Gene transcription in animals involves the assembly of RNA polymerase II at core promoters and its cell-type-specific activation by enhancers that can be located more distally1. However, how ubiquitous expression of housekeeping genes is achieved has been less clear. In particular, it is unknown whether ubiquitously active enhancers exist and how developmental and housekeeping gene regulation is separated. An attractive hypothesis is that different core promoters might exhibit an intrinsic specificity to certain enhancers2,3,4,5,6. This is conceivable, as various core promoter sequence elements are differentially distributed between genes of different functions7, including elements that are predominantly found at either developmentally regulated or at housekeeping genes8,9,10. Here we show that thousands of enhancers in Drosophila melanogaster S2 and ovarian somatic cells (OSCs) exhibit a marked specificity to one of two core promoters—one derived from a ubiquitously expressed ribosomal protein gene and another from a developmentally regulated transcription factor—and confirm the existence of these two classes for five additional core promoters from genes with diverse functions. Housekeeping enhancers are active across the two cell types, while developmental enhancers exhibit strong cell-type specificity. Both enhancer classes differ in their genomic distribution, the functions of neighbouring genes, and the core promoter elements of these neighbouring genes. In addition, we identify two transcription factors—Dref and Trl—that bind and activate housekeeping versus developmental enhancers, respectively. Our results provide evidence for a sequence-encoded enhancer–core-promoter specificity that separates developmental and housekeeping gene regulatory programs for thousands of enhancers and their target genes across the entire genome.
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Gene Expression Omnibus
All deep sequencing data are available at http://www.starklab.org and have been deposited in the Gene Expression Omnibus database under accession numbers GSE40739 and GSE57876.
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We thank L. Cochella and O. Bell for comments on the manuscript. Deep sequencing was performed at the CSF Next-Generation Sequencing Unit (http://csf.ac.at). M.A.Z. was supported by the Austrian Science Fund (FWF, F4303-B09) and C.D.A., K.S., M.R. and O.F. by a European Research Council Starting Grant (no. 242922) awarded to A.S. Basic research at the Research Institute of Molecular Pathology is supported by Boehringer Ingelheim GmbH.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Set-up of STARR-seq with different core promoters.
a, STARR-seq detects enhancers but no promoters (reproduced with permission from ref. 12). Left, STARR-seq couples the enhancer activities of candidate fragments to the sequences of the candidates in cis by placing the candidates to a position within the reporter transcript. Enhancer activities can therefore be assessed by the presence of candidates among cellular messenger RNAs, which allows the parallel assessment of millions of candidates, enabling genome-wide screens. Sequences that activate transcription from the intended core promoter of the STARR-seq vector lead to a full-length reporter transcript and can be detected by STARR-seq. Shown are the reverse transcription (RT) and nested polymerase chain reaction (PCR) steps of the STARR-seq reporter RNA processing protocol that ensure this. Right, in contrast, STARR-seq does not detect truncated transcripts that result if a candidate fragment functions as a promoter to initiate transcription. Thus, core-promoter-containing (that is, TSS-overlapping) sequences that are detected by STARR-seq exhibit enhancer activity as they can activate transcription from a remote position, in addition to their ability to serve as core promoters endogenously12. b, Luciferase signals (firefly/Renilla) assessing the intrinsic (or basal) activity of the core promoters used in this study. The luciferase reporter constructs do not contain any enhancer and differ only in the respective core promoter sequences. The basal activities differ as expected, but do not differ consistently between housekeeping (RpS12, eEF1δ, NipB, x16) and developmental (DSCP, eve (long), eve and pnr) core promoters, nor between core promoters for which the STARR-seq screens appear most similar (for example, RpS12 and eEF1δ; see Fig. 3). Note that all luciferase assays and STARR-seq screens are corrected for differences in intrinsic activity. c, Reproducibility of hkCP and dCP STARR-seq in D. melanogaster S2 cells. The reproducibility of hkCP and dCP STARR-seq as assessed by the STARR-seq enrichments (replicate 1 versus 2) at the summits of enhancer peaks called in the merged experiments (hkCP: 5,956; dCP: 5,408). Scatter plots are enlarged versions of the insets in Fig. 1d. “Enr. rep X”, STARR-seq enrichment in replicate X. Note that the raw data for dCP have been re-analysed from ref. 12.
