Drosophila poised enhancers are generated during tissue patterning with the help of repression

Abstract

Histone modifications are frequently used as markers for enhancer states, but how to interpret enhancer states in the context of embryonic development is not clear. The poised enhancer signature, involving H3K4me1 and low levels of H3K27ac, has been reported to mark inactive enhancers that are poised for future activation. However, future activation is not always observed, and alternative reasons for the widespread occurrence of this enhancer signature have not been investigated. By analyzing enhancers during dorsal-ventral (DV) axis formation in the Drosophila embryo, we find that the poised enhancer signature is specifically generated during patterning in the tissue where the enhancers are not induced, including at enhancers that are known to be repressed by a transcriptional repressor. These results suggest that, rather than serving exclusively as an intermediate step before future activation, the poised enhancer state may be a mark for spatial regulation during tissue patterning. We discuss the possibility that the poised enhancer state is more generally the result of repression by transcriptional repressors.

Figure 1.

Dorsal-ventral (DV) transcription factors occupy uninduced enhancers at intermediate levels. (A) Overview of the model system of DV patterning in the Drosophila embryo, in which homogeneous cell fates can be obtained through mutants such as Tl^10b and gd⁷, for which a large number of tissue-specific enhancers and their target genes are known. A summary of the analyzed ChIP-seq experiments of transcription factors and histone modifications is shown on the right. (AED) After egg deposition. (B) Boxplots of ChIP-seq enrichment over input for the DV transcription factors Dl, Twi, Mad, Zen, and Zld at known DV enhancers. The ChIP-seq experiments were performed in Tl^10b or gd⁷ or both, dependent on which tissue the transcription factor is expressed in. Note that DV transcription factors occupy uninduced enhancers less than active enhancers but significantly more than closed regions, indicating that uninduced enhancers are accessible. Closed regions are 100 presumptive late enhancers that are inaccessible by DHS (Thomas et al. 2011) at early stages and are enriched for H3K27ac at later embryonic stages (see Methods). Active enhancers are mesoderm enhancers (MEs) in Tl^10b embryos or dorsal ectoderm enhancers (DEEs) in gd⁷ embryos. Uninduced enhancers are MEs in gd⁷ embryos or DEEs in Tl^10b embryos. Whiskers show 1.5 times the interquartile range, and outliers are shown as dots. Asterisk indicates P < 10⁻³ using the Wilcoxon rank-sum test. (C) ChIP-seq binding profiles of the transcription factors at four DEEs and two MEs (red boxes with target genes shown in black) illustrate higher binding at active enhancers (gray shading) but some degree of binding at uninduced enhancers (white background). The DEEs of zen, dpp, and tld are known to be repressed by Dl, while the snail (sna) ME is activated by Dl (Ip et al. 1992). ChIP-seq reads are normalized to reads per million.

Figure 2.

The histone modifications at uninduced enhancers resemble the poised enhancer signature. (A) Boxplots of normalized H3K27ac and H3K4me1 ChIP-seq enrichments show that all uninduced DV enhancers (n = 59, from both mutants) have lower H3K27ac enrichment levels than the same enhancers in the active state, yet the levels of H3K4me1 are significantly above closed regions (n = 100, same as in Fig. 1B), consistent with a poised enhancer signature. Whiskers show 1.5 times the interquartile range, and outliers are shown as dots. Significance between enhancer groups was determined using the Wilcoxon rank-sum test. (B) Boxplots of the fold-change of normalized histone modification ChIP-seq enrichments between mutant embryos show that H3K27ac and H3K4me1 levels are higher at active enhancers versus uninduced enhancers: The majority of MEs (blue) have higher H3K27ac enrichment in the Tl^10b mutant than in the gd⁷ mutant (thus log₂ Tl^10b − log₂ gd⁷ above 0), while the inverse is true for dorsal ectodermal enhancers (DEEs; yellow). Significance between MEs and DEEs was determined using the Wilcoxon rank-sum test. (C) Binding profiles of histone modification ChIP-seq enrichments show higher enrichment of H3K27ac and H3K4me1 when the enhancer is active. At the four DEEs, the levels are higher in gd⁷ (yellow), while at four MEs, the levels are higher in Tl^10b (blue). The red box and the pink shading show the position of the enhancers, and the black arrow indicates the position and orientation of transcription start sites. The 1 kb scale bar shown for the tin region also applies to the sna and htl region.

Figure 3.

H3K27me3 levels are higher at uninduced enhancers but correlate more strongly with distance to the nearest PRE. (A) Boxplots show a wide range of different H3K27me3 levels at the different enhancer states, with no significant differences between them as determined by the Wilcoxon rank-sum test. (B) The fold-change difference in H3K27me3 enrichment between DV mutants shows that H3K27me3 at individual enhancers tends to be higher in the uninduced state versus active state. Significance was determined using the Wilcoxon rank-sum test. (C) H3K27me3 ChIP-seq enrichment profiles for three DEEs and three MEs illustrate clear differences between mutants (yellow vs. blue). H3K27me3 enrichment levels are highest near putative Polycomb response elements (PREs; green). Enhancers are shown as red boxes with pink shading. (D) Boxplots showing the distance of enhancers to the nearest PRE, dependent on whether they have low or high H3K27me3 enrichment levels. For DV enhancers with low H3K27me3 enrichment (below twofold enrichment, n = 39), the distances between enhancers and putative PREs are much larger than for those with high H3K27me3 levels (above twofold enrichment, n = 20). This is also true for Zld-bound regions, which include a large number of putative early Drosophila enhancers (low H3K27me3 n = 14,4425, high H3K27me3 n = 2720). Zld-bound regions are the union of Zld ChIP-seq peaks in Tl^10b and gd⁷ with at least twofold enrichment in either tissue. Putative PREs are defined as the overlap between Pc and GAF regions (n = 602). Whiskers show 1.5 times the interquartile range, and outliers are shown as dots.

Figure 4.

DV enhancers are not poised for future activation. (A) Histone modification levels at DV enhancers during the maternal-to-zygotic transition (Li et al. 2014) show that H3K27ac levels are accumulating early and gradually during development, and thus, some H3K27ac is present during enhancer priming by Zld at cell cycles 8 and 12. In contrast, H3K4me1 and H3K27me3, which mark poised enhancers, are only detectable after Dl-dependent transcription begins at stage 5 or cell cycle 14 (shaded in gray). Data are shown as average ChIP-seq signal in a 1-kb window centered on each enhancer. (B) Boxplots of DNase I hypersensitivity (DHS) at DV enhancers during embryogenesis show that all DV enhancers are most accessible during stages 5 and 9 when DV patterning takes place (shaded in gray), and become less accessible at subsequent stages. The DHS score is the average signal per enhancer region derived from the data by Thomas et al. (2011). Whiskers show 1.5 times the interquartile range, and outliers are shown as dots.

Figure 5.

Summary model showing the poised enhancer signature arising specifically during tissue patterning in the Drosophila DV system. Before DV patterning begins in the Drosophila embryo, DV enhancers are primed by the pioneer transcription factor Zld and have low levels of H3K27ac. During DV patterning, DV enhancers may be active in one tissue but repressed by sequence-specific repressors in another tissue and thus remain uninduced. As has been studied extensively, these repressors recruit histone deacetylases, remove H3K27ac, and thus produce the poised enhancer signature. After DV patterning is complete, DV enhancers gradually close, thus enhancers with the poised enhancer signature also close and are not poised for future activation.