Sumoylation of bZIP transcription factor NRL modulates target gene expression during photoreceptor differentiation

Abstract

Development of rod photoreceptors in the mammalian retina is critically dependent on the basic motif-leucine zipper transcription factor NRL (neural retina leucine zipper). In the absence of NRL, photoreceptor precursors in mouse retina produce only cones that primarily express S-opsin. Conversely, ectopic expression of NRL in post-mitotic precursors leads to a rod-only retina. To explore the role of signaling molecules in modulating NRL function, we identified putative sites of post-translational modification in the NRL protein by in silico analysis. Here, we demonstrate the sumoylation of NRL in vivo and in vitro, with two small ubiquitin-like modifier (SUMO) molecules attached to the Lys-20 residue. NRL-K20R and NRL-K20R/K24R sumoylation mutants show reduced transcriptional activation of Nr2e3 and rhodopsin promoters (two direct targets of NRL) in reporter assays when compared with wild-type NRL. Consistent with this, in vivo electroporation of the NRL-K20R/K24R mutant into newborn Nrl(-/-) mouse retina leads to reduced Nr2e3 activation and only a partial rescue of the Nrl(-/-) phenotype in contrast to the wild-type NRL that is able to convert cones to rod photoreceptors. Although PIAS3 (protein inhibitor of activated STAT3), an E3-SUMO ligase implicated in photoreceptor differentiation, can be immunoprecipitated with NRL, there appears to be redundancy in E3 ligases, and PIAS3 does not seem to be essential for NRL sumoylation. Our studies suggest an important role of sumoylation in fine-tuning the activity of NRL and thereby incorporating yet another layer of control in gene regulatory networks involved in photoreceptor development and homeostasis.

FIGURE 1.

A schematic of the human NRL protein with the sequence alignment of NRL orthologs (upper panel) and MAF family proteins (lower panel). Alignments were performed using AlignX from VectorNTI (Invitrogen). Amino acids conserved in all orthologs and across MAF family members are indicated in red, and less conserved residues are shown in blue. Arrowheads indicate lysine residues with their position in the human NRL protein. Sumoylation sites predicted with high probability are framed by a green rectangle. MTD, minimal transactivation domain; Hinge, hinge domain; EHD, extended homology domain; BM, basic motif; Leu Zipper, leucine zipper; Hsap, Homo sapiens (human); Ptro, Pan troglodyte (chimpanzee); Mmul, Macaca mulatta (rhesus monkey); Cfam, Canis familiaris (dog); Ecab, Equus caballus (horse); Btau, Bos taurus (cow); Mmus, Mus musculus (mouse); Rrat, Rattus norvegicus (rat); Brer, Brachydanio rerio (zebrafish); Xlae, Xenopus laevis (Xenopus); Ggal, Gallus gallus (chicken).

FIGURE 2.

Sumoylation of NRL in vivo and in vitro. A and B, adult mouse retina extracts were immunoprecipitated with anti-NRL IgG (A) or anti-SUMO1 (B) followed by immunoblotting with anti-SUMO1 antibody or anti-NRL IgG, respectively. The arrow indicates sumoylated NRL. IP, immunoprecipitation. C, p53 and GST control experiments in the presence of WT or mutant SUMO1. p53, used as a positive control, was conjugated with SUMO1 and not by mutant SUMO1 under our assay conditions. D and E, purified GST-tagged WT or mutant NRL proteins were sumoylated in vitro with E1 and E2 ligases in the presence of SUMO1 or mutated SUMO1 protein. D shows the immunoblot probed with anti-SUMO1, and E shows the immunoblot probed with anti-NRL antibodies. Arrowheads show sumoylated p53 control, and arrows indicate sumoylated NRL. F, HEK293T cells were co-transfected with plasmids expressing WT-NRL or lysine mutants with or without FLAG-SUMO1. Cell extracts were immunoprecipitated with anti-NRL IgG and immunoblotted with anti-FLAG antibody. The arrowhead shows sumoylated NRL. G, HEK293T cells were transfected with plasmids expressing WT-NRL, NRL-S50T, and NRL-K20R/K24R. After immunoprecipitation of cell extracts with anti-NRL IgG, immunoblot was probed with anti-FLAG antibody. H, HEK293T cells were co-transfected with plasmids expressing WT-NRL, NRL-K20R/K24R, and NRL-S50T with FLAG-SUMO1. Immunoblots of cell extracts were probed with anti-NRL IgG.

