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Genome-wide identification of Drosophila dorso-ventral enhancers by differential histone acetylation analysis

View Article: PubMed Central - PubMed

ABSTRACT

Background: Drosophila dorso-ventral (DV) patterning is one of the best-understood regulatory networks to date, and illustrates the fundamental role of enhancers in controlling patterning, cell fate specification, and morphogenesis during development. Histone acetylation such as H3K27ac is an excellent marker for active enhancers, but it is challenging to obtain precise locations for enhancers as the highest levels of this modification flank the enhancer regions. How to best identify tissue-specific enhancers in a developmental system de novo with a minimal set of data is still unclear.

Results: Using DV patterning as a test system, we develop a simple and effective method to identify tissue-specific enhancers de novo. We sample a broad set of candidate enhancer regions using data on CREB-binding protein co-factor binding or ATAC-seq chromatin accessibility, and then identify those regions with significant differences in histone acetylation between tissues. This method identifies hundreds of novel DV enhancers and outperforms ChIP-seq data of relevant transcription factors when benchmarked with mRNA expression data and transgenic reporter assays. These DV enhancers allow the de novo discovery of the relevant transcription factor motifs involved in DV patterning and contain additional motifs that are evolutionarily conserved and for which the corresponding transcription factors are expressed in a DV-biased fashion. Finally, we identify novel target genes of the regulatory network, implicating morphogenesis genes as early targets of DV patterning.

Conclusions: Taken together, our approach has expanded our knowledge of the DV patterning network even further and is a general method to identify enhancers in any developmental system, including mammalian development.

Electronic supplementary material: The online version of this article (doi:10.1186/s13059-016-1057-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Genes near putative enhancers are differentially regulated across tissues. a The closest active genes near MEs and DEEs are significantly differentially expressed in the expected tissue (Tl10b or gd7) as compared to non-differential control regions (p < 10−30, one-sided chi-squared test). The second closest genes also show a small but significant enrichment (p < 0.034, one-sided chi-squared test), while the third nearest active genes are no longer enriched over the control. b Fraction of genes with higher mesodermal expression (left) is largest among MEs, and that with higher dorsal ectoderm expression (right) is largest among DEEs. As a comparison, the top 400 regions identified by transcription factor ChIP-seq data are shown: Dl and Twi ChIP-seq experiments from Tl10b embryos, and Mad and Zen ChIP-seq experiments from gd7 embryos. The star marks significance over non-differential regions (p < 0.01, one-sided chi-squared test). c Transgenic reporter activity of Vienna Tiles (VTs) that overlap MEs, DEEs, and transcription factor ChIP regions are preferentially expressed during early embryonic stages (with annotated expression in any tissue at stages 4–10). All groups show expression far above the average of all VTs (marked as star, p < 0.01, Fisher’s exact test). d VT reporter expression is more tissue-specific for MEs and DEEs compared to transcription factor ChIP regions. ME reporter activity (left) was defined as annotations by Kvon et al. [39] containing “mesoderm,” and DEE reporter activity (right) as annotations containing “amnioserosa.” The star marks significance over non-differential regions (p < 0.01, Fisher’s exact test). Number of regions: all VTs (7705), early expressed VTs (1595), VTs overlapping putative DV enhancers (148), MEs (68), DEEs (80)
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Fig2: Genes near putative enhancers are differentially regulated across tissues. a The closest active genes near MEs and DEEs are significantly differentially expressed in the expected tissue (Tl10b or gd7) as compared to non-differential control regions (p < 10−30, one-sided chi-squared test). The second closest genes also show a small but significant enrichment (p < 0.034, one-sided chi-squared test), while the third nearest active genes are no longer enriched over the control. b Fraction of genes with higher mesodermal expression (left) is largest among MEs, and that with higher dorsal ectoderm expression (right) is largest among DEEs. As a comparison, the top 400 regions identified by transcription factor ChIP-seq data are shown: Dl and Twi ChIP-seq experiments from Tl10b embryos, and Mad and Zen ChIP-seq experiments from gd7 embryos. The star marks significance over non-differential regions (p < 0.01, one-sided chi-squared test). c Transgenic reporter activity of Vienna Tiles (VTs) that overlap MEs, DEEs, and transcription factor ChIP regions are preferentially expressed during early embryonic stages (with annotated expression in any tissue at stages 4–10). All groups show expression far above the average of all VTs (marked as star, p < 0.01, Fisher’s exact test). d VT reporter expression is more tissue-specific for MEs and DEEs compared to transcription factor ChIP regions. ME reporter activity (left) was defined as annotations by Kvon et al. [39] containing “mesoderm,” and DEE reporter activity (right) as annotations containing “amnioserosa.” The star marks significance over non-differential regions (p < 0.01, Fisher’s exact test). Number of regions: all VTs (7705), early expressed VTs (1595), VTs overlapping putative DV enhancers (148), MEs (68), DEEs (80)

Mentions: To validate the identified MEs and DEEs, we first analyzed whether the mRNA-seq levels of the nearby genes were significantly higher in the expected tissue (e.g., whether the mRNA transcripts near MEs are significantly higher in Tl10b over gd7). As a control group, we used the CBP regions without differential H3K27ac (”non-differential regions”, n = 6352). We found that 51 % of the closest active genes are differentially expressed in the predicted tissue, significantly higher than in the control group of CBP regions, which have 31 % (p < 10−30, Fig. 2a). The second nearest active genes were also slightly more differentially expressed (p < 0.034) than the control, but the third nearest genes were no longer enriched, consistent with enhancers functioning predominately locally in Drosophila [39]. Overall, we found that a large fraction of putative DV enhancers is associated with gene activation in the expected tissue.Fig. 2


