Several lines of initial evidence have suggested that the FIZ1 protein can exist in both the cell's nuclear and cytoplasmic compartments in cultured cells and in neural retina [11–13]. We desired to visualize FIZ1 location in the neural retina at the subcellular level. Current improvements to TEM-immunogold techniques, particularly the use of smaller nano-gold antibody conjugates and silver-enhancement, provide clear visualization of nano-gold particles and lower background in electron micrographs of mammalian retina . Ultra-thin sections also expose the dense chromatin matrix of the very compact nuclei of photoreceptor cells in the outer-nuclear layer.
Nano-gold TEM micrographs show that FIZ1 protein is present in the nuclei of all mature neurons in the ganglion, inner-nuclear and outer-nuclear layers (i.e. photoreceptors). This included nuclei of both rod and cone photoreceptors. Labeling of FIZ1 was present throughout the entire area of photoreceptor nuclei. While previous immunofluorescence study showed a distribution of NRL throughout rod-photoreceptor nuclei in human retina , the photoreceptors of nocturnal species (mouse) have compact nuclei with an inverted pattern of chromatin density. In mouse, dense chromatin fills the central nucleus, leaving lower density chromatin at the periphery. Evidence is mounting that this periphery corresponds to euchromatin, where active photoreceptor-specific genes are located. Fluorescent labeling of the mouse Rho gene has demonstrated a location in the periphery of most photoreceptor nuclei .
Some immunohistochemistry reports show detection of CRX throughout photoreceptor nuclei (mouse) [34, 35], while immunofluorescence labeling with confocal imaging shows an intense peripheral labeling for CRX [9, 33]. Immunofluorescence of NR2E3 in the mouse retina also shows an intense labeling in the nuclear periphery [9, 10, 36]. Mice expressing a fusion protein of GFP with OTX2, another transriptional activator present in photoreceptors, display a ring-like distribution of the GFP tag in their photoreceptors . It is clear that this region correlates with the expression of active genes in mouse photoreceptors.
Recent reports also indicate that NRL and CRX have roles in the transcriptional repression of cone-specific genes in addition to their roles as transcriptional activators of rod-specific genes. The ectopic expression of NRL in mouse cone progenitors, results in the conversion of cone-precursors to rod photoreceptors . CRX is also present on cone-opsin promoters in rod photoreceptor cells, and NR2E3 inhibits the CRX-mediated activation of cone-opsin promoters, in vitro[9, 10]. The later could explain why NR2E3 deficient mice develop extra cone-opsin expressing photoreceptors in the form of hybrid cone-rod photoreceptors . As such, NRL and CRX, two transcription factors that can bind FIZ1, could recruit FIZ1 to both euchromatin and heterochromatin regions of mouse photoreceptors.
Significant labeling of FIZ1 was also present in the inner segments of photoreceptors in mature retina. The packing of photoreceptor cells to high density is facilitated by the location of ribosomes and mitochondria in the inner-segments. As the location for much of the protein synthesis in photoreceptors, many proteins should be present in this region. We did not detect FIZ1 by immunogold staining in ultra thin sections of P-5 retina, which is likely due to the low sensitivity of this particular technique and a much lower level of FIZ1 protein in P-5 retina. This is consistent with previous findings, showing that FIZ1 concentration in immature neural retina is less than 10% of that found in the mature tissue . The inability to detect this lower level of FIZ1 does not indicate that FIZ1 is absent. The more sensitive Q-Chip analysis confirmed this. FIZ1 association with the Rho promoter at P-3 was much lower than at P-25; however, the level of FIZ1 at P-3 was detectable above the background, as represented by an untranslated control region on the same chromosome (Chr-6).
Previously we have shown that FIZ1 could alter activation of a Rho test-promoter, suggesting that FIZ1 could have some direct or indirect interactions with CRX in addition to NRL . CRX is present in all rod and cone photoreceptors, placing FIZ1 in a biologically relevant location for interaction with CRX. Our co-IP experiments from bovine retina nuclear extract, confirmed that native FIZ1 and CRX could be isolated as part of the same nuclear protein complex. Co-IP with antibody to FIZ1 captured CRX from nuclear protein preparations. Conversely, antibody to CRX co-precipitated FIZ1.
