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. 2017 Apr 20;45(7):3693-3706.
doi: 10.1093/nar/gkw1283.

Inducible super-enhancers are organized based on canonical signal-specific transcription factor binding elements

Dóra Bojcsuk  1 , Gergely Nagy  2 , Balint L Balint  1
Affiliations

Inducible super-enhancers are organized based on canonical signal-specific transcription factor binding elements

Dóra Bojcsuk et al. Nucleic Acids Res. .

Abstract

Super-enhancers are established through the interactions of several enhancers and a large number of proteins, including transcription factors and co-regulators; however, the formation of these interactions is poorly understood. By re-analysing previously published estrogen receptor alpha (ERα) ChIP-seq data sets derived from the MCF-7 cell line, we observed that in the absence of stimulation, future super-enhancers are represented by one or a few transcription factor binding event(s) and these extraordinary enhancers possess a response element largely specific to the ERα dimer. Upon hormonal stimulation, these primary binding sites are surrounded by a large amount of ERα and the critical components of active enhancers, such as P300 and MED1, and together with neighbouring sites bound by newly recruited ERα, they generate the functional super-enhancers. To further validate the role of canonical elements in super-enhancer formation, we investigated some additional signal-dependent transcription factors, confirming that certain, distinguished binding elements have a general organizer function. These results suggest that certain signal-specific transcription factors guide super-enhancer formation upon binding to strong response elements. These findings may reshape the current understanding of how these regulatory units assemble, highlighting the involvement of DNA elements instead of protein-protein interactions.

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Figures

Figure 1.
Figure 1.
MCF-7-specific estrogen receptor alpha (ERα) super-enhancers are represented by one (or a few) enhancer(s) even in the absence of estradiol treatment. (A) Integrative Genomics Viewer (IGV) snapshot of ERα ChIP-seq coverage representing six ERα super-enhancers (SEs) upon vehicle and estradiol (E2) treatment. The interval scale is 50 in both cases. (B and C) ERα tag density upon vehicle or E2 treatment in the deciles determined based on ERα recruitment at peaks within SEs. (D and E) ERα tag density upon vehicle or E2 treatment in deciles determined based on ERα recruitment at peaks not overlapping with SEs. The boxes represent the first and third quartiles, the horizontal lines indicate the median reads per kilobase per million mapped reads (RPKM) values, and the whiskers indicate the lower and upper extremes per decile. (F and G) ERα tag densities of the mother (M) and the top 6 daughter enhancers (1–6) within the same super-enhancer region in vehicle- and E2-treated MCF-7 cells. Enhancers were vertically sorted based on the RPKM values of the mother enhancers (in the first column), and the individual enhancers within a SE region were subsequently horizontally aligned based on the read enrichment of the vehicle-treated samples. (H and I) Histograms show the average tag density of mother (392) and daughter enhancers (3650) in the presence or absence of E2. (J and K) Histograms show the average tag density of mother (392) and daughter enhancers (3650) in the presence vehicle- or E2-treatment and upon tamoxifen- and fulvestrant-treatment. Samples derived from the same experiment.
Figure 2.
Figure 2.
MED1 is recruited to ERα-bound sites with an extremely high binding affinity. (A) IGV snapshot of MED1 and ERα ChIP-seq coverage, representing six ERα SEs upon vehicle and estradiol (E2) treatment. The interval scale is 50 in both cases. (B and C) MED1 tag density upon vehicle or E2 treatment in deciles determined based on ERα recruitment at peaks within SEs. The boxes represent the first and third quartiles, horizontal lines indicate the median RPKM values, and whiskers indicate the lower and upper extremes per decile. Paired t-test, * significant at P < 0.05; ** at P < 0.01; *** at P < 0.001; **** at P < 0.0001. (D) Read distribution plot of ERα, DNase I, MED1, P300, H3K27ac and BRD4 upon vehicle or E2 treatment, relative to SE peaks in 2-kb frames. Mother (392 in total) and daughter (3650 in total) peaks are sorted according to ERα tag density.
Figure 3.
Figure 3.
Canonical elements provide higher DNA-binding affinity than non-canonical elements. (A and B) Motif enrichments of mother and daughter enhancers. The P-value and target and background (Bg) percentages are included for each motif. (C) Estrogen response element (ERE) motif scores in the deciles determined based on ERα recruitment upon vehicle (in the case of mother enhancers) or E2 treatment (in the case of daughter enhancers) within SEs. (D and E) RPKM values of ERα and FoxA1 coverage at ERα mother and daughter enhancers. In the case of panels C, D and E, the boxes represent the first and third quartiles, the horizontal lines indicate the median RPKM values, and the whiskers indicate the 10th to 90th percentile ranges per decile. Paired t-test, * significant at P < 0.05, ** at P < 0.01, *** at P < 0.001, **** at P < 0.0001.
Figure 4.
Figure 4.
ERα, FoxA1 and AP2γ form distinct super-enhancers. (A–C) Read distribution plot of ERα, FoxA1 and AP2γ upon vehicle or E2 treatment, relative to each other's SE peaks in 2-kb frames. The number of mother and daughter peaks, sorted based on tag density, is indicated. (D–I) ERα, FoxA1 and AP2γ tag density of each other's mother and daughter enhancers in E2-treated MCF-7 cells. (J) Area-proportional Venn diagram illustrates the overlaps between the ERα, FoxA1 and AP2γ SEs.
Figure 5.
Figure 5.
Recruitment of FoxA1 and AP2γ at ERα super-enhancers. IGV snapshot of ERα ChIP-seq coverage, representing eight ERα SEs upon vehicle (veh) or E2 treatment and the simultaneous presence of FoxA1, AP2γ, MED1, P300, H3K27ac, BRD4 and DNase I upon vehicle and E2 treatment. The interval scales are indicated in the upper left corners. Peaks, highlighted in grey, represent the sites of ERα mother enhancers, while dashed lines indicate the location of FoxA1 peaks.
Figure 6.
Figure 6.
FoxA1 and AP2γ mother enhancers are located in active regions but are not inducible compared with ERα mother enhancers. The box plots demonstrate the accessibility of DNase I and the recruitment of MED1, P300, H3K27ac and BRD4 within the (A, D, G, J and M) ERα mother enhancers, (B, E, H, K, N) FoxA1 mother enhancers and (C, F, I, L and O) AP2γ mother enhancers. The boxes represent the first and third quartiles, the horizontal lines indicate the median RPKM values and the whiskers indicate the 10th to 90th percentile ranges per decile. Paired t-test, * significant at P < 0.05, ** at P < 0.01, *** at P < 0.001, **** at P < 0.0001.
Figure 7.
Figure 7.
Working model of an ERα-dominated SE in the presence or absence of estradiol. The upper, closed chromatin region demonstrates the presence of EREs within a SE, and one of these regions provides a canonical DNA-binding element (red box) for mother enhancers in the absence of treatment. Upon E2 treatment, the resulting SE region becomes more opened and E2 treatment recruits several ERα molecules, which may occupy the surrounding non-canonical EREs. The existence of a canonical element provides competition between ERs, and the attracted ERs likely bind to neighbouring non-canonical EREs rather than to similar non-canonical EREs in the distal regions of the genome. The SE region becomes more acetylated and MED1 and P300 bind to ERα that subsequently binds a canonical ERE.
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