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Transcriptional coupling of distant regulatory genes in living embryos

Abstract

The prevailing view of metazoan gene regulation is that individual genes are independently regulated by their own dedicated sets of transcriptional enhancers. Past studies have reported long-range gene–gene associations1,2,3, but their functional importance in regulating transcription remains unclear. Here we used quantitative single-cell live imaging methods to provide a demonstration of co-dependent transcriptional dynamics of genes separated by large genomic distances in living Drosophila embryos. We find extensive physical and functional associations of distant paralogous genes, including co-regulation by shared enhancers and co-transcriptional initiation over distances of nearly 250 kilobases. Regulatory interconnectivity depends on promoter-proximal tethering elements, and perturbations in these elements uncouple transcription and alter the bursting dynamics of distant genes, suggesting a role of genome topology in the formation and stability of co-transcriptional hubs. Transcriptional coupling is detected throughout the fly genome and encompasses a broad spectrum of conserved developmental processes, suggesting a general strategy for long-range integration of gene activity.

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Fig. 1: Pervasive long-range promoter–promoter connectivity of genes with shared enhancers.
Fig. 2: Distant interconnected genes show physical proximity and co-initiation within single nuclei.
Fig. 3: Manipulations of promoter-proximal tethering elements alter knrl/kni transcriptional dynamics.
Fig. 4: Tethering elements are important for the long-range co-regulation of scyl and chrb.

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Data availability

All Micro-C data are available at the Gene Expression Omnibus (GSE173518). The following publicly available databases and datasets were used: FlyBase r6.40 (https://flybase.org/) using the dm6 reference genome; BDGP in situ database (https://insitu.fruitfly.org/); Fly Enhancer @ Stark Lab (https://enhancers.starklab.org/); chromatin immunoprecipitation sequencing data for Zelda (GSE30757), Cohesin (GSE54529), CTCF and CP190 (GSE30740), Pc (GSE68983), Pho and Ph (GSE77342), Cg (GSE77582), CLAMP (GSE39271) and GAF (GSE152773); RAMPAGE TSS profiling (GSE36213); ATAC-seq data (GSE152771).

Code availability

Custom codes (MATLAB) used for image processing and data analysis are available on request. All details of algorithms are described in the Methods.

References

  1. Schoenfelder, S. et al. Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells. Nat. Genet. 42, 53–61 (2010).

    Article  CAS  PubMed  Google Scholar 

  2. Li, G. et al. Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation. Cell 148, 84–98 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Jung, I. et al. A compendium of promoter-centered long-range chromatin interactions in the human genome. Nat. Genet. 51, 1442–1449 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Jacob, F. & Monod, J. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3, 318–356 (1961).

    Article  CAS  PubMed  Google Scholar 

  5. Long, H. K., Prescott, S. L. & Wysocka, J. Ever-changing landscapes: transcriptional enhancers in development and evolution. Cell 167, 1170–1187 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sanyal, A., Lajoie, B. R., Jain, G. & Dekker, J. The long-range interaction landscape of gene promoters. Nature 489, 109–113 (2012).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. Furlong, E. E. M. & Levine, M. Developmental enhancers and chromosome topology. Science 361, 1341–1345 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. Lunde, K., Biehs, B., Nauber, U. & Bier, E. The knirps and knirps-related genes organize development of the second wing vein in Drosophila. Development 125, 4145–4154 (1998).

    Article  CAS  PubMed  Google Scholar 

  9. Scuderi, A., Simin, K., Kazuko, S. G., Metherall, J. E. & Letsou, A. scylla and charybde, homologues of the human apoptotic gene RTP801, are required for head involution in Drosophila. Dev. Biol. 291, 110–122 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Cheng, Y. et al. Co-regulation of invected and engrailed by a complex array of regulatory sequences in Drosophila. Dev. Biol. 395, 131–143 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Stathopoulos, A., Tam, B., Ronshaugen, M., Frasch, M. & Levine, M. pyramus and thisbe: FGF genes that pattern the mesoderm of Drosophila embryos. Genes Dev. 18, 687–699 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Rothe, M., Wimmer, E. A., Pankratz, M. J., González-Gaitán, M. & Jäckle, H. Identical transacting factor requirement for knirps and knirps-related gene expression in the anterior but not in the posterior region of the Drosophila embryo. Mech. Dev. 46, 169–181 (1994).

    Article  CAS  PubMed  Google Scholar 

  13. Zinani, O. Q. H., Keseroğlu, K., Ay, A. & Özbudak, E. M. Pairing of segmentation clock genes drives robust pattern formation. Nature 589, 431–436 (2021).