Peng and Chen (2005) have reported that NRL, CRX and NR2E3 are part of the transcriptional complex on several photoreceptor-specific genes in vivo, including Rho, Pde6b, Rbp3, M-Opsin and S-Opsin . Our chromatin immunoprecipitation results indicated that FIZ1 is also associated with the proximal promoter regions of these genes. Our results here, and those previously reported, indicate that FIZ1 can alter the activation potential of NRL and/or CRX at four of these promoters in vitro: Rho, PDE6B, M-Opsin, and S-Opsin. Additionally, ChIP analysis found that FIZ1 occupies the regulatory complex of photoreceptor-specific genes in wild-type retina, but not in rodless/coneless mice (rd1). This result indicates that FIZ1 recruitment to the regulatory protein complex at photoreceptor-specific genes is an event specific to adult photoreceptors. While FIZ1 is present in bipolar and ganglion cells, it is not associated with photoreceptor-specific gene promoters in these neurons.
Our EMSA results indicate that FIZ1 is also a component of retinal nuclear protein complexes that can bind to the NRL-binding element (NRE), and the two CRX-binding elements (BAT-1, Ret-4). NRL binds to an extended AP-1 like element, the NRE, in the Rho gene's proximal promoter region [21, 40, 41]. The CRX homeodomain can bind to two promoter elements, BAT-1 and Ret-4, that flank the NRE . Historically, the Ret-4 element was used in a yeast 1-hybrid survey to discover CRX from a bovine retina library .
Rehemtulla et al., demonstrated that the NRE probe is shifted into two major complexes with bovine nuclear protein extract, and the higher shifted complex can be removed by antibody to NRL . In our tests here, FIZ1 antibody was also able to remove the larger probe/protein complex. While this element is relatively short and will be covered by NRL, stable protein-protein interactions could involve non-DNA binding proteins in the overall complex. FIZ1 is capable of direct and stable interaction with NRL in vitro; therefore, FIZ1 may associate with the NRE element indirectly through its interaction with NRL. This would be consistent with the fact that the higher shifted band, diminished by incubation with antibody to FIZ1, is also disrupted by antibody to NRL .
Similarly, it is conceivable for FIZ1 to associate indirectly with CRX-binding sites (BAT-1 and Ret-4) by association with CRX. BAT-1 has two core elements that can bind the K50-type HD of CRX [29, 30]. Consistent with the presence of two potential binding sites, Chen et al., have demonstrated that the BAT-1 probe can be shifted into two major bands by recombinant CRX-HD . The BAT-1 site is crucial for CRX mediated activation of the Rhodopsin proximal promoter .
Here, using bovine retina nuclear extract, two major shifted bands were also seen, similar to the pattern reported for recombinant CRX-HD. It is possible that these major shifted bands represent the association of one and two molecules of CRX. In our case, a third higher shifting faint band was removed after complex formation by treatment with FIZ1 antibody, while the two major bands remained. This suggests FIZ1 was not required for formation of the smaller complexes.
In contrast to BAT-1, the Ret-4 probe contains one K50-type HD binding element, and shifts to a doublet band with bovine retina nuclear extract [5, 42]. Our EMSA here, with bovine retina nuclear extract, also resulted in a single major shifted complex. This intense band was efficiently removed by FIZ1 antibody, suggesting the presence of FIZ1. The disruption of only a higher shifting faint band with the BAT-1 probe, suggests that there could also be a different affinity for FIZ1 between a CRX dimer and monomer. This is interesting in the context of the Rho promoter, where the potential CRX-dimer binding site (BAT-1) and the monomer-binding site (Ret-4) flank the NRE site.
What could be the benefit of two different CRX-binding sites in such a small region? There is evidence that several other proteins interact with CRX at the Rho promoter, including NRL, NR2E3 and BAF [8, 9, 34]. Two CRX binding elements, binding in different stoichiometry, may provide a mechanism to have different CRX molecules participating in different protein interactions.
