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FIZ1 is part of the regulatory protein complex on active photoreceptor-specific gene promoters in vivo.

Mali RS, Peng GH, Zhang X, Dang L, Chen S, Mitton KP - BMC Mol. Biol. (2008)

Bottom Line: The quantity of transcriptionally active RNA Polymerase-II within the Rhodopsin gene (Rho) was significantly increased in the adult neural retina, compared to the immature retina.Together with previous findings, our results suggest that FIZ1 may act as a transcriptional co-regulator of photoreceptor-specific genes, recruited by at least two photoreceptor-specific transcription factors, CRX and NRL.Further studies are underway to elucidate the exact role of FIZ1 in photoreceptor gene expression, development and maintenance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Eye Research Institute, Oakland University, Rochester, MI, USA. mali2@oakland.edu

ABSTRACT

Background: FIZ1 (Flt-3 Interacting Zinc-finger) is a broadly expressed protein of unknown function. We reported previously that in the mammalian retina, FIZ1 interacts with NRL (Neural-Retina Leucine-zipper), an essential transcriptional activator of rod photoreceptor-specific genes. The concentration of FIZ1 in the retina increases during photoreceptor terminal maturation, when two key transcription factors NRL and CRX (Cone-Rod Homeobox) become detectable on the promoters of photoreceptor-specific genes (i.e. Rhodopsin, Pde6b). To determine if FIZ1 is involved in regulating CRX-mediated transcriptional activation, we examined FIZ1 subcellular location in mouse neural retina, its ability to interact with CRX, and its association with CRX/NRL target genes.

Results: FIZ1 is present in the nucleus of adult photoreceptors as well as other retinal neurons as shown by transmission electron microscopy with nano-gold labeling. FIZ1 and CRX were co-precipitated from retinal nuclear extracts with antibodies to either protein. Chromatin immunoprecipitation (ChIP) assays revealed that FIZ1 is part of the protein complex on several rod and cone gene promoters, within photoreceptor cells of the mouse retina. FIZ1 complexes with CRX or NRL on known NRL- and CRX-responsive elements, as shown by electrophoretic mobility shift assays with FIZ1 antibody. FIZ1 can directly bind to CRX, as demonstrated using yeast two-hybrid and GST pull-down assays. Co-transfection assays demonstrated that FIZ1 increases CRX-mediated activation of Opsin test promoters. Quantitative ChIP analysis revealed an increased association of FIZ1 with the Rhodopsin promoter in adult (P-25) neural retina versus immature (P-3) neural retina. The quantity of transcriptionally active RNA Polymerase-II within the Rhodopsin gene (Rho) was significantly increased in the adult neural retina, compared to the immature retina.

Conclusion: FIZ1 directly interacts with CRX to enhance CRX's transactivation activity for target genes. Developmentally, in neural retina tissue, the increased association of FIZ1 with CRX target genes corresponds to an increased association of transcriptionally active Pol-II within the Rho gene. Together with previous findings, our results suggest that FIZ1 may act as a transcriptional co-regulator of photoreceptor-specific genes, recruited by at least two photoreceptor-specific transcription factors, CRX and NRL. Further studies are underway to elucidate the exact role of FIZ1 in photoreceptor gene expression, development and maintenance.

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FIZ1 is part of retinal protein complexes that bind to NRE, BAT-1 and Ret-4 elements from the Rho proximal promoter. The [32P]-labeled oligonucleotide probes NRE, BAT-1, and Ret-4 were incubated with bovine retinal nuclear protein extract (NucEx), followed by nondenaturing PAGE. A) NRE: The binding site for the NRL bZIP domain is shown in bold face type. Removal of a shifted complex by antibody to FIZ1 is indicated (arrow). B) BAT-1: The binding sites for the Homeo-Domain of CRX are shown in bold face type. Removal of a shifted complex by antibody to FIZ1 is indicated (arrow). C) Ret-4: The core binding site for the CRX-HD is shown in bold face type. Removal of a shifted complex by antibody to FIZ1 is indicated (arrow). Presence of components are as indicated (+/-). Competition for each probe with unlabeled oligonucleotide validated the specificity of the band shift. Anti-FIZ1, or preserum IgG, was added after complex formation.
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Figure 5: FIZ1 is part of retinal protein complexes that bind to NRE, BAT-1 and Ret-4 elements from the Rho proximal promoter. The [32P]-labeled oligonucleotide probes NRE, BAT-1, and Ret-4 were incubated with bovine retinal nuclear protein extract (NucEx), followed by nondenaturing PAGE. A) NRE: The binding site for the NRL bZIP domain is shown in bold face type. Removal of a shifted complex by antibody to FIZ1 is indicated (arrow). B) BAT-1: The binding sites for the Homeo-Domain of CRX are shown in bold face type. Removal of a shifted complex by antibody to FIZ1 is indicated (arrow). C) Ret-4: The core binding site for the CRX-HD is shown in bold face type. Removal of a shifted complex by antibody to FIZ1 is indicated (arrow). Presence of components are as indicated (+/-). Competition for each probe with unlabeled oligonucleotide validated the specificity of the band shift. Anti-FIZ1, or preserum IgG, was added after complex formation.

