Integrative genomic analysis reveals novel regulatory mechanisms of eyeless during Drosophila eye development

Abstract

Eyeless (ey) is one of the most critical transcription factors for initiating the entire eye development in Drosophila. However, the molecular mechanisms through which Ey regulates target genes and pathways have not been characterized at the genomic level. Using ChIP-Seq, we generated an endogenous Ey-binding profile in Drosophila developing eyes. We found that Ey binding occurred more frequently at promoter compared to non-promoter regions. Ey promoter binding was correlated with the active transcription of genes involved in development and transcription regulation. An integrative analysis revealed that Ey directly regulated a broad and highly connected genetic network, including many essential patterning pathways, and known and novel eye genes. Interestingly, we observed that Ey could target multiple components of the same pathway, which might enhance its control of these pathways during eye development. In addition to protein-coding genes, we discovered Ey also targeted non-coding RNAs, which represents a new regulatory mechanism employed by Ey. These findings suggest that Ey could use multiple molecular mechanisms to regulate target gene expression and pathway function, which might enable Ey to exhibit a greater flexibility in controlling different processes during eye development.

Figure 1.

Genome-wide profiling of Ey-binding regions using ChIP-Seq. (A) A snapshot of Ey ChIP-Seq profiles on chromosome 2L from two independent experiments. (B) Plot showing that the two independent Ey ChIP-Seq experiments are highly reproducible. (C) ChIP-PCR validation of Ey ChIP-Seq results. Enrichment of Ey binding at 10 randomly selected regions identified by ChIP-Seq is shown. Two representative non-Ey binding regions are shown as negative controls. (D) The peak width distribution of Ey-binding regions detected by ChIP-Seq. (E) The range of fold enrichment of Ey peaks across the whole genome. (F) Computer simulation of sequencing depth of Ey ChIP-Seq. (G) Ey ChIP-Seq peaks show Ey binds to previously known Ey-binding regions. The reported Ey-binding regions are marked with red shaded region. The Ey ChIP peaks are normalized to the negative ChIP control.

Figure 2.

Ey binding at promoters is associated with active transcription of developmental or transcription-related genes. (A) Classification of Ey binding in the genome based on genomic features. (B) GO analysis of genes with Ey binding at the promoters. The top three groups enriched at three function categories are shown. (C) Snapshot of ChIP-Seq profiles at chromosome 2R showing that Ey and PolII ChIP-Seq profiles are highly correlated. (D) Pie chart showing the percentage of all Ey peaks that overlap with PolII bound regions. Ey binding is enriched at promoters with PolII occupancy. (E) Plot showing the distance between the ChIP-seq peaks of Ey and PolII at the nearest annotated TSS. (F) Examples showing Ey and PolII ChIP-Seq profiles around TSSs.

Figure 3.

Ey binding at promoters is associated with active transcription of genes at the isoform level. (A) Ey can selectively bind the promoters of specific isoforms of a gene. The left pie chart shows the fraction of genes with multiple isoforms that are bound by Ey at the promoters. The right chart shows the fraction of genes with Ey binding to the promoters of multiple isoforms versus only a single isoform. (B) Pie chart showing percentage of isoform specific Ey peaks that coincide with PolII peaks. (C–E) Examples showing the isoforms bound by Ey are transcribed in third larval instar eye discs and corresponding ChIP-Seq profiles of Ey and PolII at the promoters.

Figure 4.

Identification of Ey-associated enhancers using ChIP-Seq. (A) The ChIP-Seq peak profiles of H3K4me1 and H3K27Ac around Ey-binding peaks at non-TSS regions. The peak intensities of H3K4me1 and H3K27Ac are plotted against their distance to the center of Ey peaks. (B) Examples showing predicted enhancers based on Ey binding and histone modification marks. The red shaded areas mark the identified Ey-associated enhancers. (C) The majority of Ey binding at non-TSS regions is associated with H3K4me1 and/or H3K27Ac modification. The percentage of Ey peaks with particular histone marks are indicated. (D–F) Validation of predicted Ey-associated enhancers using in vivo reporter assays. Examples showing GFP reporter expression driven by enhancers of hipk, tsh and dpp identified by Ey ChIP-Seq. The ChIP-Seq profiles of Ey and other chromatin marks are shown. Enhancers tested in the study are marked with red shaded area; scale bar: 50 μm.

Figure 5.

Identification of Ey targets in developing eye discs. (A) Ey targets identified in the N, Dpp, Wg and Hh signaling pathways are listed. (B and C) Ey targets in the Dpp and N pathways. Identified Ey targets are marked with shaded boxes. (D) Examples of new pathways and cellular events enriched in GO analysis of Ey targets. (E) Expression of novel Ey targets in developing eye discs as detected by in situ hybridization; scale bar: 50 μm.

Figure 6.

Ey binds non-coding RNAs in the genome. (A) A portion of Ey-bound ncRNAs are miRNA primary transcripts. (B) Classification of Ey-bound ncRNAs based on the location in the genome. (C) Some ncRNA-associated Ey peaks are enriched with PolII binding. (D) In situ hybridization showed the expression of Ey-bound ncRNAs in the developing eye disc. Corresponding ChIP-Seq profiles are also shown, with red shaded areas marking Ey peaks; scale bar: 50 μm. (E) Examples showing a bimodal Ey-binding profile at some ncRNAs close to the promoter of adjacent coding genes. The coding gene-associated Ey peaks are marked as red shaded areas.

Figure 7.

A model of Ey function in transcription regulation. (A) In the absence of transcription, both promoters and enhancers are in a ‘closed’ status without any prominent active chromatin signatures. (B) Histones of nucleosomes near promoter and enhancer regions are specifically modified, which exposes regulatory regions and allows transcription factor and/or polymerase binding. (C) Ey binding at the promoter or enhancer regions can be mediated by its DNA-binding domain(s) or via interaction with other transcription factors or regulatory proteins in a dynamic protein complex. This Ey-associated protein complex may interact with PolII transcription machinery at the promoter and thus regulate target gene expression. So and Toy were shown as Ey potential binding partners that may also include other proteins yet to be characterized.