Integrative genomic analyses reveal putative cell type-specific targets of the Drosophila ets transcription factor Pointed

Abstract

The Ets domain transcription factors direct diverse biological processes throughout all metazoans and are implicated in development as well as in tumor initiation, progression and metastasis. The Drosophila Ets transcription factor Pointed (Pnt) is the downstream effector of the Epidermal growth factor receptor (Egfr) pathway and is required for cell cycle progression, specification, and differentiation of most cell types in the larval eye disc. Despite its critical role in development, very few targets of Pnt have been reported previously. Here, we employed an integrated approach by combining genome-wide single cell and bulk data to identify putative cell type-specific Pnt targets. First, we used chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) to determine the genome-wide occupancy of Pnt in late larval eye discs. We identified enriched regions that mapped to an average of 6,941 genes, the vast majority of which are novel putative Pnt targets. Next, we integrated ChIP-seq data with two other larval eye single cell genomics datasets (scRNA-seq and snATAC-seq) to reveal 157 putative cell type-specific Pnt targets that may help mediate unique cell type responses upon Egfr-induced differentiation. Finally, our integrated data also predicts cell type-specific functional enhancers that were not reported previously. Together, our study provides a greatly expanded list of putative cell type-specific Pnt targets in the eye and is a resource for future studies that will allow mechanistic insights into complex developmental processes regulated by Egfr signaling.

Keywords: Differentiation; Drosophila eye disc; Epidermal growth factor receptor; Integrative genomics; Pointed-ChIP-seq; Single cell genomics.

Fig. 1

Binding profiles of Pnt ChIP-seq replicates and snATAC-seq show clear enrichment at known targets of Pnt. (A) Schematic of Epidermal growth factor receptor (Egfr) pathway and cell type differentiation in larval eye discs. Induction of Egfr by Spitz (Spi) triggers the activation of Ras, which in turn activates Extracellular signal regulated kinase (Erk). Erk translocates to the nucleus and activates Pointed (Pnt), which mediates the transcriptional output of Egfr pathway. Except R8, all cell types undergo differentiation upon Egfr activation. (B) Schematic of tangential sections of an adult ommatidium showing different cell types. 1° = primary pigment cells; 2° = secondary pigment cells; 3° = tertiary pigment cells; and B = bristle cells. (C) Late larval scRNA-seq plot showing clusters corresponding to all major cell types present in the physical eye disc. (D) Plot showing the expression pattern of hh from single cell RNA sequencing (scRNA-seq) data. The intensity of blue is proportional to log-normalized expression levels. (E) The hedgehog (hh) locus with ChIP-seq peaks overlapping an enhancer reported to be bound by Pnt (black rectangular box). FLAG1, FLAG2, FLAG3, GFP1 and GFP2 are Pnt ChIP-seq biological replicates. (F) snATAC-seq genomic track showing the hh locus with peaks that overlap the known enhancer and peak shown in (E). Predicted Ets binding sites are shown as red triangles

Fig. 2

Putative coregulators of Pnt are expressed in overlapping patterns with pnt. (A-K) scRNA-seq plots showing the expression patterns of pnt (A), aop (B), crol (C), ttk (D), Aef1 (E), klu (F), BEAF-32 (G), med (H), mad (I), CG15812 (J), and prd (K). Other than prd, all genes show overlapping expression with pnt

Fig. 3

Pnt ChIP-seq peak profiles are found near genes with known essential roles in eye development. (A) ChIP-seq genomic tracks showing peaks in the intron of rough (ro). The peak region is highlighted in gray. The solid black bar indicates a known enhancer of ro. (B) scRNA-seq plot showing the expression of ro in the MF, R2/5 and R3/4. (C) snATAC-seq plot showing the ro locus with a peak in an intron that aligns with the ChIP-seq peak. The peak region is highlighted in gray. Four Pnt binding sites are shown as red triangles

Fig. 4

pyramus is a putative novel cell type-specific target of Pnt. (A) Genomic track showing a prominent Pnt ChIP-seq peak about 11 kb 3’ of pyramus (pyr). (B) Larval eye disc scRNA-seq plot showing the expression (blue) of pyr in R1/6, R7 and the Convergence cell clusters. (C) snATAC-seq genomic tracks at the pyr locus show prominent peaks in R1/6, R7 and the Convergence cell clusters. This peak is present at the same genomic location as the 3’ peak in the ChIP-seq data sets (A). The black box represents the peak-region DNA that was tested for enhancer activity in vivo. (D-F) Immunostaining of transgenic larval eye discs carrying the peak-region DNA in (A) and (C) driving expression of a dGFP reporter. Larval eye discs were stained with GFP (E) and Runt (F), which marks R7 and R8. Many ommatidia show coexpression of GFP and Runt antibodies (D)