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Enhancer loops appear stable during development and are associated with paused polymerase

Nature volume 512, pages 96–100 (2014)Cite this article

Subjects

A Corrigendum to this article was published on 13 July 2016

Abstract

Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer–promoter interactions and their dynamics during embryogenesis remain unclear. At the β-globin locus, enhancer–promoter interactions appear dynamic and cell-type specific1,2, whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed3,4. The extent to which preformed enhancer–promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. Here we generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. Our results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer–promoter loops through release of paused polymerase.

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Figure 1: A high-resolution view of enhancer interactions during Drosophila embryogenesis.
Figure 2: Long-range interactions are widespread in the Drosophila genome.
Figure 3: Specific loci display localized differential interactions.
Figure 4: Interactions are stable across developmental time and associated with paused polymerase.

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Accession codes

Primary accessions

ArrayExpress

European Nucleotide Archive

Data deposits

All raw data, which consists of 2,587 demultiplexed files, have been submitted to the EBI European Nucleotide Archive and ArrayExpress databases, accession numbers ERP004524 and E-MTAB-2180, respectively. To enable the community to browse through the data, 4C-seq interaction data is available in a customized web browser at http://furlonglab.embl.de/4CBrowser.

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Acknowledgements

This work was technically supported by the EMBL Genomics Core and FACS core facilities. We thank all members of the Furlong laboratory for discussions and comments, in particular D. Garfield, J. Reddington and I. Schor for important suggestions. Embryo images in Fig. 3c were used with permission from Development27 and Nature Publishing Group26 as these fly strains no longer exist. This work was supported by a DFG (FU 750) grant to E.E.M.F., an EMBO post-doctoral fellowship to Y.G.-H., and the EC FP7 project ‘Radiant’ grant to F.A.K. and W.H.

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Author notes

  1. Felix A. Klein and Tibor Pakozdi: These authors contributed equally to this work.

Authors and Affiliations

  1. European Molecular Biology Laboratory, Genome Biology Unit, D-69117 Heidelberg, Germany,

    Yad Ghavi-Helm, Felix A. Klein, Tibor Pakozdi, Lucia Ciglar, Wolfgang Huber & Eileen E. M. Furlong

  2. Swiss Federal Institute of Technology, School of Life Sciences, CH-1015 Lausanne, Switzerland,

    Daan Noordermeer

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Contributions

Y.G.-H. and E.E.M.F. designed the study, analysed the results and wrote the manuscript. Y.G.-H. performed 4C experiments, DNA in situ hybridization and imaging and performed data analysis. F.A.K., T.P. and W.H. developed and performed 4C-seq bioinformatics analysis. L.C. generated all transgenic strains and performed 4C-PCR reactions, RNA in situ hybridizations and imaging. D.N. was involved in 4C primer design. T.P. and F.A.K. contributed equally to the study. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Eileen E. M. Furlong.

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Extended data figures and tables

Extended Data Figure 1 4C-seq in Drosophila embryos.

a, Example of classification into distal and promoter-proximal enhancer viewpoints. Distal enhancers are located >1 kb away from the closest TSS and devoid of H3K4me3 chromatin signature (green), a mark characteristic of gene promoters, while promoter-proximal enhancers are located within 1 kb of the closest TSS. WE, whole embryo. b, Outline of BiTS-4C-seq: genetically modified embryos expressing a tagged nuclear protein exclusively in the mesoderm were collected, aged and crosslinked. Fixed nuclei were extracted and digested with DpnII (whole embryo analysis) or sorted by FACS before DpnII digestion (tissue-specific analysis). After 4C template preparation, ligation junctions were amplified and sequenced. Count values for each DpnII fragment were transformed and fitted using a monotone fit (red line) facilitating an analysis of significant interactions (blue arrowheads). In addition to the 103 viewpoints used at both time-points, four additional viewpoints were used for reciprocal 4C experiments in whole embryos at 6–8 h. c, e, g–l, 4C interaction maps (viewpoint, red arrowhead) at the E2f (c), pdm2 (ref. 51) (e), Con (g), eya (h), stumps (i), Mef2 (j), sli (k) and slp1 (l) loci. The expected interaction with the promoter (blue arrowhead) of the genes is observed. Known enhancers are indicated. MESO, mesoderm (generated by FACS sorting). d, f, Expression (double in situ hybridization) of the E2f (d) or pdm2 (f) genes (red) and the expression driven by their interacting enhancer (green) at stage 11.

