Dynamics of the rhomboid-like protein RHBDD2 expression in mouse retina and involvement of its human ortholog in retinitis pigmentosa.

The novel rhomboid-like protein RHBDD2 is distantly related to rhomboid proteins, a group of highly specialized membrane-bound proteases that catalyze regulated intramembrane proteolysis. In retina, RHBDD2 is expressed from embryonic stages to adulthood, and its levels show age-dependent changes. RHBDD2 is distinctly abundant in the perinuclear region of cells, and it localizes to their Golgi. A glycine zipper motif present in one of the transmembrane domains of RHBDD2 is important for its packing into the Golgi membranes. Its deletion causes dislodgment of RHBDD2 from the Golgi. A specific antibody against RHBDD2 recognizes two forms of the protein, one with low (39 kDa; RHBDD2(L)) and the other with high (117 kDa; RHBDD2H) molecular masses in mouse retinal extracts. RHBDD2(L) seems to be ubiquitously expressed in all retinal cells. In contrast, RHBDD2H seems to be present only in the outer segments of cone photoreceptors and may correspond to a homotrimer of RHBDD2(L). This protein consistently co-localizes with S- and M-types of cone opsins. We identified a homozygous mutation in the human RHBDD2 gene, R85H, that co-segregates with disease in affected members of a family with autosomal recessive retinitis pigmentosa. Our findings suggest that the RHBDD2 protein plays important roles in the development and normal function of the retina.

Sequence-specific binding of recombinant Zbed4 to DNA: insights into Zbed4 participation in gene transcription and its association with other proteins.

Zbed4, a member of the BED subclass of Zinc-finger proteins, is expressed in cone photoreceptors and glial Müller cells of human retina whereas it is only present in Müller cells of mouse retina. To characterize structural and functional properties of Zbed4, enough amounts of purified protein were needed. Thus, recombinant Zbed4 was expressed in E. coli and its refolding conditions optimized for the production of homogenous and functionally active protein. Zbed4's secondary structure, determined by circular dichroism spectroscopy, showed that this protein contains 32% α-helices, 18% ß-sheets, 20% turns and 30% unordered structures. CASTing was used to identify the target sites of Zbed4 in DNA. The majority of the DNA fragments obtained contained poly-Gs and some of them had, in addition, the core signature of GC boxes; a few clones had only GC-boxes. With electrophoretic mobility shift assays we demonstrated that Zbed4 binds both not only to DNA and but also to RNA oligonucleotides with very high affinity, interacting with poly-G tracts that have a minimum of 5 Gs; its binding to and GC-box consensus sequences. However, the latter binding depends on the GC-box flanking nucleotides. We also found that Zbed4 interacts in Y79 retinoblastoma cells with nuclear and cytoplasmic proteins Scaffold Attachment Factor B1 (SAFB1), estrogen receptor alpha (ERα), and cellular myosin 9 (MYH9), as shown with immunoprecipitation and mass spectrometry studies as well as gel overlay assays. In addition, immunostaining corroborated the co-localization of Zbed4 with these proteins. Most importantly, in vitro experiments using constructs containing promoters of genes directing expression of the luciferase gene, showed that Zbed4 transactivates the transcription of those promoters with poly-G tracts.

Regulation of retinoschisin secretion in Weri-Rb1 cells by the F-actin and microtubule cytoskeleton.