Genome-wide identification of Drosophila dorso-ventral enhancers by differential histone acetylation analysis
Genes near putative enhancers are differentially regulated across tissues. a The closest active genes near MEs and DEEs are significantly differentially expressed in the expected tissue (Tl10b or gd7) as compared to non-differential control regions (p < 10−30, one-sided chi-squared test). The second closest genes also show a small but significant enrichment (p < 0.034, one-sided chi-squared test), while the third nearest active genes are no longer enriched over the control. b Fraction of genes with higher mesodermal expression (left) is largest among MEs, and that with higher dorsal ectoderm expression (right) is largest among DEEs. As a comparison, the top 400 regions identified by transcription factor ChIP-seq data are shown: Dl and Twi ChIP-seq experiments from Tl10b embryos, and Mad and Zen ChIP-seq experiments from gd7 embryos. The star marks significance over non-differential regions (p < 0.01, one-sided chi-squared test). c Transgenic reporter activity of Vienna Tiles (VTs) that overlap MEs, DEEs, and transcription factor ChIP regions are preferentially expressed during early embryonic stages (with annotated expression in any tissue at stages 4–10). All groups show expression far above the average of all VTs (marked as star, p < 0.01, Fisher’s exact test). d VT reporter expression is more tissue-specific for MEs and DEEs compared to transcription factor ChIP regions. ME reporter activity (left) was defined as annotations by Kvon et al. [39] containing “mesoderm,” and DEE reporter activity (right) as annotations containing “amnioserosa.” The star marks significance over non-differential regions (p < 0.01, Fisher’s exact test). Number of regions: all VTs (7705), early expressed VTs (1595), VTs overlapping putative DV enhancers (148), MEs (68), DEEs (80)
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Fig2: Genes near putative enhancers are differentially regulated across tissues. a The closest active genes near MEs and DEEs are significantly differentially expressed in the expected tissue (Tl10b or gd7) as compared to non-differential control regions (p < 10−30, one-sided chi-squared test). The second closest genes also show a small but significant enrichment (p < 0.034, one-sided chi-squared test), while the third nearest active genes are no longer enriched over the control. b Fraction of genes with higher mesodermal expression (left) is largest among MEs, and that with higher dorsal ectoderm expression (right) is largest among DEEs. As a comparison, the top 400 regions identified by transcription factor ChIP-seq data are shown: Dl and Twi ChIP-seq experiments from Tl10b embryos, and Mad and Zen ChIP-seq experiments from gd7 embryos. The star marks significance over non-differential regions (p < 0.01, one-sided chi-squared test). c Transgenic reporter activity of Vienna Tiles (VTs) that overlap MEs, DEEs, and transcription factor ChIP regions are preferentially expressed during early embryonic stages (with annotated expression in any tissue at stages 4–10). All groups show expression far above the average of all VTs (marked as star, p < 0.01, Fisher’s exact test). d VT reporter expression is more tissue-specific for MEs and DEEs compared to transcription factor ChIP regions. ME reporter activity (left) was defined as annotations by Kvon et al. [39] containing “mesoderm,” and DEE reporter activity (right) as annotations containing “amnioserosa.” The star marks significance over non-differential regions (p < 0.01, Fisher’s exact test). Number of regions: all VTs (7705), early expressed VTs (1595), VTs overlapping putative DV enhancers (148), MEs (68), DEEs (80)
Mentions: To validate the identified MEs and DEEs, we first analyzed whether the mRNA-seq levels of the nearby genes were significantly higher in the expected tissue (e.g., whether the mRNA transcripts near MEs are significantly higher in Tl10b over gd7). As a control group, we used the CBP regions without differential H3K27ac (”non-differential regions”, n = 6352). We found that 51 % of the closest active genes are differentially expressed in the predicted tissue, significantly higher than in the control group of CBP regions, which have 31 % (p < 10−30, Fig. 2a). The second nearest active genes were also slightly more differentially expressed (p < 0.034) than the control, but the third nearest genes were no longer enriched, consistent with enhancers functioning predominately locally in Drosophila [39]. Overall, we found that a large fraction of putative DV enhancers is associated with gene activation in the expected tissue.Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Drosophila dorso-ventral (DV) patterning is one of the best-understood regulatory networks to date, and illustrates the fundamental role of enhancers in controlling patterning, cell fate specification, and morphogenesis during development. Histone acetylation such as H3K27ac is an excellent marker for active enhancers, but it is challenging to obtain precise locations for enhancers as the highest levels of this modification flank the enhancer regions. How to best identify tissue-specific enhancers in a developmental system de novo with a minimal set of data is still unclear.

Results: Using DV patterning as a test system, we develop a simple and effective method to identify tissue-specific enhancers de novo. We sample a broad set of candidate enhancer regions using data on CREB-binding protein co-factor binding or ATAC-seq chromatin accessibility, and then identify those regions with significant differences in histone acetylation between tissues. This method identifies hundreds of novel DV enhancers and outperforms ChIP-seq data of relevant transcription factors when benchmarked with mRNA expression data and transgenic reporter assays. These DV enhancers allow the de novo discovery of the relevant transcription factor motifs involved in DV patterning and contain additional motifs that are evolutionarily conserved and for which the corresponding transcription factors are expressed in a DV-biased fashion. Finally, we identify novel target genes of the regulatory network, implicating morphogenesis genes as early targets of DV patterning.

Conclusions: Taken together, our approach has expanded our knowledge of the DV patterning network even further and is a general method to identify enhancers in any developmental system, including mammalian development.

Electronic supplementary material: The online version of this article (doi:10.1186/s13059-016-1057-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus