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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.


The identified putative DV enhancer regions are enriched for known DV transcription factor motifs. a Known Drosophila motifs that are significantly enriched (red) or depleted (blue) at MEs or DEEs over non-differential control regions, or DEEs over MEs, are shown as sequence logos in bits on the left. The transcription factor that is known to bind the motif is shown on the right. If there are several transcription factors (TFs), all matches are shown, and the factor that is known to function during DV patterning is underlined. Significance was determined by a one-sided proportion test (* p < 0.05, ** p < 10−2, *** p < 10−3, **** p < 10−4, ***** p < 10−5 after Benjamini-Hochberg correction). b Many identified transcription factors are expressed in a DV-specific pattern themselves, e.g., localized to the mesoderm (top) or the dorsal ectoderm (bottom) in in situ hybridization images from stage 4, obtained from the Berkeley Drosophila Genome Project (BDGP) in situ hybridization database [73–75]. Among them are lov and Dll, which are expressed in the dorsal ectoderm. c Expression of lov and Dll in the dorsal ectoderm could be mediated by two enhancers identified among DEEs (lov-DEE4 and Dll-DEE-TSS). Overlapping VTs drive reporter activity similar to the endogenous genes. Both enhancers are occupied by Mad, Zen, and Zld based on ChIP-seq data, indicating direct regulation by the DV network. Dl, Twi ChIP-seq are from Tl10b embryos, Sna ChIP-seq from wild-type embryos, and Mad, Zen, Zld ChIP-seq from gd7 embryos. d DV regulatory network based on the rediscovered transcription factors and lov and Dll as added components (boxed). The shown regulatory interactions are based on literature knowledge, confirmed by our own ChIP-seq data. Shown in red are transcription factors that likely function as repressors, since the tissue in which they are expressed is distinct from the tissue of their target genes
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Fig3: The identified putative DV enhancer regions are enriched for known DV transcription factor motifs. a Known Drosophila motifs that are significantly enriched (red) or depleted (blue) at MEs or DEEs over non-differential control regions, or DEEs over MEs, are shown as sequence logos in bits on the left. The transcription factor that is known to bind the motif is shown on the right. If there are several transcription factors (TFs), all matches are shown, and the factor that is known to function during DV patterning is underlined. Significance was determined by a one-sided proportion test (* p < 0.05, ** p < 10−2, *** p < 10−3, **** p < 10−4, ***** p < 10−5 after Benjamini-Hochberg correction). b Many identified transcription factors are expressed in a DV-specific pattern themselves, e.g., localized to the mesoderm (top) or the dorsal ectoderm (bottom) in in situ hybridization images from stage 4, obtained from the Berkeley Drosophila Genome Project (BDGP) in situ hybridization database [73–75]. Among them are lov and Dll, which are expressed in the dorsal ectoderm. c Expression of lov and Dll in the dorsal ectoderm could be mediated by two enhancers identified among DEEs (lov-DEE4 and Dll-DEE-TSS). Overlapping VTs drive reporter activity similar to the endogenous genes. Both enhancers are occupied by Mad, Zen, and Zld based on ChIP-seq data, indicating direct regulation by the DV network. Dl, Twi ChIP-seq are from Tl10b embryos, Sna ChIP-seq from wild-type embryos, and Mad, Zen, Zld ChIP-seq from gd7 embryos. d DV regulatory network based on the rediscovered transcription factors and lov and Dll as added components (boxed). The shown regulatory interactions are based on literature knowledge, confirmed by our own ChIP-seq data. Shown in red are transcription factors that likely function as repressors, since the tissue in which they are expressed is distinct from the tissue of their target genes

Mentions: We then scored the relative motif enrichments among MEs or DEEs over non-differential H3K27ac control regions, or DEEs over MEs using a Fisher exact test (p < 0.05 after Benjamini-Hochberg correction, Fig. 3a). To avoid false positives, we only scored motifs for which the transcription factor was expressed in either the mesoderm (Tl10b) or dorsal ectoderm tissue (gd7). We then collapsed motifs whose occurrences substantially overlapped (see Methods). In these cases, we show the most significant motif with the corresponding transcription factor, as well as other transcription factors if they have known roles in DV patterning. In total, we found 13 independent motifs that were significantly enriched in our putative DV enhancers (Fig. 3a).Fig. 3