Mentions: To determine if FIZ1 is part of retinal nuclear protein complexes that bind to DNA elements recognized by NRL and CRX, we performed electrophoretic mobility shift assays (EMSA) using bovine retina nuclear protein extract and 32P-labeled probes derived from conserved NRL- or CRX-responsive elements from the mammalian Rho proximal promoter region [5,21]. Previous EMSA results showed that bovine nuclear extracts produced two major shifted bands on a probe containing the NRL-Response Element (NRE) [21]. The NRE probe sequence and the NRL binding site are shown in Figure 5-A. In our assay, this probe was shifted in a characteristic pattern as previously published[21]. The higher shifted DNA-protein complex known to contain NRL[21] was efficiently removed by incubation with affinity-purified antibody (rabbit IgG) specific for FIZ1 (anti-bFIZ1), after complexes were allowed to form (indicated by the arrow). Control IgG (preimmune, rabbit IgG) did not remove any of the native protein-DNA complexes. As previously seen by Rehemtulla et al., 1996, competition with cold probe was more effective on diminishing the higher shifted complex. These results suggest that FIZ1 is a part of the NRL protein complex bound to NRE.


FIZ1 is part of the regulatory protein complex on active photoreceptor-specific gene promoters in vivo.

Mali RS, Peng GH, Zhang X, Dang L, Chen S, Mitton KP - BMC Mol. Biol. (2008)

FIZ1 is part of retinal protein complexes that bind to NRE, BAT-1 and Ret-4 elements from the Rho proximal promoter. The [32P]-labeled oligonucleotide probes NRE, BAT-1, and Ret-4 were incubated with bovine retinal nuclear protein extract (NucEx), followed by nondenaturing PAGE. A) NRE: The binding site for the NRL bZIP domain is shown in bold face type. Removal of a shifted complex by antibody to FIZ1 is indicated (arrow). B) BAT-1: The binding sites for the Homeo-Domain of CRX are shown in bold face type. Removal of a shifted complex by antibody to FIZ1 is indicated (arrow). C) Ret-4: The core binding site for the CRX-HD is shown in bold face type. Removal of a shifted complex by antibody to FIZ1 is indicated (arrow). Presence of components are as indicated (+/-). Competition for each probe with unlabeled oligonucleotide validated the specificity of the band shift. Anti-FIZ1, or preserum IgG, was added after complex formation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2571102&req=5