    Article  CAS  PubMed  Google Scholar 

  14. Michalak, P. Coexpression, coregulation, and cofunctionality of neighboring genes in eukaryotic genomes. Genomics 91, 243–248 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Tomancak, P. et al. Global analysis of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 8, R145 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Hammonds, A. S. et al. Spatial expression of transcription factors in Drosophila embryonic organ development. Genome Biol. 14, R140 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Hsieh, T.-H. S. et al. Resolving the 3D landscape of transcription-linked mammalian chromatin folding. Mol. Cell 78, 539–553 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Krietenstein, N. et al. Ultrastructural details of mammalian chromosome architecture. Mol. Cell 78, 554–565 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Rowley, M. J. et al. Analysis of Hi-C data using SIP effectively identifies loops in organisms from C. elegans to mammals. Genome Res. 30, 447–458 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cusanovich, D. A. et al. The cis-regulatory dynamics of embryonic development at single-cell resolution. Nature 555, 538–542 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gaskill, M. M., Gibson, T. J., Larson, E. D. & Harrison, M. M. GAF is essential for zygotic genome activation and chromatin accessibility in the early Drosophila embryo. eLife 10, e66668 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fukaya, T., Lim, B. & Levine, M. Enhancer control of transcriptional bursting. Cell 166, 358–368 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chen, H. et al. Dynamic interplay between enhancer–promoter topology and gene activity. Nat. Genet. 50, 1296–1303 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Garcia, H. G., Tikhonov, M., Lin, A. & Gregor, T. Quantitative imaging of transcription in living Drosophila embryos links polymerase activity to patterning. Curr. Biol. 23, 2140–2145 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Ghavi-Helm, Y. et al. Enhancer loops appear stable during development and are associated with paused polymerase. Nature 512, 96–100 (2014).

    Article  ADS  CAS  PubMed  Google Scholar 

  26. Benabdallah, N. S. et al. Decreased enhancer-promoter proximity accompanying enhancer activation. Mol. Cell 76, 473–484 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Calhoun, V. C., Stathopoulos, A. & Levine, M. Promoter-proximal tethering elements regulate enhancer-promoter specificity in the Drosophila antennapedia complex. Proc. Natl Acad. Sci. USA 99, 9243–9247 (2002).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Batut, P. J. et al. Genome organization controls transcriptional dynamics during development. Science 375, 566–570 (2022).

    Article  ADS  CAS  PubMed  Google Scholar 

  29. Judd, J., Duarte, F. M. & Lis, J. T. Pioneer-like factor GAF cooperates with PBAP (SWI/SNF) and NURF (ISWI) to regulate transcription. Genes Dev. 35, 147–156 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Tsai, A. et al. Nuclear microenvironments modulate transcription from low-affinity enhancers. eLife 6, e28975 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Mir, M. et al. Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos. eLife 7, e40497 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Tsai, A., Alves, M. R. & Crocker, J. Multi-enhancer transcriptional hubs confer phenotypic robustness. eLife 8, e45325 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Li, J. et al. Single-gene imaging links genome topology, promoter-enhancer communication and transcription control. Nat. Struct. Mol. Biol. 27, 1032–1040 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Eagen, K. P., Aiden, E. L. & Kornberg, R. D. Polycomb-mediated chromatin loops revealed by a subkilobase-resolution chromatin interaction map. Proc. Natl Acad. Sci. USA 114, 8764–8769 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ogiyama, Y., Schuettengruber, B., Papadopoulos, G. L., Chang, J. M. & Cavalli, G. Polycomb-dependent chromatin looping contributes to gene silencing during Drosophila development. Mol. Cell 71, 73–88 (2018).

    Article  CAS  PubMed  Google Scholar 

  36. Kyrchanova, O. et al. The bithorax complex iab-7 Polycomb response element has a novel role in the functioning of the Fab-7 chromatin boundary. PLoS Genet. 14, e1007442 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Espinola, S. M. et al. Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development. Nat. Genet. 53, 477–486 (2021).

    Article  CAS  PubMed  Google Scholar 

  38. Ing-Simmons, E. et al. Independence of chromatin conformation and gene regulation during Drosophila dorsoventral patterning. Nat. Genet. 53, 487–499 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Di Giammartino, D. C. et al. KLF4 is involved in the organization and regulation of pluripotency-associated three-dimensional enhancer networks. Nat. Cell Biol. 21, 1179–1190 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Fanucchi, S., Shibayama, Y., Burd, S., Weinberg, M. S. & Mhlanga, M. M. Chromosomal contact permits transcription between coregulated genes. Cell 155, 606–620 (2013).

    Article  CAS  PubMed  Google Scholar 

  41. Spilianakis, C. G. & Flavell, R. A. Long-range intrachromosomal interactions in the T helper type 2 cytokine locus. Nat. Immunol. 5, 1017–1027 (2004).

    Article  CAS  PubMed  Google Scholar 

  42. Allahyar, A. et al. Enhancer hubs and loop collisions identified from single-allele topologies. Nat. Genet. 50, 1151–1160 (2018).

    Article  CAS  PubMed  Google Scholar 

  43. Montavon, T. et al. A regulatory archipelago controls Hox genes transcription in digits. Cell 147, 1132–1145 (2011).

    Article  CAS  PubMed  Google Scholar 

  44. The Alliance of Genome Resources Consortium. Alliance of Genome Resources Portal: unified model organism research platform. Nucleic Acids Res. 48, D650–D658 (2020).

    Article  CAS  Google Scholar 

  45. Dao, L. T. M. et al. Genome-wide characterization of mammalian promoters with distal enhancer functions. Nat. Genet. 49, 1073–1081 (2017).

