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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.


High-confidence DV enhancers identify novel DV target genes. a All MEs and DEEs that were confirmed by high occupancy of DV transcription factors and differential target gene expression by mRNA-seq. The occupancy of DV transcription factors Dl, Twi, Sna, Zen, Mad, and Zld is shown as a heatmap of normalized ChIP enrichment over input. The enhancers were categorized based on the function of their target genes (transcription factors, signaling, morphogenesis, other) and whether they have high occupancy of Dl, Mad (green and light brown bars, respectively), which indicates that they are likely direct targets of the signaling cascade. b A selection of MEs and DEEs that were confirmed by in vivo reporter expression of overlapping Vienna Tiles (VTs), which match the expression pattern of the assigned target gene. In situ hybridization images for VTs were obtained from Kvon et al. [39]. Unless noted otherwise, the in situ hybridization images for target genes are from the BDGP database [73–75]. The CadN expression pattern is by Biemar et al. [17], copyright (2006) National Academy of Sciences, USA; that of be is from Fly-FISH [76, 77] and was color-modified to resemble the black-and-white in situ hybridization images, reused with permission; that of Wnt2 is from Russell et al. [78] Copyright 1992 The Company of Biologists, and that of Stat92E is from Yan et al. [79], reprinted from Yan et al., Identification of a Stat gene that functions in Drosophila development. Cell, 84(3):421–430, Copyright 1996, with permission from Elsevier
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Fig5: High-confidence DV enhancers identify novel DV target genes. a All MEs and DEEs that were confirmed by high occupancy of DV transcription factors and differential target gene expression by mRNA-seq. The occupancy of DV transcription factors Dl, Twi, Sna, Zen, Mad, and Zld is shown as a heatmap of normalized ChIP enrichment over input. The enhancers were categorized based on the function of their target genes (transcription factors, signaling, morphogenesis, other) and whether they have high occupancy of Dl, Mad (green and light brown bars, respectively), which indicates that they are likely direct targets of the signaling cascade. b A selection of MEs and DEEs that were confirmed by in vivo reporter expression of overlapping Vienna Tiles (VTs), which match the expression pattern of the assigned target gene. In situ hybridization images for VTs were obtained from Kvon et al. [39]. Unless noted otherwise, the in situ hybridization images for target genes are from the BDGP database [73–75]. The CadN expression pattern is by Biemar et al. [17], copyright (2006) National Academy of Sciences, USA; that of be is from Fly-FISH [76, 77] and was color-modified to resemble the black-and-white in situ hybridization images, reused with permission; that of Wnt2 is from Russell et al. [78] Copyright 1992 The Company of Biologists, and that of Stat92E is from Yan et al. [79], reprinted from Yan et al., Identification of a Stat gene that functions in Drosophila development. Cell, 84(3):421–430, Copyright 1996, with permission from Elsevier

Mentions: First, we assembled all putative DV enhancers that have high ChIP-seq occupancy of DV transcription factors (Dl, Twi, Mad, or Zen) and have a nearby gene that is upregulated in the expected tissue based on mRNA-seq data. We then grouped the enhancers based on their pattern of DV transcription factor occupancy and the tissue in which they are active (Fig. 5a). Second, we assembled novel DV enhancers that overlapped a VT whose expression pattern matched that of the assigned nearby gene (Fig. 5b).Fig. 5


Genome-wide identification of Drosophila dorso-ventral enhancers by differential histone acetylation analysis
High-confidence DV enhancers identify novel DV target genes. a All MEs and DEEs that were confirmed by high occupancy of DV transcription factors and differential target gene expression by mRNA-seq. The occupancy of DV transcription factors Dl, Twi, Sna, Zen, Mad, and Zld is shown as a heatmap of normalized ChIP enrichment over input. The enhancers were categorized based on the function of their target genes (transcription factors, signaling, morphogenesis, other) and whether they have high occupancy of Dl, Mad (green and light brown bars, respectively), which indicates that they are likely direct targets of the signaling cascade. b A selection of MEs and DEEs that were confirmed by in vivo reporter expression of overlapping Vienna Tiles (VTs), which match the expression pattern of the assigned target gene. In situ hybridization images for VTs were obtained from Kvon et al. [39]. Unless noted otherwise, the in situ hybridization images for target genes are from the BDGP database [73–75]. The CadN expression pattern is by Biemar et al. [17], copyright (2006) National Academy of Sciences, USA; that of be is from Fly-FISH [76, 77] and was color-modified to resemble the black-and-white in situ hybridization images, reused with permission; that of Wnt2 is from Russell et al. [78] Copyright 1992 The Company of Biologists, and that of Stat92E is from Yan et al. [79], reprinted from Yan et al., Identification of a Stat gene that functions in Drosophila development. Cell, 84(3):421–430, Copyright 1996, with permission from Elsevier
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Related In: Results  -  Collection

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Fig5: High-confidence DV enhancers identify novel DV target genes. a All MEs and DEEs that were confirmed by high occupancy of DV transcription factors and differential target gene expression by mRNA-seq. The occupancy of DV transcription factors Dl, Twi, Sna, Zen, Mad, and Zld is shown as a heatmap of normalized ChIP enrichment over input. The enhancers were categorized based on the function of their target genes (transcription factors, signaling, morphogenesis, other) and whether they have high occupancy of Dl, Mad (green and light brown bars, respectively), which indicates that they are likely direct targets of the signaling cascade. b A selection of MEs and DEEs that were confirmed by in vivo reporter expression of overlapping Vienna Tiles (VTs), which match the expression pattern of the assigned target gene. In situ hybridization images for VTs were obtained from Kvon et al. [39]. Unless noted otherwise, the in situ hybridization images for target genes are from the BDGP database [73–75]. The CadN expression pattern is by Biemar et al. [17], copyright (2006) National Academy of Sciences, USA; that of be is from Fly-FISH [76, 77] and was color-modified to resemble the black-and-white in situ hybridization images, reused with permission; that of Wnt2 is from Russell et al. [78] Copyright 1992 The Company of Biologists, and that of Stat92E is from Yan et al. [79], reprinted from Yan et al., Identification of a Stat gene that functions in Drosophila development. Cell, 84(3):421–430, Copyright 1996, with permission from Elsevier
Mentions: First, we assembled all putative DV enhancers that have high ChIP-seq occupancy of DV transcription factors (Dl, Twi, Mad, or Zen) and have a nearby gene that is upregulated in the expected tissue based on mRNA-seq data. We then grouped the enhancers based on their pattern of DV transcription factor occupancy and the tissue in which they are active (Fig. 5a). Second, we assembled novel DV enhancers that overlapped a VT whose expression pattern matched that of the assigned nearby gene (Fig. 5b).Fig. 5

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.