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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.


Differential H3K27ac analysis across tissues is an effective method to identify tissue-specific enhancers. a Scatterplots of H3K27ac ChIP-seq enrichment at each known DV enhancer (1 kb centered on midpoint) against the transcript levels of the known target gene show a good but modest correlation in dorsal ectoderm precursor embryos gd7 (left) and in mesoderm precursor embryos Tl10b (right). ChIP-seq enrichment values represent fold change over the corresponding input control after normalizing for differences in read count and fragment size. MEs mesoderm enhancers, DEEs dorsal ectoderm enhancers. b The correlation between H3K27ac and transcript levels becomes stronger when the fold changes in H3K27ac levels between the two mutant embryos at each enhancer are plotted against the fold changes in transcript levels of the corresponding target genes. See Additional file 3: Figure S1 for gene names. c De novo identification of DV enhancers based on CBP candidate regions and differential H3K27ac analysis by the package DESeq2. Numerous candidate enhancers were located with CBP ChIP-seq data in wild-type embryos. DESeq2 was then used to identify significant differences in H3K27ac between gd7 and Tl10b embryos within 1-kb windows centered on the CBP peaks. The average DESeq2-normalized ChIP signal for all replicates is shown as a scatterplot on the right. CBP regions significantly different for H3K27ac are shown in blue (MEs) and yellow (DEEs), while non-differential regions are shown in gray. d Average enrichment of H3K27ac in gd7 and Tl10b embryos, as well as CBP in wild-type embryos, is shown for MEs and DEEs that were located distally, at least 1 kb from any transcription start site (TSS). The gray bar represents the 1-kb window used to calculate H3K27ac enrichments, and the red box represents the 201-bp enhancer region. e Examples of newly identified distal enhancers zfh1-ME2 and C15-DEE2. H3K27ac is shown as normalized ChIP enrichments over input, while CBP is shown as normalized ChIP reads (reads per million). The gray bar represents the 1-kb H3K27ac window, and the blue and yellow boxes represent the 201-bp ME or DEE enhancer region, respectively
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Fig1: Differential H3K27ac analysis across tissues is an effective method to identify tissue-specific enhancers. a Scatterplots of H3K27ac ChIP-seq enrichment at each known DV enhancer (1 kb centered on midpoint) against the transcript levels of the known target gene show a good but modest correlation in dorsal ectoderm precursor embryos gd7 (left) and in mesoderm precursor embryos Tl10b (right). ChIP-seq enrichment values represent fold change over the corresponding input control after normalizing for differences in read count and fragment size. MEs mesoderm enhancers, DEEs dorsal ectoderm enhancers. b The correlation between H3K27ac and transcript levels becomes stronger when the fold changes in H3K27ac levels between the two mutant embryos at each enhancer are plotted against the fold changes in transcript levels of the corresponding target genes. See Additional file 3: Figure S1 for gene names. c De novo identification of DV enhancers based on CBP candidate regions and differential H3K27ac analysis by the package DESeq2. Numerous candidate enhancers were located with CBP ChIP-seq data in wild-type embryos. DESeq2 was then used to identify significant differences in H3K27ac between gd7 and Tl10b embryos within 1-kb windows centered on the CBP peaks. The average DESeq2-normalized ChIP signal for all replicates is shown as a scatterplot on the right. CBP regions significantly different for H3K27ac are shown in blue (MEs) and yellow (DEEs), while non-differential regions are shown in gray. d Average enrichment of H3K27ac in gd7 and Tl10b embryos, as well as CBP in wild-type embryos, is shown for MEs and DEEs that were located distally, at least 1 kb from any transcription start site (TSS). The gray bar represents the 1-kb window used to calculate H3K27ac enrichments, and the red box represents the 201-bp enhancer region. e Examples of newly identified distal enhancers zfh1-ME2 and C15-DEE2. H3K27ac is shown as normalized ChIP enrichments over input, while CBP is shown as normalized ChIP reads (reads per million). The gray bar represents the 1-kb H3K27ac window, and the blue and yellow boxes represent the 201-bp ME or DEE enhancer region, respectively

Mentions: We first tested whether the H3K27ac enrichment levels at each enhancer could be used as an absolute marker for enhancer activity in each tissue. By using the transcript levels of the corresponding target genes as a proxy for each enhancer’s activity, we found a reasonably high correlation between H3K27ac enrichment and enhancer activity in both tissues (R2 = 0.36 and R2 = 0.51, Fig. 1a, see Additional file 3: Figure S1 for gene names). This general trend is consistent with previous reports [28–30].Fig. 1


