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Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development

Abstract

Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (CRMs), for example, enhancers and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly within multicellular developing organisms. In the present study, we employ Hi-M, a single-cell spatial genomics approach, to detect CRM–promoter looping interactions within topologically associating domains (TADs) during early Drosophila development. By comparing cis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses reveal that multiple CRMs spatially coalesce to form hubs. Loops and CRM hubs are established early during development, before the emergence of TADs. Moreover, CRM hubs are formed, in part, via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach provides insight into the role of CRM–promoter interactions in defining transcriptional states, as well as distinct cell types.

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Fig. 1: Hi-M reveals widespread cis-regulatory chromatin loops and hubs within TADs.
Fig. 2: CRM–CRM and CRM–P loop frequencies are similar between cell types.
Fig. 3: CRM loops and hubs precede TAD formation and gene expression.
Fig. 4: Formation of CRM loops and hubs in the doc-TAD requires the pioneer factor Zld.

Data availability

The Oligopaint public database (http://genetics.med.harvard.edu) was used to select Oligopaints. Publicly available datasets used in the present study (accession nos. GSE86966, GSE25180, E-MTAB-4918, GSM763062, GSE58935, GSE16245, GSE68983, GSE68654, E-MTAB-1673, GSE62904 and GSE65441) are detailed in Supplementary Table 9. Data for matrices in Figs. 14 and in Extended Data Figs. are publicly available at https://github.com/NollmannLab/Espinola-Goetz-2021. Source data are provided with this paper.

Code availability

Code used in this manuscript is available at https://github.com/NollmannLab/Espinola-Goetz-2021.

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Acknowledgements

We thank N. Benabdallah, G. Cavalli, T. Forne, T. Robert, J. Bonnet and members of the Lagha and Nollmann laboratories for their critical reading of the manuscript. We thank A. Makrini and D. Cattoni for help with bioinformatic analysis. This project was funded by an ERC Consolidator Grant from the European Union’s Horizon 2020 Research and Innovation Program (grant no. 724429 to M.N.). We thank the Bettencourt–Schueller Foundation for their prize Coup d’élan pour la recherche Française, the France-BioImaging infrastructure supported by the French National Research Agency (grant no. ANR-10-INBS-04, Investments for the Future), the Labex EpiGenMed (ANR-10-LABX-12-01) and the Drosophila facility (BioCampus Montpellier, CNRS, INSERM, Université de Montpellier, France). M.G. was funded by the Deutsche Forschungsgemeinschaft (German Research Foundation; project no. 431471305). M.L.’s laboratory is supported by an ERC Starting Grant (SyncDev, grant no. 679792) and CNRS. M.B. and O.M. are supported by an FRM PhD fellowship.

Author information

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Authors

Contributions

A.M.C.G., M.L. and M.N. conceived the study and the design. S.M.E., C.H., M.B. and I.R. acquired the data. M.G., S.M.E., M.B., O.M., I.R. and M.N. analyzed the data. M.G., M.N., J.B.F. and O.M. provided the software. S.M.E., M.G., O.M., I.R., M.B., M.N. and M.L. interpreted the data. M.L. and M.N. wrote the manuscript. J.-B.F., C.H. and I.R. provided the reagents. S.M.E., M.G. and M.N. did the visualization of the study. M.N. and M.L. supervised the study. M.N. and M.L. acquired funds.

Corresponding authors

Correspondence to Mounia Lagha or Marcelo Nollmann.

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The authors declare no competing interests.

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

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Hi-M allows high-resolution chromatin tracing in the doc-TAD.

a, Schematic representation of the genomic positions of barcodes for the low (green) and high (red) resolution doc Hi-M libraries. Triangles demarcate the two TADs registered in this genomic region90. b. Chip-seq profiles for architectural proteins in the doc-TAD54,90. Cis-regulatory modules (CRMa-d) from Fig. 1c are highlighted by blue bars. c. Typical maximum intensity projection displaying the fluorescence emission signal from a single barcode in a section of an embryo (outline in red). Emissions from individual barcodes appear as diffraction-limited spots. d. Map of pairwise distance distributions for all barcode combinations. The order of the distributions follow that in the Hi-M matrix (Fig. 1f). Blue shade represents a kernel density estimation with a bandwidth of 0.2 μm, red line represents the maximum of the distribution, and black vertical lines on the x-axis represent individual data points. e. Efficiency of barcode detection and distribution of number of barcodes detected per cell. f. To verify that uneven barcode efficiencies did not affect our results, we plotted the pairwise distance distributions for the full dataset (right) and half the data (left, here cells were randomly chosen). Map of pairwise distributions is centered at the barcode bin (4,13). g. Hi-M contact probability map (left) and inverse pairwise distance map (right) for the same experiment (doc-TAD, all cells). N = 37129, n = 29. h, Pearson correlation coefficient of the contact probability of the full doc-TAD Hi-M dataset (nc14, dorsal cells displaying doc1 expression) against subsets with a fraction of cells. One hundred random subsets were generated for each tested subset size. The central bar indicates the mean and the error bars indicate the extreme values of the distribution. i. Distribution of radii of gyration for the doc-TAD calculated from single cells. Blue shade represents a kernel density estimation with a bandwidth of 0.1 μm, black vertical lines on the x-axis represent individual data points. Dashed line represents the maximum of the distribution. The size of the doc-TAD, as estimated from its radius of gyration (0.27 ± 0.1 μm), was comparable with that of TADs of similar genomic sizes96.