Figure 5: FIZ1 is part of retinal protein complexes that bind to NRE, BAT-1 and Ret-4 elements from the Rho proximal promoter. The [32P]-labeled oligonucleotide probes NRE, BAT-1, and Ret-4 were incubated with bovine retinal nuclear protein extract (NucEx), followed by nondenaturing PAGE. A) NRE: The binding site for the NRL bZIP domain is shown in bold face type. Removal of a shifted complex by antibody to FIZ1 is indicated (arrow). B) BAT-1: The binding sites for the Homeo-Domain of CRX are shown in bold face type. Removal of a shifted complex by antibody to FIZ1 is indicated (arrow). C) Ret-4: The core binding site for the CRX-HD is shown in bold face type. Removal of a shifted complex by antibody to FIZ1 is indicated (arrow). Presence of components are as indicated (+/-). Competition for each probe with unlabeled oligonucleotide validated the specificity of the band shift. Anti-FIZ1, or preserum IgG, was added after complex formation.
Mentions: To determine if FIZ1 is part of retinal nuclear protein complexes that bind to DNA elements recognized by NRL and CRX, we performed electrophoretic mobility shift assays (EMSA) using bovine retina nuclear protein extract and 32P-labeled probes derived from conserved NRL- or CRX-responsive elements from the mammalian Rho proximal promoter region [5,21]. Previous EMSA results showed that bovine nuclear extracts produced two major shifted bands on a probe containing the NRL-Response Element (NRE) [21]. The NRE probe sequence and the NRL binding site are shown in Figure 5-A. In our assay, this probe was shifted in a characteristic pattern as previously published[21]. The higher shifted DNA-protein complex known to contain NRL[21] was efficiently removed by incubation with affinity-purified antibody (rabbit IgG) specific for FIZ1 (anti-bFIZ1), after complexes were allowed to form (indicated by the arrow). Control IgG (preimmune, rabbit IgG) did not remove any of the native protein-DNA complexes. As previously seen by Rehemtulla et al., 1996, competition with cold probe was more effective on diminishing the higher shifted complex. These results suggest that FIZ1 is a part of the NRL protein complex bound to NRE.

Bottom Line: The quantity of transcriptionally active RNA Polymerase-II within the Rhodopsin gene (Rho) was significantly increased in the adult neural retina, compared to the immature retina.Together with previous findings, our results suggest that FIZ1 may act as a transcriptional co-regulator of photoreceptor-specific genes, recruited by at least two photoreceptor-specific transcription factors, CRX and NRL.Further studies are underway to elucidate the exact role of FIZ1 in photoreceptor gene expression, development and maintenance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Eye Research Institute, Oakland University, Rochester, MI, USA. mali2@oakland.edu

ABSTRACT

Background: FIZ1 (Flt-3 Interacting Zinc-finger) is a broadly expressed protein of unknown function. We reported previously that in the mammalian retina, FIZ1 interacts with NRL (Neural-Retina Leucine-zipper), an essential transcriptional activator of rod photoreceptor-specific genes. The concentration of FIZ1 in the retina increases during photoreceptor terminal maturation, when two key transcription factors NRL and CRX (Cone-Rod Homeobox) become detectable on the promoters of photoreceptor-specific genes (i.e. Rhodopsin, Pde6b). To determine if FIZ1 is involved in regulating CRX-mediated transcriptional activation, we examined FIZ1 subcellular location in mouse neural retina, its ability to interact with CRX, and its association with CRX/NRL target genes.

Results: FIZ1 is present in the nucleus of adult photoreceptors as well as other retinal neurons as shown by transmission electron microscopy with nano-gold labeling. FIZ1 and CRX were co-precipitated from retinal nuclear extracts with antibodies to either protein. Chromatin immunoprecipitation (ChIP) assays revealed that FIZ1 is part of the protein complex on several rod and cone gene promoters, within photoreceptor cells of the mouse retina. FIZ1 complexes with CRX or NRL on known NRL- and CRX-responsive elements, as shown by electrophoretic mobility shift assays with FIZ1 antibody. FIZ1 can directly bind to CRX, as demonstrated using yeast two-hybrid and GST pull-down assays. Co-transfection assays demonstrated that FIZ1 increases CRX-mediated activation of Opsin test promoters. Quantitative ChIP analysis revealed an increased association of FIZ1 with the Rhodopsin promoter in adult (P-25) neural retina versus immature (P-3) neural retina. The quantity of transcriptionally active RNA Polymerase-II within the Rhodopsin gene (Rho) was significantly increased in the adult neural retina, compared to the immature retina.

Conclusion: FIZ1 directly interacts with CRX to enhance CRX's transactivation activity for target genes. Developmentally, in neural retina tissue, the increased association of FIZ1 with CRX target genes corresponds to an increased association of transcriptionally active Pol-II within the Rho gene. Together with previous findings, our results suggest that FIZ1 may act as a transcriptional co-regulator of photoreceptor-specific genes, recruited by at least two photoreceptor-specific transcription factors, CRX and NRL. Further studies are underway to elucidate the exact role of FIZ1 in photoreceptor gene expression, development and maintenance.

Show MeSH
Related in: MedlinePlus