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Quantitative genome-wide enhancer activity maps for five Drosophila species show functional enhancer conservation and turnover during cis-regulatory evolution

Nature Genetics volume 46, pages 685–692 (2014)Cite this article

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Abstract

Phenotypic differences between closely related species are thought to arise primarily from changes in gene expression due to mutations in cis-regulatory sequences (enhancers). However, it has remained unclear how frequently mutations alter enhancer activity or create functional enhancers de novo. Here we use STARR-seq, a recently developed quantitative enhancer assay, to determine genome-wide enhancer activity profiles for five Drosophila species in the constant trans-regulatory environment of Drosophila melanogaster S2 cells. We find that the functions of a large fraction of D. melanogaster enhancers are conserved for their orthologous sequences owing to selection and stabilizing turnover of transcription factor motifs. Moreover, hundreds of enhancers have been gained since the D. melanogaster–Drosophila yakuba split about 11 million years ago without apparent adaptive selection and can contribute to changes in gene expression in vivo. Our finding that enhancer activity is often deeply conserved and frequently gained provides functional insights into regulatory evolution.

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Figure 1: Functional conservation of D. melanogaster S2 cell enhancers.
Figure 2: Compensatory enhancers stabilize total enhancer strengths for gene loci.
Figure 3: Motif conservation by positional sequence constraints.
Figure 4: Compensatory motif turnover in functionally conserved enhancers.
Figure 5: Species-specific gained enhancers and associated sequence changes.
Figure 6: Evolution of enhancer activity in OSCs and gene expression in follicle cells in vivo.

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Acknowledgements

We thank the Drosophila Species Stock Center (DSSC) at the University of California, San Diego for genomic DNA, I. Sinitsyn and Ł.M. Boryń for help with library cloning, M. Rath, K. Schernhuber and O. Frank for help with experiments, J.O. Yáñez-Cuna and M.A. Zabidi for help with data analysis, and S. Westermann, L. Cochella, I. Crisostomo, J. Brennecke, J. Knoblich, U. Technau, K. Tessmar-Raible and F. Raible for comments on the manuscript. Deep sequencing was performed at the CSF (Campus Science Support Facilities) Next-Generation Sequencing Unit and the Brandeis Genomics Core Facility (laboratory of M. Rosbash). C.D.A. is supported by a European Research Council (ERC) Starting Grant (242922) awarded to A.S. This work was partly supported by the Austrian Science Fund (FWF), F4303-B09. The work of N.C.L. was supported by the Searle Scholars foundation and the US National Institutes of Health (HD057298). Basic research at IMP is supported by Boehringer Ingelheim.

Author information

Author notes

  1. Daniel Gerlach & Jessica A Matts

    Present address: Present addresses: Boehringer Ingelheim, Vienna, Austria (D.G.) and Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma, USA (J.A.M.).,

  2. Cosmas D Arnold and Daniel Gerlach: These authors contributed equally to this work.

Authors and Affiliations

  1. Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Vienna, Austria

    Cosmas D Arnold, Daniel Gerlach, Daniel Spies, Michaela Pagani & Alexander Stark

  2. Department of Biology, Brandeis University, Waltham, Massachusetts, USA

    Jessica A Matts, Yuliya A Sytnikova & Nelson C Lau

  3. Rosenstiel Basic Medical Science Research Center at Brandeis University, Waltham, Massachusetts, USA

    Jessica A Matts, Yuliya A Sytnikova & Nelson C Lau

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Contributions

C.D.A. and A.S. conceived the project. C.D.A., D.S. and M.P. performed the experiments. J.A.M., Y.A.S. and N.C.L. generated the follicle cell RNA-seq data. D.G. and A.S. conducted all computational experiments and bioinformatics analyses. C.D.A., D.G. and A.S. wrote the manuscript.

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Correspondence to Alexander Stark.

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Integrated supplementary information

Supplementary Figure 1 High quantitative and qualitative reproducibility of STARR-seq in D. melanogaster S2 cells.