    Article  CAS  PubMed  Google Scholar 

  46. Diao, Y. et al. A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells. Nat. Methods 14, 629–635 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Pachano, T. et al. Orphan CpG islands amplify poised enhancer regulatory activity and determine target gene responsiveness. Nat. Genet. 53, 1036–1049 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Schroeder, M. D., Greer, C. & Gaul, U. How to make stripes: deciphering the transition from non-periodic to periodic patterns in Drosophila segmentation. Development 138, 3067–3078 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Kvon, E. Z. et al. Genome-scale functional characterization of Drosophila developmental enhancers in vivo. Nature 512, 91–95 (2014).

    Article  ADS  CAS  PubMed  Google Scholar 

  50. Wieschaus, E. & Nusslein-Volhard, C. The Heidelberg screen for pattern mutants of Drosophila: a personal account. Annu. Rev. Cell Dev. Biol. 32, 1–46 (2016).

    Article  CAS  PubMed  Google Scholar 

  51. Lim, B., Heist, T., Levine, M. & Fukaya, T. Visualization of transvection in living Drosophila embryos. Mol. Cell 70, 287–296 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Rogers, W. A., Goyal, Y., Yamaya, K., Shvartsman, S. Y. & Levine, M. S. Uncoupling neurogenic gene networks in the Drosophila embryo. Genes Dev. 31, 634–638 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ren, X. et al. Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proc. Natl Acad. Sci. USA 110, 19012–19017 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  54. Dubuis, J. O., Samanta, R. & Gregor, T. Accurate measurements of dynamics and reproducibility in small genetic networks. Mol. Syst. Biol. 9, 639 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Fukaya, T., Lim, B. & Levine, M. Rapid rates of Pol II elongation in the Drosophila embryo. Curr. Biol. 27, 1387–1391 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Abdennur, N. & Mirny, L. A. Cooler: scalable storage for Hi-C data and other genomically labeled arrays. Bioinformatics 36, 311–316 (2020).

    Article  CAS  PubMed  Google Scholar 

  58. Kerpedjiev, P. et al. HiGlass: web-based visual exploration and analysis of genome interaction maps. Genome Biol. 19, 125 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  59. Kruse, K., Hug, C. B. & Vaquerizas, J. M. FAN-C: a feature-rich framework for the analysis and visualisation of chromosome conformation capture data. Genome Biol. 21, 303 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Wood, A. M. et al. Regulation of chromatin organization and inducible gene expression by a Drosophila insulator. Mol. Cell 44, 29–38 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Larkin, A. et al. FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res. 49, D899–D907 (2021).

    Article  CAS  PubMed  Google Scholar 

  62. Bothma, J. P. et al. Enhancer additivity and non-additivity are determined by enhancer strength in the Drosophila embryo. eLife 4, e07956 (2015).

    Article  PubMed Central  Google Scholar 

  63. Harrison, M. M., Li, X. Y., Kaplan, T., Botchan, M. R. & Eisen, M. B. Zelda binding in the early Drosophila melanogaster embryo marks regions subsequently activated at the maternal-to-zygotic transition. PLoS Genet. 7, e1002266 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Van Bortle, K. et al. Insulator function and topological domain border strength scale with architectural protein occupancy. Genome Biol. 15, R82 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  65. Koenecke, N., Johnston, J., He, Q., Meier, S. & Zeitlinger, J. Drosophila poised enhancers are generated during tissue patterning with the help of repression. Genome Res. 27, 64–74 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. De, S., Mitra, A., Cheng, Y., Pfeifer, K. & Kassis, J. A. Formation of a Polycomb-domain in the absence of strong polycomb response elements. PLoS Genet. 12, e1006200 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Ray, P. et al. Combgap contributes to recruitment of Polycomb group proteins in Drosophila. Proc. Natl Acad. Sci. USA 113, 3826–3831 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  68. Soruco, M. M. et al. The CLAMP protein links the MSL complex to the X chromosome during Drosophila dosage compensation. Genes Dev. 27, 1551–1556 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Wilk, R., Hu, J., Blotsky, D. & Krause, H. M. Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs. Genes Dev. 30, 594–609 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Lecuyer, E. et al. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131, 174–187 (2007).

    Article  CAS  PubMed  Google Scholar 

  71. Couderc, J. L. et al. The bric a brac locus consists of two paralogous genes encoding BTB/POZ domain proteins and acts as a homeotic and morphogenetic regulator of imaginal development in Drosophila. Development 129, 2419–2433 (2002).

    Article  CAS  PubMed  Google Scholar 

  72. Baanannou, A. et al. Drosophila distal-less and Rotund bind a single enhancer ensuring reliable and robust bric-a-brac2 expression in distinct limb morphogenetic fields. PLoS Genet. 9, e1003581 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Kuhnlein, R. P., Bronner, G., Taubert, H. & Schuh, R. Regulation of Drosophila spalt gene expression. Mech. Dev. 66, 107–118 (1997).