Extended Data Figure 2 Enhancers have a complex interaction network with multiple active enhancers and promoters.

a, Histogram showing the frequency of viewpoint interactions, averaged over all conditions per viewpoint (n = 107). b, Frequency of 4C unique interacting regions that overlap enhancers or promoters when using distal enhancers (left) or promoter-proximal elements (right) as viewpoints (defined in Extended Data Fig. 1a). Whole-embryo RNA-seq9 was used to define active and inactive promoters. Enrichments over a background set of interactions (Fisher’s Exact Test) are indicated. The background set (or expectation regions) was based on random sampling of all DpnII fragments throughout the genome that have matched mappability, G+C content and size to the observed interactions. c–e, Mesoderm-specific chromatin signatures6 at whole embryo 6–8 h 4C interacting regions. c, H3K27ac, H3K4me3, H3K79me3 and Pol II signals6 are enriched at promoters interacting with promoter-proximal viewpoints. Note, we only consider interactions between promoter-proximal viewpoints and distal promoters (>1 kb away; Extended Data Fig. 1a). d, H3K27ac, H3K4me1, H3K79me3 and Pol II signals6 are enriched at enhancers interacting with promoter-proximal viewpoints. e, H3K27ac, H3K4me3, H3K79me3 and Pol II signals6 are enriched at promoters interacting with distal enhancer viewpoints. Shading indicates 95% confidence intervals estimated by non-parametric bootstrapping, grey lines indicate background signal.

Extended Data Figure 3 4C interactions at insulator protein-bound and unbound regions.

a, Histogram showing the observed and expected percentage of 4C interacting regions (shown for whole embryo 6–8 h) that overlap with insulator-binding protein occupancy17 (6 factors, including N- and C-term CTCF). A significantly higher percentage of interactions overlap the occupancy of at least 4 insulator-binding proteins (Fisher’s Exact Test). b, Percentage of genomic regions occupied by insulator-binding proteins (outer circle), insulator-bound 4C-interacting regions (middle circle) and non-insulator-bound 4C-interacting regions (inner circle) that overlap enhancers or promoters. c, Histogram representing the percentage of insulator-bound interacting regions for all combinations of insulator proteins that overlap with at least 20 interacting regions. d, Log2 odds ratio for an interacting region bound by a given combination of insulator proteins to be more enriched on enhancers or promoters than non-insulator bound interacting regions (Fisher’s Exact Test). Red asterisks in d: *FDR <10%; **FDR <0.1%. e, Box plot showing the genomic distance to the viewpoint of non-insulator-bound and insulator-bound interacting regions (P value from a two-sided Wilcoxon test). f, Box plot showing the interaction strength (log2 fold change of the 4C signal) on non-insulator-bound and insulator-bound 4C interacting regions (P value from a two-sided Wilcoxon test). g, Histogram showing, for each of the 107 viewpoints (103 plus 4 reciprocal viewpoints), the percentage of 4C interacting regions (blue) overlapping with the binding of insulator proteins17.

Extended Data Figure 4 Chromatin 3D interactions at the pdm2 and en loci.

a, c, Reciprocal 4C interaction map (viewpoint, red arrowhead) around the nub and pdm2 (a) or en and inv (c) loci. Top 2 lanes: 4C interactions with a region surrounding the pdm2 or nub (a) and en or inv (c) genes (blue arrowhead). Top lane: 4C using the nub (a) or inv (c) promoter as the viewpoint; bottom lane: using a promoter-proximal region in the vicinity of the pmd2 (a) or en (c) promoter as the viewpoint. Significant 4C interactions and known enhancers are indicated. The pdm2 CE8012 enhancer is indicated. WE, whole embryo. b, d, Expression (double in-situ hybridization) of the pdm2 or inv (green) and nub or en (red) at stage 11, the same stage shown for the 4C interaction in a and c (6–8 h).