Retinoschisin is encoded by the gene responsible for X-linked retinoschisis (XLRS), an early onset macular degeneration that results in a splitting of the inner layers of the retina and severe loss in vision. Retinoschisin is predominantly expressed and secreted from photoreceptor cells as a homo-oligomer protein; it then associates with the surface of retinal cells and maintains the retina cellular architecture. Many missense mutations in the XLRS1 gene are known to cause intracellular retention of retinoschisin, indicating that the secretion process of the protein is a critical step for its normal function in the retina. However, the molecular mechanisms underlying retinoschisin's secretion remain to be fully elucidated. In this study, we investigated the role of the F-actin cytoskeleton in the secretion of retinoschisin by treating Weri-Rb1 cells, which are known to secrete retinoschisin, with cytochalasin D, jasplakinolide, Y-27632, and dibutyryl cGMP. Our results show that cytochalasin D and jasplakinolide inhibit retinoschisin secretion, whereas Y-27632 and dibutyryl cGMP enhance secretion causing F-actin alterations. We also demonstrate that high concentrations of taxol, which hyperpolymerizes microtubules, inhibit retinoschisin secretion. Our data suggest that retinoschisin secretion is regulated by the F-actin cytoskeleton, that cGMP or inhibition of ROCK alters F-actin structure enhancing the secretion, and that the microtubule cytoskeleton is also involved in this process.

Sp4 is expressed in retinal neurons, activates transcription of photoreceptor-specific genes, and synergizes with Crx.

To investigate the molecular mechanisms of photoreceptor-specific gene transcription, we examined the role of the neuronal-enriched Sp4 nuclear protein in transcription from the rod-specific beta-PDE and rod opsin gene promoters and compared it to the ubiquitous members of the Sp family, Sp1 and Sp3. Sp4 activates both the rod opsin and beta-PDE promoters, whereas Sp1 activates only the rod opsin promoter and Sp3 activates neither promoter. Interestingly, Sp1 and Sp3 competitively repress Sp4-mediated activation of the beta-PDE promoter. In addition, Sp4, Sp1, and Sp3 each show functional synergy with the photoreceptor-enriched Crx transcriptional regulator on the rod opsin promoter but not the beta-PDE promoter, although Sp4-mediated activation was the most significant. Sp4, Sp1, and Sp3 bind Crx in co-immunoprecipitation experiments, and their zinc finger domains as well as the Crx homedomain are necessary and sufficient for these interactions. Chromatin immunoprecipitation showed that the rod opsin and beta-PDE promoters are targets of both Sp4 and Crx, which further supports Sp4-Crx interactions in vivo in the context of retinal chromatin environment. In situ hybridization and immunohistochemistry demonstrated that Sp4 is abundantly expressed in various neurons of all retinal layers, and thus co-localizes or overlaps with multiple retina-restricted and -enriched genes, its putative targets. Our results indicate that photoreceptor-specific gene transcription is controlled by the combinatorial action of Sp4 and Crx. The other Sp family members may be involved in photoreceptor-specific transcription directly or through their competition with Sp4. These data suggest the potential importance of Sp4 in retinal neurobiology and pathology.

The rod cGMP-phosphodiesterase beta-subunit promoter is a specific target for Sp4 and is not activated by other Sp proteins or CRX.

The beta-subunit of cGMP-phosphodiesterase (beta-PDE) is a key protein in phototransduction expressed exclusively in rod photoreceptors. It is necessary for visual function and for structural integrity of the retina. beta-PDE promoter deletions showed that the -45/-23 region containing a consensus Crx-response element (CRE) was necessary for low level transcriptional activity. Overexpressed Crx modestly transactivated this promoter in 293 human embryonic kidney cells; however, mutation of CRE had no significant effect on transcription either in transfected Y79 retinoblastoma cells or Xenopus embryonic heads. Thus, Crx is unlikely to be a critical beta-PDE transcriptional regulator in vivo. Interestingly, although the beta/GC element (-59/-49) binds multiple Sp transcription factors in vitro, only Sp4, but not Sp1 or Sp3, significantly enhanced beta-PDE promoter activity. Thus, the Sp4-mediated differential activation of the beta-PDE transcription defines the first specific Sp4 target gene reported to date and implies the importance of Sp4 for retinal function. Further extensive mutagenesis of the beta-PDE upstream sequences showed no additional regulatory elements. Although this promoter lacks a canonical TATA box or Inr element, it has the (T/A)-rich beta/TA sequence located within the -45/-23 region. We found that it binds purified TBP and TFIIB in gel mobility shift assays with cooperative enhancement of binding affinity.