(a) The quantitative reproducibility of STARR-seq in two independent biological replicates was assessed at enhancer peak summits (D. melanogaster (D.mel), 2,325 peaks; D. yakuba (D.yak), 2,293 peaks; D. ananassae (D.ana), 2,096 peaks; D. pseudoobscura (D.pse), 3,469 peaks; D. willistoni (D.wil), 2,860 peaks) and for 100,000 positions randomly sampled from the genome (common D. melanogaster coordinates for all species). Each data point represents the fragment density for both replicates normalized to 1 million mapped fragments (FPM). The Pearson correlation coefficient (PCC) and the coefficient of determination (R2) for the linear fit (plus the regression line) are indicated in each subplot. The open black circle shows median values of coverage for replicate 1 versus replicate 2. (b) Qualitative reproducibility of STARR-seq measuring the consistency of enhancer calls between enhancers called in replicate 1 evaluated with enrichment data from replicate 2 equivalently to the assessment of conservation (bar height, relaxed settings with P ≤ 0.05; white line, P ≤ 0.001). The second bar for each species evaluates replicate 2 against replicate 1. (c,d) The same data are shown as for a and b, but using peak calls and fragment densities in the respective, original Drosophila genomes before coordinate translation.

Supplementary Figure 2 Similar genomic distribution of STARR-seq enhancers for all five Drosophila species in D. melanogaster S2 cells.

The pie charts show the absolute genomic distribution of enhancers across different functional regions, and the bar charts show enrichment or depletion relative to overall region sizes in the genome (a, D. melanogaster; b, D. yakuba; c, D. ananassae; d, D. pseudoobscura; e, D. willistoni). Globally, the majority of identified enhancers were located within introns (53.2–59.4%) and in intergenic regions (18.7–26.3%), as described in ref. 1. Overall the genomic distribution of enhancers is comparable among the five Drosophila species. These data show that enhancer location with respect to different genomic regions is similar for all five Drosophila species.

Supplementary Figure 3 Functional conservation of open and closed D. melanogaster S2 cell enhancers in D. melanogaster S2 cells.

D. melanogaster S2 cell enhancers were classified as open or closed depending on their accessibility in DNase I hypersensitivity (DHS) sequencing assays as described previously1. The functional conservation rates of (a) 1,554 open and (b) 771 closed D. melanogaster enhancers in the 4 other Drosophila species are shown (see Figure 1c for details of the conservation rate analysis). The conservation rate of open enhancers is roughly twice as high as for closed enhancers, whereas both show similar reproducibility in independent replicates (D. melanogaster bars, marked by an asterisk).

Supplementary Figure 4 Positional and compensatory conservation of enhancers.

The number of positionally conserved D. melanogaster enhancers declines with evolutionary distances. The drop in positionally conserved enhancers, however, is balanced by an increasing number of compensatory enhancers within the same respective gene loci, leading to a similar number of enhancers per gene locus. This might stabilize gene expression levels or confer regulatory robustness2–4. Note that this plot shows data from analysis of the two biological replicates combined. The white numbers inside the bars indicate the fraction of enhancers of each category as percentage of the total number of D. melanogaster enhancers (1,552) assigned to 1,201 gene loci.

Supplementary Figure 5 Motif conservation by positional sequence constraints.

(a) Pairwise sequence identity for functionally conserved (colored) and non-conserved (gray) enhancers along the entire 500-bp enhancer sequence or (c) for a 100-bp core enhancer sequence (boxes depict the median and the interquartile range, and whiskers depict the 10th and 90th percentiles). (b,d) Sequence identity as in a and c, respectively, but restricted to positions that overlap with motifs of the transcription factor Serpent (Srp). Note: a and b show the same data as Figure 3a,b to allow for a comparison with c and d, demonstrating that the results are robust with respect to the lengths of the analyzed regions (for b–d, n = 214, 338, 413, 196, 361, 216, 366 and 174, respectively). (e) Sequence identity as in b, but for the motif of the transcription factor Buttonhead (Btd), which is not expressed in S2 cells. The largely overlapping sequence identities of the Btd motifs in conserved and non-conserved enhancers indicate that the motifs for Btd in conserved enhancers in S2 cells are not under constraint. (f) Position weight matrix logos for the Srp and Btd motifs.

Supplementary Figure 6 Positional and compensatory conservation of TF motifs in functionally conserved and non-conserved enhancers.