    Article  CAS  PubMed  Google Scholar 

  74. Skeath, J. B., Panganiban, G., Selegue, J. & Carroll, S. B. Gene regulation in two dimensions: the proneural achaete and scute genes are controlled by combinations of axis-patterning genes through a common intergenic control region. Genes Dev. 6, 2606–2619 (1992).

    Article  CAS  PubMed  Google Scholar 

  75. Estella, C. & Mann, R. S. Non-redundant selector and growth-promoting functions of two sister genes, buttonhead and Sp1, in Drosophila leg development. PLoS Genet. 6, e1001001 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  76. Patel, M. et al. The appendage role of insect disco genes and possible implications on the evolution of the maggot larval form. Dev. Biol. 309, 56–69 (2007).

    Article  CAS  PubMed  Google Scholar 

  77. Erclik, T., Hartenstein, V., Lipshitz, H. D. & McInnes, R. R. Conserved role of the Vsx genes supports a monophyletic origin for bilaterian visual systems. Curr. Biol. 18, 1278–1287 (2008).

    Article  CAS  PubMed  Google Scholar 

  78. Svendsen, P. C., Ryu, J. R. & Brook, W. J. The expression of the T-box selector gene midline in the leg imaginal disc is controlled by both transcriptional regulation and cell lineage. Biol. Open 4, 1707–1714 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Svendsen, P. C. et al. The selector genes midline and H15 control ventral leg pattern by both inhibiting Dpp signaling and specifying ventral fate. Dev. Biol. 455, 19–31 (2019).

    Article  CAS  PubMed  Google Scholar 

  80. Perry, M. W., Boettiger, A. N. & Levine, M. Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo. Proc. Natl Acad. Sci. USA 108, 13570–13575 (2011).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  81. Schroeder, M. D. et al. Transcriptional control in the segmentation gene network of Drosophila. PLoS Biol. 2, e271 (2004).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  82. Yao, L. et al. Genome-wide identification of Grainy head targets in Drosophila reveals regulatory interactions with the POU domain transcription factor Vvl. Development 144, 3145–3155 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Batut, P., Dobin, A., Plessy, C., Carninci, P. & Gingeras, T. R. High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression. Genome Res. 23, 169–180 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Otsu, N. A threshold selection method from gray-level histograms. IEEE Trans. Syst. Man. Cybern. 9, 62–66 (1979).

    Article  Google Scholar 

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Acknowledgements

We thank all of the members of the Levine and Gregor laboratories for discussions and comments on the manuscript; E. Wieschaus for suggestions at various stages of the project; M. J. Rowley for his assistance with the SIP algorithm that was used for the automatic detection of focal contacts; E. Gatzogiannis for his help with live imaging microscopy; and B. Zoller for his contribution to the imaging analysis pipeline. This work was supported in part by the US National Science Foundation, through the Center for the Physics of Biological Function (PHY-1734030), and by National Institutes of Health Grants R01GM097275 (to T.G.), U01DA047730 (to T.G. and M.S.L.) and U01DK127429 (to T.G. and M.S.L.). The work was also supported by National Institutes of Health grant R35 GM118147 (to M.S.L.). M.L. is the recipient of a Human Frontier Science Program fellowship (LT000852/2016-L), EMBO long-term postdoctoral fellowship (ALTF 1401-2015) and the Rothschild postdoctoral fellowship.

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Authors and Affiliations

Authors

Contributions

M.L., J.R., T.G. and M.S.L. designed experiments. M.L., J.R. and P.J.B. devised imaging procedures. M.L. performed all of the experiments related to knrl/kni and J.R. performed all of the experiments related to scyl/chrb (cloning, fly generation and live imaging). M.L. performed image analysis. J.R. curated published literature for enhancer and gene expression patterns. Z.S. assisted with cloning and fly generation. S.R. assisted with cloning. M.L., J.R. and X.Y.B. fixed material for Micro-C. X.Y.B. prepared Micro-C libraries and analysed sequencing data. M.L., J.R., X.Y.B. and P.J.B. performed the genome-wide focal contact analysis. M.L., J.R., T.G. and M.S.L. wrote the manuscript. T.G. and M.S.L. secured funding and supervised the work.

Corresponding authors

Correspondence to Thomas Gregor or Michael S. Levine.

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The authors declare no competing interests.

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Nature thanks Justin Crocker and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Long range promoter-promoter connectivity is a pervasive feature of the Drosophila genome.

a, Promoter-promoter interaction distances distribution of connected genes. bg, Micro-C contact map of the inv/en (b), slp1/slp2 (c), odd/sob/drm48 (d), pyr/ths11 (e), E5/ems (f) and nub/pdm2 (g) loci. Below, aligned to the map, are auto-scaled ChIP-seq tracks for Zelda (3h embryo)63 in red, Cohesin RAD21 (Kc167 cells)64 in blue, CP190 (Kc167 cells60), CTCF (Kc167 cells)60 in green, and in orange: Pc (2-4h embryos)65, Pho (3rd instar larva)66, Ph (3rd instar larva66), Cg (3rd instar larva)67, CLAMP (Kc167 cells)68 and GAF (2–4 h embryo21). The orange tracks correspond to proteins that show binding at the anchors of promoter-proximal regions displaying high connectivity (tethering elements). A schematic representation (to scale) of the locus is displayed below, with in situ images showing the overlapping expression pattern between the paralogue genes15,16,69,70 and a reporter line of the putative shared enhancers49.

