Nr2e3-directed transcriptional regulation of genes involved in photoreceptor development and cell-type specific phototransduction

Abstract

The retinal transcription factor Nr2e3 plays a key role in photoreceptor development and function. In this study we examine gene expression in the retina of Nr2e3(rd7/rd7) mutants with respect to wild-type control mice, to identify genes that are misregulated and hence potentially function in the Nr2e3 transcriptional network. Quantitative candidate gene real time PCR and subtractive hybridization approaches were used to identify transcripts that were misregulated in Nr2e3(rd7/rd7) mice. Chromatin immunoprecipitation assays were then used to determine which of the misregulated transcripts were direct targets of NR2E3. We identified 24 potential targets of NR2E3. In the developing retina, NR2E3 targets transcription factors such as Ror1, Rorg, and the nuclear hormone receptors Nr1d1 and Nr2c1. In the mature retina NR2E3 targets several genes including the rod specific gene Gnb1 and cone specific genes blue opsin, and two of the cone transducin subunits, Gnat2 and Gnb3. In addition, we identified 5 novel transcripts that are targeted by NR2E3. While mislocalization of proteins between rods and cones was not observed, we did observe diminished concentration of GNB1 protein in adult Nr2e3(rd7/rd7) retinas. These studies identified novel transcriptional pathways that are potentially targeted by Nr2e3 in the retina and specifically demonstrate a novel role for NR2E3 in regulating genes involved in phototransduction.

Figure 1. Genes misregulated in the developing and mature Nr2e3^rd7/rd7 retina

Relative fold change of genes misregulated in Nr2e3^rd7/rd7 retinas compared to control, B6, retinas. Panel A are genes misregulated at P2, Panel B are genes misregulated at P21. Standard errors are indicated for each gene. All genes met a minimum significance of p ≤ 0.05.

Figure 2. Direct targets for NR2E3

Chromatin immunoprecipitation assays to identify direct targets of NR2E3 at P2 and P21. Genes for each amplicon are indicated at the bottom of each gel. Marker (100 base pair ladder, Roche), I (input DNA, positive control) +NR2E3 (NR2E3 precipitated chromatin sample), +IgG (negative control, goat IgG precipitated chromatin) Mock (negative, no chromatin control).

Figure 3. NR2E3 directly regulates rod specific genes that are not misexpressed in Nr2e3^rd7/rd7 retinas

P30 B6 and Nr2e3^rd7/rd7 retinas labeled for GNB1 in rod photoreceptor cells. A–C: B6 retina double labeled with (A) GNB1 and (B) rhodopsin, and (C) merged image shows co-localization of both GNB1 and rhodopsin with DAPI nuclear stain (blue). D–F: Nr2e3^rd7/rd7 retina double labeled with GNB1 (D) and rhodopsin (E), and merged image with GNB1, rhodopsin, and DAPI nuclear stain (blue) (F) shows co-localization of both proteins. G–I: B6 retina double labeled with GNB1 (G) and cone marker PNA (H), and merged image with GNB1, PNA, and DAPI nuclear stain (blue) (I) shows no co-localization. Inset is a higher magnification view of cells indicated with asterisks. J–L Nr2e3^rd7/rd7 retina double labeled with GNB1 (J) and cone marker PNA (K), and merged image with GNB1, PNA, and DAPI nuclear stain (blue) (L) shows no co-localization. Inset is a higher magnification view of cells indicated with asterisks. GNB1 is expressed in both the inner and outer segments of B6 retina; expression appears to be restricted to the outer segments in Nr2e3^rd7/rd7 retinas.