Extended Data Figure 2 Transcription initiates within the core promoter of the STARR-seq construct.
a–d, 5′ Rapid amplification of cDNA ends (5′ RACE) demonstrates that transcription initiates at the TCT and Inr motifs within the hkCP and dCP, respectively. a, Set-up of the 5′ RACE experiment, including the STARR-seq plasmid, used here with two defined enhancers, the STARR-seq transcript and the location of all primers used to specifically amplify 5′-capped STARR-seq transcripts. b, 5′ RACE nested PCR products separated on a 1% agarose gel. c, Screenshot of Sanger sequencing results (chromatogram and called bases) compared with the template sequence. Annotations are shown in green, in the following order: 5′ RACE adaptor, hkCP with TCT motif (only the part downstream of the TSS is annotated, as the 5′ part is not present in the sequenced complementary DNA), spliced intron, green fluorescent protein (GFP); the sequencing primer is shown in red (top). Also shown is a version that displays the template and Sanger sequencing results for the core promoter region only (zoom in). d, Same as in c but for the dCP for which transcription initiates within the Inr motif.
Extended Data Figure 3 Specificity of hkCP and dCP enhancers to the hkCP and dCP assessed by luciferase assays.
a, Luciferase reporter set-up with the hkCP or dCP (see also Fig. 1e). b, Luciferase signals of 24 hkCP-specific enhancers tested in a hkCP- (purple bars) as well as in a dCP-containing (brown bars) luciferase reporter. Twenty-one out of 24 hkCP enhancers showed luciferase activity (>1.5 fold over negative, P < 0.05 via one-sided unpaired Student’s t-test, n = 3) with the hkCP, while only 1 out of 24 showed activity with the dCP (error bars are s.d. of three biological replicates, ‘x’ indicates candidates that are not active with the correct core promoter, and ‘+’ indicates candidates for which the activity with the wrong core promoter is above the threshold (note that the activity with the correct core promoter is still higher in all three cases). c, As in b but testing dCP-specific enhancers. Ten out of 12 are positive with the dCP whereas only 2 out of 12 are positive with the hkCP. d, As in b and c but testing shared enhancers that were found by STARR-seq with hkCP and dCP; 6 out of 7 are active with both core promoters. See Supplementary Table 17 for the genomic coordinates of the enhancers and the primers used to amplify them.
Extended Data Figure 4 hkCP and dCP STARR-seq signal in S2 cells around different core promoter types.
Average hkCP (top) and dCP (bottom) S2 STARR-seq enrichment in 40 kb intervals around TSSs that contain different combinations of known core promoter motifs. Shown are (left to right) TATA box–Inr (179 TSSs), Inr (that do not contain either TATA box or DPE; 1,901), Inr–DPE (100), TCT (303) and motif 1–motif 6 (266). According to their motif contents, the first three are developmental-type core promoters and the last two are housekeeping-type core promoters. Indeed, only the housekeeping-type core promoters show a strong enrichment of hkCP S2 STARR-seq signals at the TSS, which is not seen for the dCP STARR-seq signal (owing to enhancer–core-promoter specificity) nor for the developmental-type core promoters (owing to the dCP enhancers location at more distal sites).
Extended Data Figure 5 TSS-overlapping hkCP enhancers function independent of their orientation.
Luciferase signals for all 17 TSS-overlapping hkCP enhancers (that is, containing one TSS or two divergent TSSs; see Supplementary Table 17) from Extended Data Fig. 3 cloned in the second orientation with respect to the TSS of the luciferase gene (bottom bar plot; the top bar plot corresponds to the initial orientation as in Extended Data Fig. 3 and is shown for comparison). In both orientations, 15 out of 17 enhancers showed activity towards the hkCP (details as in Extended Data Fig. 3). These results together with the findings in Extended Data Fig. 3 challenge the widespread notion that TSS-proximal sequences are promoters and even the concept of promoters more generally: sequences that autonomously activate gene expression—and are therefore often termed promoters—might in fact be the combination of a core promoter and a proximal enhancer. The TSS-proximal location of many housekeeping enhancers might be evolutionarily more ancient, consistent with regulatory mechanisms in simple eukaryotes such as yeast. In contrast, enhancers of genes with more complex regulation are typically located more distally, potentially simply because the several different cell-type-specific enhancers of these genes would not all fit to positions near TSSs. Consistently, such genes frequently have larger intergenic and intragenic regions40 known to accommodate enhancers with diverse activity patterns41.