Extended Data Fig. 2 Long range promoter-promoter connectivity is a pervasive feature of the Drosophila genome (cont’d).

af, Same as Extended Data Fig. 1 for dan/danr (a), NetA/NetB (b), comm/comm2 (c), bab1/bab271,72 (d), Doc1/Doc2/Doc3 (e) and ara/caup (f) loci.

Extended Data Fig. 3 Long range promoter-promoter connectivity is a pervasive feature of the Drosophila genome (cont’d).

af, Same as Extended Data Fig. 1 for B-H2/B-H1 (a), drl/dnt (b), fd96Ca/fd96Cb (c), gcm/gcm2 (d), salr/salm73 (e) and eya (f) loci.

Extended Data Fig. 4 Long range promoter-promoter connectivity is a pervasive feature of the Drosophila genome (cont’d).

af, Same as Extended Data Fig. 1 for ac/sc74 (a), toe/eyg (b), btd/Sp175 (c), disco-r/disco76 (d), Vsx2/Vsx177 (e) and H15/mid78,79 (f) loci.

Extended Data Fig. 5 Time averaged distance measurements and co-initiation controls.

a, Shown are the X,Y,Z distances (mean ± STD, N=3, n > 1.2x104) between the MS2 and PP7 based foci in a control reporter line, with interlaced stem loops, measured in the same imaging conditions as the scyl/chrb / chrb/CG11652 data (see methods). Spot localization errors are the presented STD values of this co-localization control. bd, fh, Distribution of average/95% percentile distances across non overlapping time windows in individual nuclei (with both foci detected for at least half of the timepoints in the window). Boxplots within the violin plots, show median, edges are 25th and 75th percentiles, whiskers extend to non-outlier data points (for the comparison of any two distributions within the same panel Mann Whitney or KS tests p value << 1*\({10}^{-4}\)). b, c, Distributions of time averaged distance measurements between fluorescent foci marking transcribing genes (corresponding to the instantaneous data plotted in Fig. 2). From left to right: for a co-localization control reporter gene with interlaced MS2 and PP7 stem loops driven by the Hunchback (hb) p2promoter/enhancer, for the scyl/chrb tagged genes and for the chrb/CG11652 tagged genes. Across non-overlapping 5min time windows (b), or 25 min windows (c). d, Distribution of the 95% percentile distance across 25min windows from individual nuclei. e, Same as a but for co-localization control measured in the same imaging conditions (see methods) as the knrl/kni data (mean ± STD, N = 3, n > 1.6x104). fh, Same as bd except that knrl/kni is compared to the corresponding co-localization control. i, Instantaneous distances for a control reporter gene with interlaced MS2 and PP7, and for knrl/kni tagged genes measured in the anterior stripe domain (as in Fig. 2a), and posterior domain (regulated by enhancers proximal to kni). Boxplots within the violin plots, show median, edges are 25th, 75th percentiles, whiskers extend to non-outlier data points. j, Micro-C map encompassing the scyl/chrb/CG11652 region. Arrows mark the focal contact between scyl and chrb and the lack of such focal contact between chrb and CG11652. k, Computed frequency of co-initiation events (within 1.5 min) out of knrl initiation events, across all measured nuclei in individual embryos, for embryos where the genes are tagged in cis (purple) or in trans (blue) is shown. The pooled data from these embryos are presented in Fig. 2b. A boxplot showing the distribution of such frequencies computed by 100 random shuffling of the single-nucleus associations between green and red traces in the cis tagged embryos (see methods), is shown in grey (centre is median, edges are 25th, 75th percentiles, whiskers extend to non-outlier data points). As an additional control frequency of co-initiation events (within 1.5 min) is also computed for embryos where the kni gene is tagged with an interlaced MS2-PP7 cassette. To serve as an appropriate control, imaging was done with the same condition as the knrl/kni tagged embryos imaging (consequently green signal is slightly stronger). l, Mean transcriptional activity in the anterior stripe domain (arbitrary units) ± SEM over time in nc14 for the cis-tagged (purple, N = 7) and trans-tagged (blue, N = 6) embryos shown in k.