Genome-wide identification of Drosophila dorso-ventral enhancers by differential histone acetylation analysis
Differential H3K27ac analysis across tissues is an effective method to identify tissue-specific enhancers. a Scatterplots of H3K27ac ChIP-seq enrichment at each known DV enhancer (1 kb centered on midpoint) against the transcript levels of the known target gene show a good but modest correlation in dorsal ectoderm precursor embryos gd7 (left) and in mesoderm precursor embryos Tl10b (right). ChIP-seq enrichment values represent fold change over the corresponding input control after normalizing for differences in read count and fragment size. MEs mesoderm enhancers, DEEs dorsal ectoderm enhancers. b The correlation between H3K27ac and transcript levels becomes stronger when the fold changes in H3K27ac levels between the two mutant embryos at each enhancer are plotted against the fold changes in transcript levels of the corresponding target genes. See Additional file 3: Figure S1 for gene names. c De novo identification of DV enhancers based on CBP candidate regions and differential H3K27ac analysis by the package DESeq2. Numerous candidate enhancers were located with CBP ChIP-seq data in wild-type embryos. DESeq2 was then used to identify significant differences in H3K27ac between gd7 and Tl10b embryos within 1-kb windows centered on the CBP peaks. The average DESeq2-normalized ChIP signal for all replicates is shown as a scatterplot on the right. CBP regions significantly different for H3K27ac are shown in blue (MEs) and yellow (DEEs), while non-differential regions are shown in gray. d Average enrichment of H3K27ac in gd7 and Tl10b embryos, as well as CBP in wild-type embryos, is shown for MEs and DEEs that were located distally, at least 1 kb from any transcription start site (TSS). The gray bar represents the 1-kb window used to calculate H3K27ac enrichments, and the red box represents the 201-bp enhancer region. e Examples of newly identified distal enhancers zfh1-ME2 and C15-DEE2. H3K27ac is shown as normalized ChIP enrichments over input, while CBP is shown as normalized ChIP reads (reads per million). The gray bar represents the 1-kb H3K27ac window, and the blue and yellow boxes represent the 201-bp ME or DEE enhancer region, respectively
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Fig1: Differential H3K27ac analysis across tissues is an effective method to identify tissue-specific enhancers. a Scatterplots of H3K27ac ChIP-seq enrichment at each known DV enhancer (1 kb centered on midpoint) against the transcript levels of the known target gene show a good but modest correlation in dorsal ectoderm precursor embryos gd7 (left) and in mesoderm precursor embryos Tl10b (right). ChIP-seq enrichment values represent fold change over the corresponding input control after normalizing for differences in read count and fragment size. MEs mesoderm enhancers, DEEs dorsal ectoderm enhancers. b The correlation between H3K27ac and transcript levels becomes stronger when the fold changes in H3K27ac levels between the two mutant embryos at each enhancer are plotted against the fold changes in transcript levels of the corresponding target genes. See Additional file 3: Figure S1 for gene names. c De novo identification of DV enhancers based on CBP candidate regions and differential H3K27ac analysis by the package DESeq2. Numerous candidate enhancers were located with CBP ChIP-seq data in wild-type embryos. DESeq2 was then used to identify significant differences in H3K27ac between gd7 and Tl10b embryos within 1-kb windows centered on the CBP peaks. The average DESeq2-normalized ChIP signal for all replicates is shown as a scatterplot on the right. CBP regions significantly different for H3K27ac are shown in blue (MEs) and yellow (DEEs), while non-differential regions are shown in gray. d Average enrichment of H3K27ac in gd7 and Tl10b embryos, as well as CBP in wild-type embryos, is shown for MEs and DEEs that were located distally, at least 1 kb from any transcription start site (TSS). The gray bar represents the 1-kb window used to calculate H3K27ac enrichments, and the red box represents the 201-bp enhancer region. e Examples of newly identified distal enhancers zfh1-ME2 and C15-DEE2. H3K27ac is shown as normalized ChIP enrichments over input, while CBP is shown as normalized ChIP reads (reads per million). The gray bar represents the 1-kb H3K27ac window, and the blue and yellow boxes represent the 201-bp ME or DEE enhancer region, respectively
Mentions: We first tested whether the H3K27ac enrichment levels at each enhancer could be used as an absolute marker for enhancer activity in each tissue. By using the transcript levels of the corresponding target genes as a proxy for each enhancer’s activity, we found a reasonably high correlation between H3K27ac enrichment and enhancer activity in both tissues (R2 = 0.36 and R2 = 0.51, Fig. 1a, see Additional file 3: Figure S1 for gene names). This general trend is consistent with previous reports [28–30].Fig. 1

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.