Extended Data Fig. 2 doc genes are highly co-expressed and doc CRMs spatially cluster, as do CRMs in the sna locus.

a, RNA-FISH staining for doc1, doc2 and doc3 in late nc14 embryos. Scale bars: 50 𝜇m/ 5 𝜇m (inset). b, Percentage of one or two active transcription sites/nuclei for doc1 and doc2. c. Percentage of cells displaying active transcription spots from 2-color RNA-FISH imaging of doc1-doc2 and doc2-doc3. Most nuclei (>70%) displayed co-activation of doc1 + doc2 and of doc2 + doc3. For this latter, a larger percentage of nuclei expressed only doc2 (~40%), because of the low efficiency of labeling of doc3 nascent mRNA (small intronic size). d, Comparison of contact maps from nuclei displaying at least one active doc1 RNA-FISH spot (top right matrix) and from a subset (33%) of nuclei displaying the strongest doc1 RNA-FISH signals. e, 4 M profiles derived from Hi-M maps of dorsal ectoderm cells in nc14. f, 4 M virtual profile for nuclei displaying at least one active doc1 RNA-FISH spot (solid pink line) and from a subset of nuclei (33%) with the highest doc1 signals (dashed dark pink). g, Epigenetic profile of selected regions around the esg and sna genes within the sna-TAD. Accessibility (ATAC-seq), pioneer factor binding (Zelda), transcriptional activity (RNA-seq), chromatin marks for active enhancers marks (H3K4me1), and for the transcriptional activators Dorsal, Zen and Mad are shown. A subset of barcodes were annotated as cis-regulatory modules (shown in cyan): CRM169 harbors the canonical H3K4me1 active enhancer mark; CRM160, and shadow sna enhancer were described in the RedFly database. Magenta barcode harbours the esg promoter and the blue barcode contains the sna promoter and its primary enhancer. See Supplementary Table 1 for more details. h, Hi-M contact probability map of the sna locus. Yellow arrow shows interactions between CRMs, red arrow between CRMs and promoters, and green arrow between promoters. i, Multi-way interactions between promoters (panels (i-iii) and CRMs (panels iv-vi). Number of nuclei and embryos examined as in panel r. j, 3D topological representation of the sna-TAD. Bead colors are as in panel d. Barcode 44 contains the wor promoter.

Extended Data Fig. 3 doc CRM loops are similar between three presumptive tissues.

a, Comparison of 4 M profiles between DE (magenta) and NE (orange) for different anchors within the doc-TAD (Panels i-iii: promoters. Panel iv: control. Panels v-viii: CRMs). Anchors are indicated by vertical purple lines. Peaks in the profiles are annotated with the corresponding CRMs (a-d) b, Comparison of 4 M profiles between DE (magenta) and M (green) for different anchors within the doc-TAD (Panels i-iii: promoters. Panel iv: control. Panels v-viii: CRMs). Anchors are indicated by vertical purple lines. Peaks in the profiles are annotated with the corresponding CRMs (a-d). Right panel: scheme indicating the three presumptive tissues.

Extended Data Fig. 4 sna CRM loops are similar between three presumptive tissues.

a, Panel i: Hi-M contact probability map of the sna locus for M (upper-right map) versus DE (lower-left map). Inset show a magnification of the region around sna. Panel ii: Same but for the difference between M and DE Hi-M maps. Blue indicates larger contact probabilities in M whereas red indicates larger contact probabilities in DE. Panel iii: Similar to panel i, but for M (upper-right map) versus NE (lower-left map). Panel iv: Similar to panel ii, but for M versus NE. N: number of nuclei. n: number of embryos. b, Comparison of 4 M profiles between M (green) and DE/NE(orange). Anchors within the sna-TAD are indicated in each panel by vertical purple lines. A subset of peaks is annotated using the nomenclature from Fig. 1d. c, Comparisons of multi-way maps for M (upper-right map) versus DE (lower-left map) in the sna locus using the anchors indicated in each panel by pictograms and dark blue crosses. Maps are color-coded according to the scale bar on the right. Number of embryos and nuclei as in panel c. d, Similar to panel e, but for M (upper-right map) versus NE (lower-left map). Number of embryos and nuclei as in panel c.