Extended Data Fig. 6 Detailed characterization of knrl/kni and scyl/chrb upstream regions displaying connectivity.

a, b, Micro-C contact map of knrl/kni (a) and scyl/chrb (b) loci. Below, aligned to the map, are auto-scaled ChIP-seq tracks of ATAC-seq (2–4 h embryo)10, for Zelda (3h embryo)63 in red, Cohesin RAD21 (Kc167 cells)64 in blue, CP190 (Kc167 cells), CTCF (Kc167 cells)60 in green, and in orange: Pc (2–4 h embryos)65, Pho (3rd instar larva)66, Ph (3rd instar larva)66, CLAMP (Kc167 cells)68 and GAF (2–4 h embryo)21. The orange tracks correspond to proteins that show binding at the anchors of promoter-proximal regions displaying high connectivity (‘tethering elements’). A schematic representation (to scale) of the locus, including the genes, the defined ‘tethering elements’ and putative shared enhancers is shown below. For knrl/kni an additional enhancer is illustrated (light blue) upstream of kni, this represents known enhancers driving an abdominal domain of kni transcription and to a lesser extent also knrl80. This region is also thought to encompass an enhancer contributing to an anterior cap pattern displayed by both genes81. In this study we focus on the shared anterior stripe enhancer illustrated in dark blue. Below these schematics is a zoom-in image of the focal contact in the Micro-C maps, aligned with the same set of above described ChIP-seq tracks. c, Images of live transcription measurements (mid nuclear cycle 14, nc14) of a reporter, with either the extended tethering region upstream of knrl (left) or the putative knrl/kni shared enhancer (right), placed upstream an eve-core promoter-MS2-yellow gene. See corresponding supplemental videos 3 and 4. The extended tether reporter has no pronounced transcription during the majority of nc14 (none detected up to ~55 min into nc14, and <5 nuclei showing brief transcription as the cephalic furrow is forming). In contrast, the enhancer reporter recapitulates the endogenous anterior stripe pattern of knrl/kni. At later stages of embryonic development a sequence encompassing the large majority of the tethering elements and extending (~360 bp) into the enhancer showed transcriptional activity in a reporter assay82, but such activity is not seen during nc14. d, In situ images49 of an embryo at mid nc14 from a reporter line for the putative scyl/chrb shared enhancer (left) showing expression across the dorsal midline, corresponding the domain of activity of scyl and chrb (VT29052). Reporters containing the sequences of tethering elements upstream of scyl (centre and right) show no detectable transcription (VT29054, VT29056). e, Virtual 4C contact maps computed from Micro-C data for two replicates of control lines with the viewpoint (1.8kb) anchored at the knrl promoter-proximal tethering elements on top and at the enhancer on the bottom, centre to centre shift of 2.3 kb (t-test p value comparing area under the virtual 4c curves encompassing the kni promoter-proximal region, [71 to 79 kb] for the tether view point and [68 to 76 kb] for the enhancer view point = 0.0065). f, Virtual 4C contact maps for two replicates of control lines with the viewpoint (6.4 kb) anchored at the scyl tethering elements on top and at the enhancer on the bottom, centre to centre shift of 8.8 kb (t-test p value comparing area under the virtual 4c curves encompassing the chrb promoter-proximal region, [232 to 245 kb] for the tether view point and [241 to 254 kb] for the enhancer view point = 0.0013).

Extended Data Fig. 7 Loss of long-range connectivity upon removal of tethering elements.

a, Micro-C contact map of a CRISPR-edited line, with a replacement of the knrl-proximal tethering elements (corresponding to the orange line in Fig. 3). Inset shows the control line map for comparison. Two replicates are combined for better visualization in these maps. Note loss of focal contact between the genes in the mutant. Insulation score (see methods) along the knrl/kni loci is shown below for the control line (2 replicates in purple) and the tether deletion (2 replicates orange). Note the similar insulation landscape outside of the replaced region, and specifically maintenance of TAD boundaries upstream of knrl and downstream of kni. b, Micro-C contact map of a CRISPR-edited line, with a replacement of the scyl tethering elements (corresponding to the orange line in Fig. 4). Inset shows the control line map for comparison. Two replicates are combined for better visualization in these maps. Note loss of focal contact between the genes in the mutant. Insulation score (see methods) along the scyl/chrb loci is shown below for the control line (2 replicates in purple) and the tether deletion (2 replicates orange). Note the similar insulation landscape outside of the replaced region. c, Virtual 4C contact maps computed based on Micro-C data for the control line (2 replicates in purple), a line with the knrl tethering elements replaced (2 replicates in orange), a line with an extended replacement of the knrl tethers encompassing also the adjacent CTCF (2 replicates in grey), a line with kni tethering element replaced (2 replicates in blue). The viewpoint is anchored on the kni tethering element region (see exact coordinated in methods). Interaction frequency over the knrl promoter-proximal region (encompassing the tethering elements) is significantly reduced in all mutants compared to wt (t-test p value comparing each mutant genotype to wt by the area under the virtual 4 curve between [−79 to −71 kb] = 0.0036 – 0.0041). d, Virtual 4C contact maps computed based on Micro-C data for the control line (2 replicates in purple), and a line with the scyl tethers replaced (2 replicates in orange). The viewpoint is anchored on the chrb tethering element region (see exact coordinated in methods). Interaction frequency over the scyl upstream region (encompassing the tethering elements) is significantly reduced in the mutant compared to wt (t-test p value comparing mutant genotype to wt by the area under the virtual 4c curve between [−246 to −234 kb] = 0.0004). e, Similar to c, but with viewpoint anchored on the knrl tethers. Interaction frequency over the kni promoter-proximal region (encompassing the tethering element) is reduced in all mutants compared to wt (t-test p value comparing each mutant genotype to wt by the area under the virtual 4c curve between [68 to 76 kb] = 0.0042–0.0049). Inset shows data for a slightly shifted viewpoint (2.3 kb), from the adjacent shared enhancer, as in Extended Data Fig. 6e. f, knrl and kni mean transcriptional activity (arbitrary units) ± SEM over time in nc14, in the anterior stripe domain for a control line (in purple, N = 7), a line with a replacement of the knrl tethering elements (in orange, N = 7, as in Fig. 3) and a line with an extended replacement encompassing also the adjacent CTCF (in grey, N = 4), corresponding to the micro-C data in c, e. g, kni mean transcriptional activity (arbitrary units) ± SEM over time, in nc14, in the anterior stripe domain (for flies with only kni intronic MS2 stem loops). Shown are transcriptional measurements for a line with the kni tether element replaced (in green, N = 6), with a corresponding micro-C map (matching the virtual 4c profiles in c, e) and a full embryo image. The latter shows kni transcriptional activity in the posterior domain is retained, in contrast to loss of activity in the anterior domain (see also supplemental video 7). Also shown are transcriptional measurements from a line in which in addition to the replacement of the kni tether deletion a copy of the shared enhancer was introduced upstream of kni (in blue, N = 5), recovering kni transcriptional activity in the anterior stripe domain.

