Regulation of Drosophila eye development by the transcription factor Sine oculis

Abstract

Homeodomain transcription factors of the Sine oculis (SIX) family direct multiple regulatory processes throughout the metazoans. Sine oculis (So) was first characterized in the fruit fly Drosophila melanogaster, where it is both necessary and sufficient for eye development, regulating cell survival, proliferation, and differentiation. Despite its key role in development, only a few direct targets of So have been described previously. In the current study, we aim to expand our knowledge of So-mediated transcriptional regulation in the developing Drosophila eye using ChIP-seq to map So binding regions throughout the genome. We find 7,566 So enriched regions (peaks), estimated to map to 5,952 genes. Using overlap between the So ChIP-seq peak set and genes that are differentially regulated in response to loss or gain of so, we identify putative direct targets of So. We find So binding enrichment in genes not previously known to be regulated by So, including genes that encode cell junction proteins and signaling pathway components. In addition, we analyze a subset of So-bound novel genes in the eye, and find eight genes that have previously uncharacterized eye phenotypes and may be novel direct targets of So. Our study presents a greatly expanded list of candidate So targets and serves as basis for future studies of So-mediated gene regulation in the eye.

Figure 1. Peak distribution and motif enrichment of the So ChIP-seq data set.

(A) Genome distribution of So-enriched regions (peaks) with respect to annotated transcription start sites (TSS). 52.4% of So peaks are <1 kb from the nearest TSS, and 78.9% of peaks are no more than 10 kb from the nearest TSS. (B) Transcription factor binding motifs enriched in the So ChIP-seq peak set. The name of the TF and the percentage of peaks that have at least one occurrence of the TF motif are listed under each motif. Abd-B, Abdominal-B; Cad, Caudal; Dref, DNA Replication Element factor; Hb, Hunchback; Hr78, Hormone receptor 78; Iro-C, Iroquois Complex; Trl, Trithorax-like; Usp, Ultraspiracle.

Figure 2. So ChIP-seq peak in the So target gene atonal maps to a previously known So-regulated enhancer.

The top panel (“So”) shows the So binding profile, which is significantly enriched compared with the negative control (“Control” panel). The bottom panel shows the atonal (ato) protein coding region in green. The yellow bar in the bottom panel marks the 3′ ato enhancer, which is necessary for the onset of ato in the eye and is directly activated by So . The So ChIP-seq peak overlaps this previously identified 3′ ato enhancer.

Figure 3. Novel eye phenotypes of putative So target genes.

Adult eye phenotypes were analyzed using ey-FLP; FRT goi/FRT cl. Homozygous mutant tissue has less pigmentation than heterozygous tissue in omd, CG13192, blot, CG8108, and Syp. In CG2747, CG12007, and l(3)j2D3, homozygous vs. heterozygous tissue cannot be distinguished by color. omd, CG2747, CG12007, and Syp are on chromosome 3R (FRT82B); blot, l(3)j2D3, and CG8108 are on 3L (FRT80B); and CG13192 is on 2R (FRT42D). Bottom panel shows control external eye phenotypes of ey-FLP; FRT/FRT cl flies.

Figure 4. Ommatidial defects in putative So target gene mutants.

Top panel: tangential sections through adult eyes of ey-FLP; FRT goi/FRT cl flies reveal defects ranging from loss of the inner photoreceptor (CG13192) to complete loss of rhabdomeres (blot). Bottom panel: Control sections through the eyes of ey-FLP; FRT/FRT cl flies.