Extended Data Figure 5 Long-range interactions in Drosophila.

a, Zoom-in of the 4C-seq transformed and fitted counts around the apterous (ap) viewpoint (mesoderm 6–8 h). The first fragments around the viewpoint are not included in the monotone fit (red line). b, Frequency of the first identified interaction (green) and the first valid fragment (red) as a function of distance to their respective viewpoints. c, 4C interaction map of the unc-5 and sli loci52,53. An interaction (blue arrowhead) between the viewpoint (red arrowhead) and the promoter of sli, over half a megabase away, is observed. Inset shows a zoomed-in view of the sli promoter. The location of significant 4C interactions and known enhancers is represented below. WE, whole embryo. d, Double in situ hybridization showing the overlap between sli (green) and unc-5 (red) heart expression (arrowhead) at stage 14. e, 4C interaction map at the scyl and chrb loci23. Independent of the location of the viewpoint (red arrowhead), the same interacting regions are recovered (blue arrowheads). The location of known enhancers is represented below. The two cloned regions are indicated as ‘new enhancer’ 1 and 2, respectively. WE, whole embryo. f, A non-parametric two-sample Kolmogorov–Smirnov test was used to assess the significance of differences between DNA FISH distance distributions. g, Double in situ hybridization of the chrb gene (red) and the expression driven by two of its interacting regions (green) at stage 11 and 14, showing that both regions recapitulate part of the expression of chrb. The overlap (shown by a white arrowhead) is located in the trunk visceral mesoderm and later in the longitudinal visceral mesoderm for enhancer 1 and in the central nervous system for enhancer 2. New enhancer 1 is overlapping a ChIP-defined CRM (CRM4311), whereas new enhancer 2 is partially overlapping (<10%) an enhancer in the sgs3 locus. Neither region was previously identified as charybde enhancers, and their spatio-temporal activity during development was unknown.

Extended Data Figure 6 Location of 4C interactions with respect to topological domains and block of microsynteny.

a, 900-kb region around the scyl and chrb loci. Top to bottom: regions of conserved synteny (grey)24; location of previously identified Hi-C interactions (light blue)15; 4C interaction map around the scyl and chrb loci (viewpoint location, red arrowhead); location of significant 4C interactions and known enhancers. The strong interaction with the scyl gene is highlighted (blue arrowhead). WE, whole embryo. b, Percentage of 4C interactions, averaged over all conditions per viewpoint, located in the same topological domain (TAD) as the viewpoint, or in adjacent TADs, if the viewpoint is on the edge of the domain (top) or in the middle (bottom). TADs were defined based on HiC data at 16–18 h of development15. Significant difference from the background (asterisk) and corresponding P values using a two-sided Mann–Whitney U-test are indicated. c, Percentage of interactions located in the same block of microsynteny as the viewpoint, or in adjacent blocks, if the viewpoint is on the edges of the block (top) or in the middle (bottom). Microsynteny was previously defined by Engström et al.24 based on 5 species spanning ∼50 million years of evolution. Significant differences between the observed and expected interactions (asterisk) were assessed using a two-sided Mann–Whitney U-test.

Extended Data Figure 7 Hierarchical clustering of 4C interactions.

a, b, Based on the 92 viewpoints used for 4C in all three conditions (whole embryo 3–4 h, whole embryo 6–8 h and mesoderm 6–8 h). Hierarchical clustering of the quantitative 4C signal of all interactions (n = 1,389) (a) and the subset of interactions with promoters (n = 396) (b), across 3 conditions (whole embryo 3–4 h, whole embryo 6–8 h and mesoderm 6–8 h). The expression level of the corresponding gene is shown on the right hand side in b, using RNA-seq data from Graveley et al.9. Clustering was performed by Euclidean distance and Ward agglomeration method on the significant interaction regions defined at whole embryo 6–8 h using DESeq2 fit reference-normalized read counts. If the interacting region was associated with more than one promoter, expression for only one transcript is shown. Red horizontal bars indicate differential interactions. c, d, Hierarchical clustering of the quantitative 4C signal for all 140 differential interactions (c) and the subset of differential interactions at promoters (n = 39) with the expression level of the corresponding gene (d) across 3 conditions (whole embryo 3–4 h, whole embryo 6–8 h and mesoderm 6–8 h). Clustering was performed using Euclidean distance and Ward agglomeration method on the significant interaction regions defined at whole embryo 6–8 h. WE, whole embryo; MESO, mesoderm.