Extended Data Fig. 8 Impact of manipulations of the knrl upstream region on both knrl and distal kni.

a, Schematic illustrations of CRISPR-edited fly lines; introducing stem loops to monitor real-time transcription of the co-regulated genes knrl and kni (‘control line’ in purple), with a replacement of the putative shared enhancer (in blue) or promoter-proximal tethering elements (in orange). b, knrl mean transcriptional activity in the anterior stripe domain (21–34% egg length) as shown in Fig. 3b, but with STD (instead of SEM) over time in nc14, for the lines illustrated in a (N = 7,7,6 respectively). c, Number of knrl transcriptionally active nuclei (mean ± SEM) over time in nc14, in the domain. Inset shows mean knrl transcriptional activity per active nucleus (mean ± SEM) over time from 30 min into nc14. d, kni mean transcriptional activity in the anterior stripe domain as shown in Fig. 3c, but with STD (instead of SEM) over time in nc14 (N = 6 for enhancer replacement and 7 for others). e, Distribution of the fraction of time ON per nucleus for all kni transcriptionally active nuclei, for the control (purple) and the tether replacement (orange) lines. For each nucleus ON durations from first robust onset are summed and divided by the overall duration of activity (from first onset to 60min into nc14). Boxplots within violins, show median, edges are 25th, 75th percentiles, whiskers extend to non-outlier data points. P value of two sided Mann Whitney or KS test comparing the two distributions <= 1.9*\({10}^{-17}\). f, Distribution of the number of ‘OFF-to-ON’ transitions per nucleus (normalized to a 30min period, see methods) on the same nuclei as in e, for the control line (purple) and the tether replacement (orange). Boxplots within violins, show median, edges are 25th, 75th percentiles, whiskers extend to non-outlier data points. P value of two sided Mann Whitney or KS test comparing the two distributions <= 8.3*\({10}^{-27}\). g, Distribution of OFF durations pooled from all kni active nuclei of the control and the tether replacement. Inset shows the cumulative distribution of OFF durations on all pooled nuclei (line) and on individual embryos (mean ± SEM, N = 7). Complementary to ON durations distributions in Fig. 3j. h, For the lines illustrates in a, the area under the curve of kni mean transcriptional activity (the data used in Fig. 3c, d) is shown for individual embryos. Mann Whitney p value for all comparison <= 0.0012. i, Same as h, but shown is the averaged activity in a window of maximal activity, between 38–46 min into nc14. Mann Whitney p value for all comparison <= 0.0012. j, Same as h but for the number of active nuclei. Mann Whitney p value for all comparison <= 0.0012. k, Same as i but for the number of active nuclei. Mann Whitney p value for all comparison <= 0.0012. l, Schematic illustrations of CRISPR-edited fly lines; the control line with both knrl and kni tagged (in purple), a partial tether replacement, encompassing the knrl downstream tether (dark green), a partial tether replacement, encompassing the knrl upstream tether and the adjacent CTCF site (in yellow) and a line with the knrl transcription start site (TSS) region (170 bp encompassing knrl TSS83) deleted (in black). m, knrl mean transcriptional activity (arbitrary units) ± SEM, in the anterior stripe domain over time in nc14, for the lines illustrated in l (N = 7,7,5,4 respectively). n, Same as m but for kni. o, Viability score (see methods) for the line with knrl TSS region deleted, a line with a replacement encompassing the upstream region of knrl and extending into the gene, a line with this same replacement but with the enhancer repositioned upstream of kni, crossed to a deficiency allele lacking the entire knrl/kni locus. Shown is mean ± STD across (N = 4,3,5) independent crosses, each with >90 progeny scored. p, Viability score for a wt allele crossed to a deficiency allele lacking the entire knrl/kni locus, and a line with the replacement of knrl upstream region, extending into the gene, on one allele and a replacement of the kni upstream and gene region on the other allele. Shown is mean ± STD across N = 4,6 independent crosses, each with >90 progeny scored.

