Protein kinase CK2 modulates NRL-mediated gene expression in rod photoreceptors of mammalian retina

Maf-family basic motif leucine zipper protein NRL specifies rod photoreceptor cell fate during retinal development and, in concert with homeodomain protein CRX and other regulatory factors, controls the expression of most rod-expressed genes including the visual pigment gene RHO (rhodopsin). Transcriptional regulatory activity of NRL is modulated by post-translational modifications (PTMs), specially phosphorylation, and mutations in phosphosites can lead to retinal degeneration. During our studies to elucidate NRL-mediated transcriptional regulation, we identified Protein Kinase CK2 in NRL-enriched complexes bound to RHO promoter-enhancer regions and in NRL-enriched high molecular mass fractions from the bovine retina. The presence of CK2 in NRL complexes was confirmed by co-immunoprecipitation from developing and adult mouse retinal extracts. Bioinformatic and mass spectrometry analysis indicated potential phosphorylation of NRL at Ser117 residue by CK2. Co-transfection of Csnk2a1 cDNA encoding CK2 with NRL and CRX reduced the bovine Rho promoter-driven luciferase expression in HEK293 cells, and mutagenesis of NRL-Ser117 residue to Ala restored the reporter gene expression. In concordance, overexpression of CK2 in the mouse retina in vivo by electroporation resulted in reduction of Rho promoter-driven DsRed reporter gene expression. RNA-seq analysis of CK2-electroporated retina revealed altered expression of many transcriptional targets of NRL. Thus, our studies demonstrate an important role of CK2 in context-dependent fine-tuning of NRL function, consequently modulating the expression of rod photoreceptor genes. Electroporated areas of the retinas were collected at postnatal day 8 and disassociated with dissociation solution (30 U/mL papain, HBSS pH 7.4, 1 mg/mL glucose, 10 mM Hepes, 100 U/mL DNase I, 5 µg/mL superoxide dismutase, 5 µg/mL catalase, 10 µg/mL D-alpha-tocopherol acetate, 1 mg/mL glucoscysteine-HCl, 5 µg/mL superoxide dismutase, 50 µg/mL gentamycin). Intact GFP positive rod photoreceptors were manually collected with a micromanipulator. Libraries were generated from 50-100 isolated rod cells using SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian (Takara Bio USA, Inc). We prepared three independent RNA samples for each treatment and performed paired-end sequencing using HiSeq2500 (Illumina, San Diego, USA).