Genome-wide identification of Drosophila dorso-ventral enhancers by differential histone acetylation analysis
The identified putative DV enhancer regions are enriched for known DV transcription factor motifs. a Known Drosophila motifs that are significantly enriched (red) or depleted (blue) at MEs or DEEs over non-differential control regions, or DEEs over MEs, are shown as sequence logos in bits on the left. The transcription factor that is known to bind the motif is shown on the right. If there are several transcription factors (TFs), all matches are shown, and the factor that is known to function during DV patterning is underlined. Significance was determined by a one-sided proportion test (* p < 0.05, ** p < 10−2, *** p < 10−3, **** p < 10−4, ***** p < 10−5 after Benjamini-Hochberg correction). b Many identified transcription factors are expressed in a DV-specific pattern themselves, e.g., localized to the mesoderm (top) or the dorsal ectoderm (bottom) in in situ hybridization images from stage 4, obtained from the Berkeley Drosophila Genome Project (BDGP) in situ hybridization database [73–75]. Among them are lov and Dll, which are expressed in the dorsal ectoderm. c Expression of lov and Dll in the dorsal ectoderm could be mediated by two enhancers identified among DEEs (lov-DEE4 and Dll-DEE-TSS). Overlapping VTs drive reporter activity similar to the endogenous genes. Both enhancers are occupied by Mad, Zen, and Zld based on ChIP-seq data, indicating direct regulation by the DV network. Dl, Twi ChIP-seq are from Tl10b embryos, Sna ChIP-seq from wild-type embryos, and Mad, Zen, Zld ChIP-seq from gd7 embryos. d DV regulatory network based on the rediscovered transcription factors and lov and Dll as added components (boxed). The shown regulatory interactions are based on literature knowledge, confirmed by our own ChIP-seq data. Shown in red are transcription factors that likely function as repressors, since the tissue in which they are expressed is distinct from the tissue of their target genes
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Fig3: The identified putative DV enhancer regions are enriched for known DV transcription factor motifs. a Known Drosophila motifs that are significantly enriched (red) or depleted (blue) at MEs or DEEs over non-differential control regions, or DEEs over MEs, are shown as sequence logos in bits on the left. The transcription factor that is known to bind the motif is shown on the right. If there are several transcription factors (TFs), all matches are shown, and the factor that is known to function during DV patterning is underlined. Significance was determined by a one-sided proportion test (* p < 0.05, ** p < 10−2, *** p < 10−3, **** p < 10−4, ***** p < 10−5 after Benjamini-Hochberg correction). b Many identified transcription factors are expressed in a DV-specific pattern themselves, e.g., localized to the mesoderm (top) or the dorsal ectoderm (bottom) in in situ hybridization images from stage 4, obtained from the Berkeley Drosophila Genome Project (BDGP) in situ hybridization database [73–75]. Among them are lov and Dll, which are expressed in the dorsal ectoderm. c Expression of lov and Dll in the dorsal ectoderm could be mediated by two enhancers identified among DEEs (lov-DEE4 and Dll-DEE-TSS). Overlapping VTs drive reporter activity similar to the endogenous genes. Both enhancers are occupied by Mad, Zen, and Zld based on ChIP-seq data, indicating direct regulation by the DV network. Dl, Twi ChIP-seq are from Tl10b embryos, Sna ChIP-seq from wild-type embryos, and Mad, Zen, Zld ChIP-seq from gd7 embryos. d DV regulatory network based on the rediscovered transcription factors and lov and Dll as added components (boxed). The shown regulatory interactions are based on literature knowledge, confirmed by our own ChIP-seq data. Shown in red are transcription factors that likely function as repressors, since the tissue in which they are expressed is distinct from the tissue of their target genes
Mentions: We then scored the relative motif enrichments among MEs or DEEs over non-differential H3K27ac control regions, or DEEs over MEs using a Fisher exact test (p < 0.05 after Benjamini-Hochberg correction, Fig. 3a). To avoid false positives, we only scored motifs for which the transcription factor was expressed in either the mesoderm (Tl10b) or dorsal ectoderm tissue (gd7). We then collapsed motifs whose occurrences substantially overlapped (see Methods). In these cases, we show the most significant motif with the corresponding transcription factor, as well as other transcription factors if they have known roles in DV patterning. In total, we found 13 independent motifs that were significantly enriched in our putative DV enhancers (Fig. 3a).Fig. 3

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.