FIGURE 3.

Modulation of transcriptional regulatory activity of NRL by sumoylation. HEK293 cells were co-transfected with a construct containing bovine Rho (A) or mouse Nr2e3 promoter (B) driving firefly luciferase reporter gene simultaneously with increasing concentrations (0.01–0.3 μg) of WT- or mutant NRL expression constructs, either alone or in association with CRX and NR2E3. -Fold change is relative to the mock expression vector control. The blue box indicates NRL-responsive element (NRE). Error bars show S.E. Asterisks indicate p value <0.05. Black lines and red, blue, and green lines and asterisks correspond to WT-NRL, NRL-K20R, NRL-K24R, and NRL-K20R/K24R, respectively.

FIGURE 4.

Partial rescue of the Nrl^−/− phenotype and reduced expression of Nr2e3 by NRL-K20R/K24R sumoylation mutant. A, representative retinal photographs of P21 Nrl^−/− mouse retinas: unelectroporated region. onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer. B, C, E, F, and G, electroporated at P0 with Ub-GFP and either Ub-WT-NRL (B, E, and F) or Ub-NRL-K20R/K24R (C and G). A–C, GFP is green, cone arrestin is red, and rhodopsin is gray. White and yellow arrows show GFP+ electroporated cells with or without rhodopsin staining, respectively. Lower panels in B and C show higher magnification images with arrowheads indicating GFP- and rhodopsin-positive photoreceptors. Cone arrestin signal is observed in these cells only with NRL-K20R/K24R mutant. Scale bar: 20 μm. D, quantification of GFP-positive cells expressing rhodopsin after electroporation of WT-NRL (gray) or NRL-K20R/K24R (white). Error bars show S.E. from seven independent electroporated retinas for each construct. **, p < 0.01 by Student's t test. E–G, GFP is green, and NR2E3 immunostaining is red. White and yellow arrows show GFP+ electroporated cells with or without NR2E3 staining, respectively. E and F show different regions of the retina electroporated with WT-NRL. Scale bar: 20 μm. H, quantification of GFP-positive cells in ONL expressing NR2E3 after electroporation of WT-NRL (gray) or NRL-K20R/K24R (white). Error bars show S.E. from four independent electroporated retinas for each construct. **, p < 0.01 by Student's t test.

FIGURE 5.

A, expression of Pias3 in rod photoreceptors. Dissociated cells from adult Nrl-GFP mouse retina were stained with anti-PIAS3-antibody and 4′,6-diamidino-2-phenylindole (DAPI). Arrows indicate colocalization of PIAS3 (red) in GFP-positive rods (green). B, co-immunoprecipitation of PIAS3 and NRL from retinal extracts. Immunoblots of anti-NRL immunoprecipitated (IP) proteins from P0, P4, P10, and adult (Ad) retina were probed with anti-PIAS3 antibody. Normal rabbit IgG served as negative control. C, immunoblot analysis of mock- and PIAS3-transfected HEK293T extracts with anti-PIAS3 antibody. D, conjugation of SUMO1 to NRL in the transfected cells in the absence or presence of PIAS3. HEK293T cell extracts transfected with WT-NRL, FLAG-SUMO and PIAS3 were immunoprecipitated with anti-NRL, and immunoblots were probed with anti-NRL or anti-FLAG antibody. Black arrows indicate sumoylated NRL protein.