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.
Highlights
Spatiotemporal control of gene expression is critical for development and homeostasis [1]
Differentiation and homeostasis of photoreceptors are tightly controlled by a set of key transcriptional regulatory proteins, which include nuclear receptors (such as ROR [15], thyroid hormone receptor 2 (TR2) [16], and NR2E3 [17,18,19,20,21]), homeodomain proteins (such as orthodenticle homeobox 2 (OTX2) [22] and CRX [23, 24], signal transducers (including STAT3 [25], PIAS3 [26], glycogen synthase kinase 3 (GSK3) [27]), and NRL, a basic motif-leucine zipper protein of Maf subfamily [28]
Because sumoylation can depend on the phosphorylation state of the target protein [51, 52], we examined the consequence of the S50T mutation, which affects NRL phosphorylation [45, 46]
Summary
Loss of Nr2e3 in mice results in rod photoreceptors that express cone genes and eventually degenerate [17, 18, 36, 37]. The expression of rod-specific visual pigment protein, rhodopsin, reveals a substantial “delay” with two distinct phases [41] despite the presence of key activator proteins, NRL, CRX, and NR2E3 [40]. PIAS3, an E3-SUMO ligase, has been shown to interact with CRX and NR2E3 and play a significant role in rod differentiation by sumoylating the NR2E3 protein [26]. Chromatin remodeling and post-translational modifications can contribute to photoreceptor development by modulating cell type-specific transcription. To gain insights into the role of extrinsic signaling molecules in guiding retinal development and homeostasis, we are exploring the impact of post-translational modifications on the transcriptional regulatory function of NRL. We show that the NRL protein is disumoylated in vivo and in vitro and that sumoylation of NRL modifies its activity toward two distinct target promoters, Nr2e3 and rhodopsin. Our studies further strengthen the growing role of posttranslational mechanisms in influencing photoreceptor development and function
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