(a) Rate of positionally conserved and compensatory D. melanogaster Srp motifs in relation to all four other Drosophila species in functional conserved enhancer regions. The total number of D. melanogaster Srp motifs for each comparison is shown above the bars (black), and percent conservation values are shown within the bars (white). (b) Plot as in a, but in functionally non-conserved enhancer regions. (c,d) Plots as in a and b, but limiting the evaluation to a subset of well-aligned enhancer regions that have no undefined nucleotides (Ns) in the pairwise alignments and have non-gapped orthologous ends. Together this shows that motif turnover is common and that the loss of positionally conserved Srp motifs can be compensated by the gain of Srp motifs at different positions within the same enhancer. Further, Srp motifs are conserved at much higher levels in conserved enhancers compared to non-conserved enhancers, suggesting that they are important for S2 cell enhancer function. (Supplementary Fig. 5). When assessing well-aligned sequences (c), motif turnover maintains the number of serpent motifs at high levels of around 80%, even over large evolutionary distances. (e,f) Relative contribution of compensatory motif turnover increases with evolutionary distance. (e) Fraction of functionally conserved enhancers with the same number of Srp motifs between species with positionally conserved motifs (blue), motifs conserved within an individual enhancer but not at the same position (compensatory; red) or a mix of positionally conserved and compensatory conserved Srp motifs (yellow). The total number of enhancers for each comparison is shown above each bar; percentages per category are plotted in white within the bars. (f) Plot as in e, but considering only a subset of enhancers that are well aligned (c,d). For the vast majority of conserved enhancers, the motifs are exclusively positionally conserved in closely related species such as D. yakuba in e. However, the fraction of enhancers with compensatory motifs increases significantly at larger evolutionary distances.

Supplementary Figure 7 Phylogeny of enhancer gain and loss events.

(a) Enhancer occurrences at non-redundant positions across species (binary representation in which blue boxes indicate enhancer presence/function). (b) Gain (blue triangles) and loss (red triangles) events assigned by parsimony to different branches of the phylogenetic tree. We assigned a gain event if two or more loss events would otherwise have to be assumed, but indicate the gain-versus-loss ratios below the trees (e.g., 1:4 = one gain or four losses; unclear events are shown in gray). Overall, the phylogeny of all 8,180 non-redundant enhancers identified in the genomes of the 5 species are shown on 31 (= 52 – 1) different trees.

Supplementary Figure 8 Sequence changes of D. melanogaster and D. yakuba gained enhancers are similar to expected neutral substitutions between the two species.

(a) Same data as Figure 4e (boxes depict the median and the interquartile range, and whiskers depict the 10th and 90th percentiles; outliers are shown individually). (b) As in a, but for 100-bp core enhancer sequences. This shows that the patterns of sequence conservation in gained and lost enhancers are consistent between 500-bp enhancer sequences and shorter regions of 100 bp centered on the enhancer peak summit.

Supplementary Figure 9 Newly gained enhancers in D. melanogaster are associated with expressed genes.

UCSC Genome Browser screenshots of expressed genes in S2 cells that are exclusively associated with a newly gained (D. melanogaster–specific) enhancer (inputs in gray; y-axis labels depict normalized fragment counts). (a) Sxn6 (RPKM 98.4). (b) roq (RPKM 25.9). (c) Hira (RPKM 12.3). RNA-seq data in D. melanogaster S2 cells are from ref. 1.

Supplementary Figure 10 High quantitative and qualitative reproducibility of STARR-seq in D. melanogaster ovarian somatic cells (OSCs).

(a) The quantitative reproducibility of STARR-seq in OSCs in two independent biological replicates was assessed at combined enhancer peak summits (D. melanogaster, 3,342 peaks; D. yakuba, 3,233 peaks; D. ananassae, 2,859 peaks) as in Supplementary Figure 1a. (b) Same data as in a, but using peak calls and fragment densities in the respective Drosophila genome coordinates before coordinate translation. (c) Qualitative reproducibility of STARR-seq in OSCs as in Supplementary Figure 1b. (d) Same as c, but using peak calls and fragment densities in the respective Drosophila genome coordinates before coordinate translation.

Supplementary Figure 11 Genomic distribution, functional conservation and sequence changes of STARR-seq OSC enhancers.