Extended Data Fig. 5 doc CRM loops are established early in development.

a, Comparison of 4 M profiles between embryos in nc14 (blue lines) and nc11 (orange) for different anchors within the doc-TAD (Panels i-iii: promoters. Panel iv: control. Panels v-viii: CRMs). The position of the anchor is indicated by a vertical purple line. Peaks in the profiles are annotated with the corresponding CRMs (a-d). b, Similar to panel A, but comparing 4 M profiles between embryos in nc14 (blue lines) and nc12 (orange). c, Similar to panel A, but comparing 4 M profiles between embryos in nc14 (blue lines) and nc13 (orange). d, Comparison of multi-way interaction matrices of nc14 (upper-right map) and nc11 (lower-left map). Anchors (dark blue crosses) are as follows: doc3, doc3, doc1 promoters (panels i-iii), control region (panel iv), CRMa-d (panels v to viii). Representative image of DAPI-stained nuclei for nc11 is shown on top. Barcodes are shown on the left and bottom of multi-way maps. Number of nuclei (nc11): N = 1320, number of embryos (nc11); n = 4. Number of nuclei (nc14): N = 37129, number of embryos (nc14); n = 29. e, Similar to panel d, but for nc14 (upper-right map) and nc12 (lower-left map). Representative image of DAPI-stained nuclei for nc12 is shown on top. Number of nuclei (nc12): N = 2154, number of embryos (nc12); n = 4. f, Similar to panel d, but for nc14 (upper-right map) and nc13 (lower-left map). Representative image of DAPI-stained nuclei for nc13 is shown on top. Number of nuclei (nc13): N = 7597, number of embryos (nc13); n = 8.

Extended Data Fig. 6 sna CRM loops are established early in development.

a, Comparison of Hi-M contact probability maps in the sna locus for nc14 (upper-right map) and nc11 (panel i), nc12 (panel ii), nc13 (panel iii) and 14 (panel iv) (lower-left maps). Maps are color-coded according to the scale bar on the right. Inset on the bottom of each map shows a magnification of the region around esg and sna CRMs (see Extended Data Fig. 2 and Supplementary Table 1). b, Comparison of multi-way contact maps between nc14 (upper-right maps) and nc11 (lower-left maps). Maps are color-coded according to the scale bar on the right. The position of anchors are indicated by dark blue crosses. White boxes indicate contacts already present at nc11 that persist through nc14. Number of nuclei and embryos examined as indicated in panel a. c, Similar to panel b, but comparing nc14 to nc12. Green boxes indicate contacts that emerge at nc12 and persist at nc14. Number of nuclei and embryos examined as indicated in panel a. d, Similar to panel b, but comparing nc14 to nc13. Yellow boxes indicate interactions that appear at nc13 (at the TAD border). Number of nuclei and embryos examined as indicated in panel a.

Extended Data Fig. 7 Perturbation of gene expression and CRM loops by enhancer deletion and Zld depletion.

a. Scheme of the wild type doc locus (+/+) and the doc locus after CRISPR/Cas9 genome editing (ΔCRMc/ΔCRMc). doc enhancer/CRMc, FRT sequence and primers used for genotyping are in teal, yellow and blue, respectively. Genotyping PCR products on agarose gel electrophoresis are shown in the lower panel. Orange and green stars correspond to the bands of the expected sizes after amplification using primers flanking the doc enhancer/CRMc sequence. See Methods for further details. b, RNA-FISH imaging of doc1 and doc2 in the CRMc-deletion mutant. Scale bars: 50 𝜇m/ 5 𝜇m (inset). c, RNA-FISH imaging of doc1, doc2 and sna in control (RNAi white) and RNAi Zld embryos. Black arrows show the doc1 and doc2 expression patterns in the anterior part of the embryo. Grey arrows indicate the absence (doc1, doc2) or perturbation (sna) of gene expression patterns in RNAi-Zld embryos. Scale bar: 50 𝜇m. d, Tracks for pioneer factor binding (Zelda) and RNA Pol2 binding in the doc-TAD. See Supplementary Table 1 for assignment of CRMb-d. e, Transcription levels (RNAseq) of doc1, doc2 and doc3 in wild-type versus zld- embryos69. f, Hi-C matrix for a genomic region containing doc-TAD in wild-type and Zld-depleted embryos. Data from Hug et al. (2017)40. g, Distribution of radius of gyration for the doc-TAD in Zld-depleted embryos (see Extended Fig. 1i for wild-type). h-j, Log2(observed/expected) average contact frequencies between Zelda bound regions at long-range distances (> 250 kb) ranked by increasing Zelda enrichment in nc14 (panel g), nc14 zld-RNAi (panel h) and at short-range ( 250 kb) in nc14 triptolide-treated embryos (panel i). k, Violin plot of intragroup Log2(observed/expected) distribution between 62 selected pre-MBT enhancers and neighbouring sequences (± 5 kb) in nc14 (upper panel) and nc14 zld-RNAi (lower panel). The central white marker indicates the median and the vertical black lines indicate the extreme values of the distribution. The coordinates of enhancers and closest pre-MBT genes are listed in Supplementary Table 8. Source data

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Espinola, S.M., Götz, M., Bellec, 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). https://doi.org/10.1038/s41588-021-00816-z

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