Extended Data Fig. 9 Impact of manipulations of scyl upstream region on both scyl and distal chrb.

a, Schematic illustrations of CRISPR-edited fly lines; introducing stem loops to monitor real-time transcription of the co-regulated genes scyl and chrb (‘control line’ in purple), with a partial (upstream tether) replacement of the tethering elements (in green) or a full replacement of the tethering elements (in orange). b, scyl mean transcriptional activity in the dorsal midline (see Fig. 4b), but with STD (instead of SEM) over time in nc14 (N = 5 embryos), for the lines illustrated in a. c, Number of scyl transcriptionally active nuclei (mean ± SEM) over time in nc14, in the dorsal midline domain. Inset shows mean scyl transcriptional activity per active nucleus (mean ± SEM) over time from 30 min into nc14. d, chrb mean transcriptional activity in the domain (see Fig. 4c), but with STD (instead of SEM), over time in nc14 (N = 5 embryos). e, Distribution of the fraction of time ON per nucleus for all chrb transcriptionally active nuclei, for the control (purple) and, upstream tether replacement (green) and tethers replacement (orange) lines. For each nucleus ON durations from first robust onset are summed and divided by the overall duration of activity (from first onset to 60min into nc14). Boxplots within violins, show median, edges are 25th, 75th percentiles, whiskers extend to non-outlier data points. P value of two sided Mann Whitney or KS test comparing the control to the tethers replacements <= 2.7*\({10}^{-11}\), for upstream tether replacements vs tethers replacements < 0.061. f, For the lines illustrates in a, the area under the curve of chrb mean transcriptional activity (the data used in Fig. 4c, d) is shown for individual embryos. Mann Whitney p value for control vs replacements lines = 0.0079, for upstream tether replacements vs tethers replacements = 0.056. g, Same as f, but shown is the averaged activity in a window of maximal activity, between 50-58 min into nc14. Mann Whitney p value for all comparison = 0.0079. h, Same as f but for the number of active nuclei. Mann Whitney p value for control vs replacements lines = 0.0079, for upstream tether replacements vs tethers replacements = 0.056. k, Same as g but for the number of active nuclei. Mann Whitney p value for all comparison = 0.0079. j, Schematic illustrations of CRISPR-edited fly lines; the control line with both scyl and chrb tagged (in purple), a line with the downstream tether replaced (in black) and a line with the CTCF site replaced (in light green). k, scyl mean transcriptional activity (arbitrary units) ± SEM, in the midline dorsal band, over time in nc14 (N = 4–5 embryos), for the lines illustrated in j. l, Same as k, but for chrb.

Supplementary information

Supplementary Tables 1–5.

Reporting Summary

Supplementary Video 1

Simultaneous live imaging of knrl and kni transcription. Simultaneous live imaging of knrl-PP7 (red) and kni-MS2 (green) transcription on a laterally oriented embryo during nuclear cycle 14, as in Fig. 1g (see the Methods for the image settings).

Supplementary Video 2

Simultaneous live imaging of scyl and chrb transcription. Simultaneous live imaging of scyl-MS2 (green) and chrb-PP7 (red) transcription on a dorsally oriented embryo during nuclear cycle 14, as in Fig. 1h.

Supplementary Video 3

Live transcription imaging of a reporter gene with the knrl/kni shared enhancer. Live imaging of a reporter gene (green) transcription with the knrl/kni shared enhancer placed upstream of the eve-core promoter-MS2-yellow gene in the anterior region of a laterally oriented embryo during nuclear cycle 14, as in Extended Data Fig. 6c (right).

Supplementary Video 4

Live transcription imaging of a reporter gene with the knrl tethering element. Live imaging of a reporter (green) transcription of the extended tethering region of knrl upstream of the eve-core promoter-MS2-yellow gene in the anterior region of a laterally oriented embryo during nuclear cycle 14, as in Extended Data Fig. 6c (left).

Supplementary Video 5

Simultaneous live imaging of knrl and kni transcription in the anterior stripe region. Simultaneous live imaging of knrl-PP7 (red) and kni-MS2 (green) transcription in the anterior stripe domain (encompassing 21–34% egg length) of a laterally oriented embryo during nuclear cycle 14, as in Fig. 1g.

Supplementary Video 6

Simultaneous live imaging of scyl and chrb transcription in the midline dorsal band. Simultaneous live imaging of scyl-MS2 (green) and chrb-PP7 (red) transcription in the midline dorsal band (encompassing 40–60% egg length) of a dorsally oriented embryo during nuclear cycle 14, as in Fig. 1h.

Supplementary Video 7

Live imaging of kni transcription with the kni tethering element replaced. Live imaging of kni-MS2 (green) transcription of a line with a replacement of the kni tethering element, in a laterally oriented embryo during nuclear cycle 14.

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Levo, M., Raimundo, J., Bing, X.Y. et al. Transcriptional coupling of distant regulatory genes in living embryos. Nature 605, 754–760 (2022). https://doi.org/10.1038/s41586-022-04680-7

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