Extended Data Figure 8 4C interactions under different developmental conditions.

a, MA plot of interaction signal between whole embryo 6–8 h and mesoderm 6–8 h. b, 4C interaction map at the Antp locus. Top to bottom: H3K4me3 ChIP-seq signal (RPGC) in whole embryo at 0–4 h and 4–8 h (ref. 49) (green), RNA-seq signal (RPKM, black) in whole embryo at 2–4 h and 6–8 h (ref. 9), 4C interaction map (viewpoint, red arrowhead) in whole embryo at 3–4 h (mauve) and 6–8 h (blue). The differential 4C signal is plotted in between in red with significant differential 4C interactions indicated (asterisk). WE, whole embryo. c, Expression (in situ hybridization) of Antp (red) and the expression driven by a new Antp enhancer (green) at stage 11 (6–8 h). The enhancer’s activity overlaps expression of Antp at 6–8 h; however, the 4C contact between the enhancer and promoter is already present, and at even higher levels, at 3–4 h. d, f, Interaction map at the pdm2 (d) and E2f (f) loci. Top to bottom: Pol II signal (RPGC) in mesoderm at 6–8 h (orange)6, RNA-seq signal (RPKM) in mesoderm and whole embryo at 6–8 h (black)9,54, 4C interaction map (viewpoint location, red arrowhead) in mesoderm (light blue) and whole embryo (dark blue) at 6–8 h. The differential 4C signal is plotted in between in red. Note that the 4C interaction is stronger in mesoderm compared to whole embryo, although those genes are not expressed in the mesoderm. Significant 4C interactions and known enhancers are indicated. WE, whole embryo, MESO, mesoderm (generated by FACS sorting). e, g, Expression (double in situ hybridization) of the pdm2 (e) or E2f (g) genes (green) with a mesoderm-specific marker (mef2, red) at stage 11.

Extended Data Figure 9 Characterization of DS genes.

a, Defining off-to-on genes. Relative frequency of genes exhibiting a given RPKM value from RNA-seq in whole embryo at 2–4 h and 6–8 h (ref. 9). The threshold (vertical lines) between non-active (off) and active genes was selected based on the local minima in the log-RPKM distribution, as described previously44. DESeq was used to determine if non-active 2–4 h genes had a significant change in their expression at 6–8 h. b, DS-paused genes are expressed at very low levels at 2–4 h, or not at all. Log2 gene expression signal (RPKM) in whole embryo at 2–4 h and 6–8 h (ref. 9) of different categories of genes. Paused DS genes (15 genes, using the stringent criteria for pausing) are significantly less expressed than the top 25% of paused genes (1,776 genes)28 at 2–4 h and are also significantly less expressed at 2–4 h compared to 6–8 h (Mann–Whitney U-test). c, In situ hybridization showing available expression data for DS genes at stage 4–6 (2–4 h) and 11–12 (Berkeley Drosophila Genome Project55). Note, for all 8 genes there is no detectable specific expression at the early time point. d, Log2 GRO-seq expression signal (RPKM) in whole embryo at 2–2.5 h (ref. 28) at the promoter and gene body of different categories of genes. RPKM was defined by Saunders et al.28. e, Histogram (grey bars) of the expected distribution for a gene to be paused (using ‘all’ paused genes defined by Saunders et al.28) by random sampling 20 genes 10,000 times from the 459 differentially expressed off-to-on genes. The red dot indicates the percentage of observed paused genes for all DS genes (differentially expressed but with stable loops). Using this test, the percentage of paused DS genes is highly significant (P 0.0071).

Extended Data Figure 10 Stable enhancer loops prefigure gene expression and are associated with paused Pol II.

Interaction map at three known Pol II paused genes: dve (a), Rx (b) and CG6959 (c). Top to bottom: Pol II signal (RPGC) in whole embryo at 2–4 h (orange)48, GRO-seq signal in whole embryo at 2–2.5 h (plus strand, red; minus strand, blue)28, RNA-seq signal (RPKM) in whole embryo at 2–4 h and 6–8 h (black)9, 4C interaction map (viewpoint, red arrowhead) in whole embryo at 3–4 h (purple) and 6–8h (dark blue). The ratio between whole embryo 3–4 h and whole embryo 6–8 h 4C signal is plotted in between in red. Significant 4C interactions and known enhancers are indicated. WE, whole embryo. Note, dve and Rx are transcribed on the plus strand, whereas CG6959 is transcribed on the minus. In each case, the enhancer–promoter interactions, Pol II occupancy in the absence of full-length transcript production (indicative of Pol II pausing56) and short nascent RNA transcription are already present at the promoter at 2–4 h, whereas the gene becomes highly expressed at 6–8 h. As the viewpoint in c is located 80 kb away from the promoter of CG6959, a zoomed-in view of the promoter is shown for clarity.

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Ghavi-Helm, Y., Klein, F., Pakozdi, T. et al. Enhancer loops appear stable during development and are associated with paused polymerase. Nature 512, 96–100 (2014). https://doi.org/10.1038/nature13417

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