Extended Data Figure 6 hkCP and dCP enhancers in S2 cells are associated with genes of different functions and core promoter elements.
a, GO analysis of genes next to hkCP- and dCP-specific enhancers in S2 cells using different enhancer-to-gene assignment strategies (top left, ‘closest TSS’ as in Fig. 2; top right, ‘1 kb TSS’; bottom left, ‘gene loci’; see Methods for details). Shown are 20 non-redundant GO categories selected from the 100 most significantly enriched categories associated with each enhancer class (see Supplementary Tables 2–4 for all categories). b, Enrichment of core promoter elements at genes next to hkCP- and dCP-specific enhancers in S2 cells. Similar analysis as in Fig. 2e, but using different enhancer-to-gene assignment strategies (see Methods for details). Consistent with Fig. 2e, core promoters of genes assigned to hkCP-specific enhancers are enriched in motifs 1, 5, 6, 7 and DRE, while core promoters of genes assigned to dCP-specific enhancers are enriched for TATA box, Inr, MTE and DPE motifs, irrespective of the assignment strategy.
Extended Data Figure 7 Housekeeping and developmental core promoters differ characteristically in their global enhancer preferences.
As in Fig. 3b but including biological replicates with independently cloned focused bacterial artificial chromosome (BAC) libraries covering around 5 Mb of genomic sequence (BAC) and assessing the PCC at each position along these regions. GW, genome-wide screens as in Fig. 3b. The similarity observed for the TATA box- and DPE-containing core promoters (Hsp70, pnr and DSCP (dCP)) suggest that differences related to these core promoter elements might be more subtle or related to alternative mechanisms, including the potential preferences of more proximal or distal enhancers42 or RNA polymerase II pausing and the dynamics versus stochasticity of initiation and elongation43,44,45.
Extended Data Figure 8 hkCP and dCP enhancers differ in OSCs.
a, b, Different enhancers activate transcription from hkCP and dCP in OSCs. As Fig. 1c, d but for OSCs rather than S2 cells (data in bottom inset of b are re-analysed from ref. 12). c, Genomic distribution of hkCP and dCP enhancers in OSCs. As Fig. 2a but for OSCs rather than S2 cells. d, hkCP and dCP STARR-seq signal in OSCs around different core promoter types. As Extended Data Fig. 4 but for OSCs rather than S2 cells.
Extended Data Figure 9 Differences between hkCP and dCP enhancers in OSCs.
a, GO analysis of genes next to hkCP- and dCP-specific enhancers in OSCs. As Extended Data Fig. 6a but for OSCs rather than S2 cells (see Supplementary Tables 8–10 for all categories). b, Enrichment of core promoter elements at genes next to hkCP- and dCP-specific enhancers in OSCs. As Fig. 2e and Extended Data Fig. 6b but for OSCs rather than S2 cells. NS, not significant (hypergeometric P > 0.05). c, Heat maps of hkCP (top) and dCP (bottom) STARR-seq enrichments in S2 cells and OSCs. Heat maps on the left and right are centred on the summits of core-promoter-type-specific enhancers in S2 and OSCs, respectively.
Extended Data Figure 10 The activities of hkCP and dCP enhancers are dependent on DRE and GAGA motifs, respectively.
a, Differential motif enrichment in distally located hkCP- and dCP-specific enhancers (as in Fig. 5a but assessing enrichments of the same motif PWMs exclusively at distal enhancers >500 bp away from the closest TSSs). Key motifs including DRE and GAGA are also differentially enriched in distal hkCP- and dCP-specific enhancers. NS, not significant (FDR-corrected hypergeometric P > 0.01). S2 cells: hkCP n = 790, dCP n = 3,013; OSCs: hkCP n = 556, dCP n = 2,555. b, Distal hkCP- and dCP-specific enhancers are differentially bound by Dref and Trl, respectively. ChIP enrichments of Dref (left) and Trl (right) at S2 hkCP- and dCP-specific enhancers that are distal (>500 bp) from the closest TSSs. Equivalent to Fig. 5b, but considering exclusively TSS-distal enhancers to exclude potentially confounding effects for TSS-proximal enhancers for which it is not possible to discern whether binding occurs due to the enhancer sequence or core promoter function. The differential binding between Dref and Trl to hkCP- and dCP-specific enhancers, respectively, is also found in Kc167 cells, in which the Dref ChIP-seq experiment had been performed (data not shown). c, Addition of DRE motifs to dCP enhancers increases their activity towards hkCP. Relative luciferase activity values (firefly/Renilla) for 11 dCP enhancers without DRE motifs (wild type (WT), light purple) and with 3 DRE motifs flanking the enhancers on each side (+DRE, dark purple). *P < 0.05, one-sided unpaired Student’s t-test; error bars denote the s.d. of three biological replicates.
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Zabidi, M., Arnold, C., Schernhuber, K. et al. Enhancer–core-promoter specificity separates developmental and housekeeping gene regulation. Nature 518, 556–559 (2015). https://doi.org/10.1038/nature13994
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