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

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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.


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Transcription factor binding motifs are conserved in novel DV enhancers. a Examples of conserved motif instances within a mesoderm enhancer (if-ME3, left), close to the gene inflated (if), and a dorsal ectoderm enhancer (Btk29A-DEE1, right), close to the Btk29A gene. H3K27ac is shown as normalized ChIP enrichment over input in Tl10b and gd7 embryos. ChIP-seq occupancy is shown for Twi and Dl (left) and Zen and Sna (right) as ChIP-seq reads normalized to reads per million. A close-up of the if-ME3 sequence shows a canonical Twi binding motif (E-box) and two Dl binding sites that are conserved across several Drosophila species. A close-up of the Btk29A-DEE1 sequence shows two canonical Sna binding motifs and a canonical Zen binding motif that reside in islands of conservation. Conservation data are phastCons data obtained from the UCSC genome browser [72]. b Conservation of all identified DV motifs among all MEs and DEES. The average phastCons score for all putative DV enhancers (”MEs + DEEs” in dark gray) is significantly higher compared to control regions (”Random non-TSS” in light gray). The average phastCons score of each DV motif (”Motifs only” in red) is in many cases higher than that of the surrounding regions (”MEs + DEEs with motif” in light red). This confirms that motifs like that of Zen and Sna are indeed preferentially found in islands of conservation. Significance was determined by Wilcoxon rank-sum test and marked with a star (p < 0.05)
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Fig4: Transcription factor binding motifs are conserved in novel DV enhancers. a Examples of conserved motif instances within a mesoderm enhancer (if-ME3, left), close to the gene inflated (if), and a dorsal ectoderm enhancer (Btk29A-DEE1, right), close to the Btk29A gene. H3K27ac is shown as normalized ChIP enrichment over input in Tl10b and gd7 embryos. ChIP-seq occupancy is shown for Twi and Dl (left) and Zen and Sna (right) as ChIP-seq reads normalized to reads per million. A close-up of the if-ME3 sequence shows a canonical Twi binding motif (E-box) and two Dl binding sites that are conserved across several Drosophila species. A close-up of the Btk29A-DEE1 sequence shows two canonical Sna binding motifs and a canonical Zen binding motif that reside in islands of conservation. Conservation data are phastCons data obtained from the UCSC genome browser [72]. b Conservation of all identified DV motifs among all MEs and DEES. The average phastCons score for all putative DV enhancers (”MEs + DEEs” in dark gray) is significantly higher compared to control regions (”Random non-TSS” in light gray). The average phastCons score of each DV motif (”Motifs only” in red) is in many cases higher than that of the surrounding regions (”MEs + DEEs with motif” in light red). This confirms that motifs like that of Zen and Sna are indeed preferentially found in islands of conservation. Significance was determined by Wilcoxon rank-sum test and marked with a star (p < 0.05)

Mentions: Phylogenetic sequence conservation (“phylogenetic footprinting”) of regulatory regions, and specifically of the transcription factor binding motifs within them, has long been used to identify functional enhancers and motifs [61, 62]. When we analyzed the newly identified enhancers, we noticed that the identified transcription factor motifs were often found within sequence blocks of high conservation across the Drosophila phylogeny, interspersed by more diverged sequences within the same enhancer region. For example, a canonical Twi binding motif in the newly identified if-ME3 enhancer is in a highly conserved sequence block, while a partially conserved sequence block right next to it contains two Dl binding motifs (Fig. 4a left). Likewise, a putative DEE, Btk29A-DEE1, has two conserved, presumably repressive, Sna binding motifs next to a conserved canonical Zen binding motif, each in a conserved sequence block (Fig. 4a right).Fig. 4


Genome-wide identification of Drosophila dorso-ventral enhancers by differential histone acetylation analysis
Transcription factor binding motifs are conserved in novel DV enhancers. a Examples of conserved motif instances within a mesoderm enhancer (if-ME3, left), close to the gene inflated (if), and a dorsal ectoderm enhancer (Btk29A-DEE1, right), close to the Btk29A gene. H3K27ac is shown as normalized ChIP enrichment over input in Tl10b and gd7 embryos. ChIP-seq occupancy is shown for Twi and Dl (left) and Zen and Sna (right) as ChIP-seq reads normalized to reads per million. A close-up of the if-ME3 sequence shows a canonical Twi binding motif (E-box) and two Dl binding sites that are conserved across several Drosophila species. A close-up of the Btk29A-DEE1 sequence shows two canonical Sna binding motifs and a canonical Zen binding motif that reside in islands of conservation. Conservation data are phastCons data obtained from the UCSC genome browser [72]. b Conservation of all identified DV motifs among all MEs and DEES. The average phastCons score for all putative DV enhancers (”MEs + DEEs” in dark gray) is significantly higher compared to control regions (”Random non-TSS” in light gray). The average phastCons score of each DV motif (”Motifs only” in red) is in many cases higher than that of the surrounding regions (”MEs + DEEs with motif” in light red). This confirms that motifs like that of Zen and Sna are indeed preferentially found in islands of conservation. Significance was determined by Wilcoxon rank-sum test and marked with a star (p < 0.05)
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Fig4: Transcription factor binding motifs are conserved in novel DV enhancers. a Examples of conserved motif instances within a mesoderm enhancer (if-ME3, left), close to the gene inflated (if), and a dorsal ectoderm enhancer (Btk29A-DEE1, right), close to the Btk29A gene. H3K27ac is shown as normalized ChIP enrichment over input in Tl10b and gd7 embryos. ChIP-seq occupancy is shown for Twi and Dl (left) and Zen and Sna (right) as ChIP-seq reads normalized to reads per million. A close-up of the if-ME3 sequence shows a canonical Twi binding motif (E-box) and two Dl binding sites that are conserved across several Drosophila species. A close-up of the Btk29A-DEE1 sequence shows two canonical Sna binding motifs and a canonical Zen binding motif that reside in islands of conservation. Conservation data are phastCons data obtained from the UCSC genome browser [72]. b Conservation of all identified DV motifs among all MEs and DEES. The average phastCons score for all putative DV enhancers (”MEs + DEEs” in dark gray) is significantly higher compared to control regions (”Random non-TSS” in light gray). The average phastCons score of each DV motif (”Motifs only” in red) is in many cases higher than that of the surrounding regions (”MEs + DEEs with motif” in light red). This confirms that motifs like that of Zen and Sna are indeed preferentially found in islands of conservation. Significance was determined by Wilcoxon rank-sum test and marked with a star (p < 0.05)
Mentions: Phylogenetic sequence conservation (“phylogenetic footprinting”) of regulatory regions, and specifically of the transcription factor binding motifs within them, has long been used to identify functional enhancers and motifs [61, 62]. When we analyzed the newly identified enhancers, we noticed that the identified transcription factor motifs were often found within sequence blocks of high conservation across the Drosophila phylogeny, interspersed by more diverged sequences within the same enhancer region. For example, a canonical Twi binding motif in the newly identified if-ME3 enhancer is in a highly conserved sequence block, while a partially conserved sequence block right next to it contains two Dl binding motifs (Fig. 4a left). Likewise, a putative DEE, Btk29A-DEE1, has two conserved, presumably repressive, Sna binding motifs next to a conserved canonical Zen binding motif, each in a conserved sequence block (Fig. 4a right).Fig. 4

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