Our Co-IP and EMSA results indicated that FIZ1 might interact directly with CRX; therefore we explored this with yeast two-hybrid and GST pull-down assays. Both assay methods confirmed the ability for a direct and stable interaction of FIZ1 and CRX in vitro. This suggests that FIZ1 has at least two interaction partners in photoreceptor neurons (NRL and CRX) that could recruit it into transcription factor complexes on photoreceptor specific genes. Multiple protein-protein interactions are consistent with our knowledge of other proteins that complex at the Rho promoter. CRX participates in interactions with NRL and NR2E3, resulting in activation of rod genes and repression of cone genes [8, 9]. NR2E3 also has multiple interactions, including CRX and the circadian clock protein NR1D1 .
While we previously demonstrated that FIZ1 could modify NRL's transactivation potential at a PDE6B test promoter, it only modified transactivation potential at the Rho promoter when both NRL and CRX were present . To examine FIZ1 effect on CRX, we used two test promoters that are activated by CRX, but not NRL: M- and S-Opsin . Alone, FIZ1 effect on the activation of either promoter was insignificant compared to empty vectors. However, FIZ1 was able to modify CRX's transactivation potential at both test promoters by over 100% of the activity seen with CRX alone. It is possible that FIZ1's presence could influence the overall activation potential of promoter complexes involving CRX.
The scale of FIZ1's effect on CRX-mediated activation is biologically significant. Mature photoreceptors must continue to precisely regulate the average expression of genes like Rho to maintain viable photoreceptors. In mouse neural retina, Rhodopsin mRNA levels vary at least 25% in a circadian fashion, to coordinate with the rhythm of outer-segment formation . Heterozygous knockout of Rho reduces its average expression by 50%, which results in a slow degeneration of photoreceptors. Likewise, transgenic mice with elevated expression of normal Rhodopsin, also have a slow photoreceptor degeneration .
Developmentally, the increased availability of FIZ1 does concur with maturation of the neural retina and the activation of many photoreceptor-specific genes. While mRNAs for M- and S-Opsin are detected a few days earlier than for Rho , the initial formation of photoreceptor outer-segments of both rods and cones begins in the maturation period. This increases the demand for all three Opsin proteins as well as Pde6b, Arrestin, and other phototransduction proteins.
Test promoters are not perfect models for native gene promoters in their chromatin context, and FIZ1 can promote or inhibit NRL/CRX mediated activation of the Rho promoter in vitro, depending on the relative amount of FIZ1 compared to NRL and CRX [11, 13]. With this in mind, it would be premature to state unequivocally whether FIZ1's role is that of promoting activation of these promoters in vivo, or if it may have a role in maintaining a precise level of their expression in the mature retina.
Our ChIP analysis indicates that FIZ1 is part of the regulatory protein complex on genes that become more active in adult photoreceptors. Rho is a fundamental marker of photoreceptor-specific gene expression; its expression increases dramatically as photoreceptor neurons mature to the adult state . Is there an increase in the relative quantity of FIZ1 recruited to the Rho promoter complex in the adult retina? This would be expected if a prior association of NRL and CRX recruits FIZ1. Relative association of FIZ1 at the Rho promoter region was 7-fold greater in adult neural retina (P-25), as compared to the immature tissue (P-3) when only a small fraction of photoreceptor precursors are starting to express Rho.
The increased association of FIZ1 at the Rho promoter also correlated with a 23-fold increase in the amount of actively transcribing Pol-II in adult neural retina (P-25) compared to the immature tissue (P-3). The total Pol-II associated with the Rho proximal-promoter region increases during maturation of the mouse retina . While the presence of Pol-II in the promoter region often correlates to transcription, it is possible for a gene to be pre-loaded with inactive Pol-II, as recently reported for many genes in human embryonic stem cells . Our assays here specifically monitored actively transcribing Pol-II (serine-2 phosphorylated) in intron-2, about 3 Kb downstream of the Rhodopsin gene's transcription start site.
The increased expression of several photoreceptor genes, during maturation of photoreceptors, certainly involves regulation at the level of transcription. Rhodopsin and Arrestin mRNA levels increase dramatically during this process, and this is documented in mammalian species that complete retinal development postnatal (mouse) or prenatal (bovine) [44, 47]. A previous study of bovine retinal development, using nuclear run-on assays, suggested that elevated transcription has a prominent role during this maturation period . Our quantitative measurement of transcriptionally active Pol-II within the Rho's transcript region, confirms that the amount of activated Pol-II enzyme is elevated.