By employing this approach, we characterize, at the teins bind the corresponding nascent stem-loops on transcription, single-cell level, a dynamic interplay between enhancer–promoter providing a dynamic readout of gene activity (Fig. 1a). Owing to topology and transcriptional activity. the strong transcriptional activity of the eve gene, the corresponding fluorescent focus further serves as a marker for the nuclear posi- Results tion of the eve enhancers, which are located within 10 kb of the eve Live imaging of chromatin topology and transcription. To exam- promoter (Supplementary Fig. 1a). In addition, we took advantage ine long-range transcriptional activation, we placed a reporter gene of a recently developed DNA labeling system27,28 to mark the posi- 142 kb from the well-studied Drosophila even-skipped (eve) locus, tion of the reporter gene in a manner that is independent of its 1Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA. 2Department of Molecular Biology, Princeton University, Princeton, NJ, USA. 3Department of Biochemistry and Molecular Biology, and the Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA. 4Joseph Henry Laboratories of Physics, Princeton University, Princeton, NJ, USA. 5Department of Developmental and Stem Cell Biology, Institut Pasteur, Paris, France. *e-mail: [email protected] NATURE GENETICS | www.nature.com/naturegenetics ARTICLES NATURE GENETICS ab 100 µm eve-MS2 parS homie evePr PP7 enhancers eve MS2 enhancers homie 142 kb Chr2R homie 10 kb PP7 nos NLS PCP mKate2 mKate2 mKate2 vas MS2 NLS MCP mTagBFP2 mTagBFP2 mTagBFP2 5 µm nos ParB EGFP parS c 0:00 0:30 1:00 1:30 2:00 2:30 3:00 3:30 Red-OFF 2 m Red-ON µ d e Red-OFF Red-ON control Red-OFF λ m) µ Red-ON ( 1 1.2 ) 2 0.8 1 m m) µ 1 ( µ ( 0 m) 0.8 µ ( 0 dz 0.6 –1 dz –1 1 dy 1 3D MSD 0 1 ( –1 0 dy 0 1 µm) –1 m) 0.4 ( 0 (µ 0.4 µm)–1 –1 m) dx dx (µ Enhancer–promoter distance 0.2 50 100 150 200 0400 8001,200 Time (s) ∆t (s) Fig. 1 | Three-color live imaging of enhancer–promoter movement and transcriptional activity. a, Male flies carrying the modified eve locus are crossed with females carrying maternally expressed blue, red and green fluorescent proteins that are fused to MS2 coat protein (MCP), PP7 coat protein (PCP), and ParB DNA binding protein, respectively. In the male flies, a reporter with an eve promoter (evePr) driving PP7 transcription is integrated at − 142!kb upstream of an MS2-tagged endogenous eve locus in the Drosophila genome. An ectopic homie insulator sequence is also included in the reporter to force loop formation through homie-homie pairing. Furthermore, a parS sequence is integrated near the homie-evePr-PP7 reporter. b, Snapshot of a representative embryo generated from crosses shown in a. The embryo displays fluorescent foci for MS2, PP7, and parS in the corresponding channels. c, Eight snapshots of a time course following two nuclei for ~4!min. The lower nucleus displays PP7 activity (Red-ON), the upper has none (Red-OFF). d, Instantaneous physical enhancer–promoter distance between endogenous eve enhancers (blue signal) and the PP7 reporter (green signal) as a function of time for the Red-OFF and Red-ON nuclei in c. Error bar corresponds to measurement error estimated from the co-localization control experiments (see Supplementary Fig. 3). e, Population-averaged MSD calculated from enhancer–promoter distance trajectories obtained from all Red-ON (n!=!720) and Red-OFF (n!=!7,163) nuclei, as well as for a control construct where homie in the reporter is replaced by phage λ DNA (λ control, n!=!1,453). Inset shows two representative trajectories for a Red-OFF nucleus (blue) and a Red-ON (red) nucleus, respectively. activity. Namely, Burkholderia parS DNA sequences were included These three florescent foci thus provide the means to measure in the reporter gene, nucleating the binding of ParB-GFP fusion the physical distance between the enhancers and the reporter, as proteins (Fig. 1a). well as to monitor the reporter’s transcriptional activity. To ascer- Using three-color time-lapse confocal microscopy, we captured tain our ability to accurately measure these properties, several stacks of optical sections of the surface of two-hour-old (nuclear control experiments were performed. To estimate the precision of cycle 14, nc14) embryos carrying the tagged eve locus and the our distance measurements, we generated a synthetic construct parS-homie-evePr-PP7 reporter (Supplementary Video 2). In these (localization control) in which all three fluorescent proteins are stacks, we can clearly identify individual fluorescent foci in 70–100 co-localized within a genomic distance of 2.0 kb (Supplementary nuclei simultaneously (Fig. 1b). In the blue channel, we observed Fig. 3a). By analyzing embryos carrying this construct, we were the endogenous transcriptional activity of the eve gene in its charac- able to calibrate chromatic aberrations from the microscope and teristic seven-striped pattern. This pattern is quantitatively identi- to estimate measurement errors in spot localization (180 ± 6 nm cal to that observed from the endogenous eve gene (Supplementary (mean ± s.e.m.), that is, ~75 nm in the x/y directions and ~150 nm Fig. 2c–g, Supplementary Video 1).
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