(a–c) Similar genomic distribution of STARR-seq enhancers for three Drosophila species in D. melanogaster OSCs. Genomic distribution analysis for (a) D. melanogaster, (b) D. yakuba and (c) D. ananassae enhancers in OSCs as in Supplementary Figure 2. Globally, the majority of identified enhancers were located in introns (44.1–52.8%) and in intergenic regions (23.7–26.5%; see ref. 1). (d,e) Functional conservation of open and closed D. melanogaster OSC enhancers in D. melanogaster OSCs. D. melanogaster OSC enhancers were classified as open and closed as described previously1. The conservation rates of (d) 2,269 open and (e) 1,073 closed D. melanogaster OSC enhancers in D. yakuba and D. ananassae (see Figs. 1c and 6a for details). (f,g) Number of sequence changes in D. melanogaster and D. yakuba gained, lost or deeply conserved S2 or OSC enhancers are similar. (f) Analysis as in Figure 4e, however, based on three species only (D. melanogaster, D. yakuba, D. ananassae) to allow the direct comparison between S2 cells and OSCs (boxes depict the median and the interquartile range, and whiskers depict the 10th and 90th percentiles). (g) As in f, but for OSC enhancers. The numbers of sequence changes for the different enhancer categories are highly similar between f and g, confirming the results shown in Figure 4e and suggesting that the reported numbers hold more generally, independent of the respective cell types.

Supplementary Figure 12 Changes in OSC enhancer activities and follicle cell in vivo gene expression between D. melanogaster and D. yakuba correlate globally.

Same data and heat-map presentation as in Figure 6d, but with matrix cells colored according to enrichments irrespective of their significance.

Supplementary Figure 13 Differences in quantitative enhancer strength follow a molecular clock.

Enhancer strength diverges with increasing evolutionary distance linearly, with the number of substitutions per neutral site (branch length) similar to qualitative enhancer conservation (Fig. 1c). The strong correlation of evolutionary distance and the fraction of enhancers with at (a) 2-fold, (b) 4-fold, (c) 6-fold and (d) 8-fold change in enhancer strength on non-redundant loci between D. melanogaster and other Drosophila species shows that enhancer strength is also conserved and follows a molecular clock. Note that the D. melanogaster replicate comparison considers replicate 1 against replicate 2 within the same species.

Supplementary Figure 14 Global range of sequence identities for functionally conserved and non-conserved enhancers.

Sequence identity distributions for functionally conserved (colored lines) and non-conserved (dark gray lines) enhancers between D. melanogaster and other Drosophila species. The distributions are largely overlapping, suggesting that there is no selective pressure on the overall enhancer sequence. In addition, the two extreme boundaries of the distributions indicate that sequences can be up to 95% identical (between D. melanogaster–D. yakuba) yet without conserved function (only active in the D. melanogaster genome), whereas enhancer function can be conserved despite as little as 39% sequence identity between D. melanogaster–D. willistoni.

Supplementary Figure 15 S2 cell and OSC enhancer gains are nearly additive.

Enhancers gained in S2 cells or OSCs show only limited overlap (right column), such that the number of gained enhancers is nearly additive for both cell types in D. melanogaster (top) and in D. yakuba (bottom; enhancer gains for both cell types are defined on the basis of three-way analyses considering only D. melanogaster, D. yakuba and D. ananassae as outgroup). The overlap of the gained enhancers is of the same magnitude as for the overlap of all enhancers in S2 cells and OSCs (left column), which suggests that different enhancers are gained in different cell types and the total number of enhancer gains in more complex tissues or organisms scales with the number of cell types and, presumably, the difference between cell types. (As the overlap of gained enhancers is even slightly lower than the overlap of all enhancers (1.8- to 3.6-fold), one could speculate that gained enhancers might have 'more unusual' sequence properties that are less likely to be shared by different cell types.)

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1, 3 and 5, and Supplementary Figures 1–15 (PDF 3751 kb)

Supplementary Table 2

TF motif conservation in functionally conserved and D. melanogaster–specific S2 cell enhancers. (XLSX 73 kb)

Supplementary Table 4

RNA-seq in follicle cells—gene expression (RPKM) values. (XLSX 456 kb)

Supplementary Data Set 1

STARR-seq peak calls (ZIP 3342 kb)

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Arnold, C., Gerlach, D., Spies, D. et al. Quantitative genome-wide enhancer activity maps for five Drosophila species show functional enhancer conservation and turnover during cis-regulatory evolution. Nat Genet 46, 685–692 (2014). https://doi.org/10.1038/ng.3009

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