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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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Nano-gold TEM visualization of FIZ1's subcellular distribution in the P-35 mouse neural retina. A) Light micrograph (400X) of the adult mouse neural retina, illustrating the relative layers and their location: the Ganglion Cell Layer (GCL), whose neurons form the optic nerve; the Inner Nuclear Layer (INL), nuclei of bipolar, horizontal and amacrine cells; the Outer Nuclear Layer (ONL), containing photoreceptor nuclei; the photoreceptor Inner Segments (IS); and the photoreceptor Outer Segments (OS), the location of disk membranes and phototransduction proteins, including Rhodopsin. (Lowicryl embedded section, Toluidine blue stain.) A) and B) Rod nuclei display dense chromatin staining throughout most of their nuclear volume (arrowhead). Cones have dense chromatin staining in a much smaller proportion of their nuclear volume (arrow). C) Nano-gold labeling of FIZ1 in the central ONL (arrowhead). Rod photoreceptors dominate. D) FIZ1 labeling in rod (arrowhead) and cone (arrow) nuclei near the ONL/IS boundary. E) ONL, pre-adsorbed antigen control. F) FIZ1 labeling of the photoreceptor IS region. The outer limiting membrane at the ONL/IS interface is indicated (arrow). G) IS, pre-adsorbed antigen control. H) The OS region did not display label for FIZ1. Pigment granules from the Retinal Pigment Epithelium (RPE) are present (arrow). I) FIZ1 labeling of the GCL, nuclei are positive for FIZ1 (arrowhead). J) GCL, pre-adsorbed antigen control. K) The INL layer, nuclei are positive for FIZ1 (arrowhead). L) INL, pre-adsorbed antigen control.
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Figure 1: Nano-gold TEM visualization of FIZ1's subcellular distribution in the P-35 mouse neural retina. A) Light micrograph (400X) of the adult mouse neural retina, illustrating the relative layers and their location: the Ganglion Cell Layer (GCL), whose neurons form the optic nerve; the Inner Nuclear Layer (INL), nuclei of bipolar, horizontal and amacrine cells; the Outer Nuclear Layer (ONL), containing photoreceptor nuclei; the photoreceptor Inner Segments (IS); and the photoreceptor Outer Segments (OS), the location of disk membranes and phototransduction proteins, including Rhodopsin. (Lowicryl embedded section, Toluidine blue stain.) A) and B) Rod nuclei display dense chromatin staining throughout most of their nuclear volume (arrowhead). Cones have dense chromatin staining in a much smaller proportion of their nuclear volume (arrow). C) Nano-gold labeling of FIZ1 in the central ONL (arrowhead). Rod photoreceptors dominate. D) FIZ1 labeling in rod (arrowhead) and cone (arrow) nuclei near the ONL/IS boundary. E) ONL, pre-adsorbed antigen control. F) FIZ1 labeling of the photoreceptor IS region. The outer limiting membrane at the ONL/IS interface is indicated (arrow). G) IS, pre-adsorbed antigen control. H) The OS region did not display label for FIZ1. Pigment granules from the Retinal Pigment Epithelium (RPE) are present (arrow). I) FIZ1 labeling of the GCL, nuclei are positive for FIZ1 (arrowhead). J) GCL, pre-adsorbed antigen control. K) The INL layer, nuclei are positive for FIZ1 (arrowhead). L) INL, pre-adsorbed antigen control.

Mentions: Regions of the adult mouse neural retina are illustrated by the light micrographs in Figure 1A, B. The ganglion cell layer (GCL), Inner Nuclear Layer (INL), and the Outer Nuclear Layer (ONL) form the three major cell layers in the mature tissue. The photoreceptor Inner Segments (IS) and Outer Segments (OS, contain visual transduction proteins) are restricted to the outer-most layer of the neural retina. Note that rod photoreceptor nuclei in the ONL are extremely compact compared to neurons of the INL and GCL. Most of the photoreceptor nuclei represent rods, while about 3–5% are cones. Rod nuclear cross-sectional areas are almost entirely densely staining chromatin. Cone nuclei are localized on the outer-most edge of the ONL layer, viewed here just below the IS, and display a much smaller proportion of densely staining cross-sectional area in comparison to rod nuclei. Examples of a rod (arrowhead) and cone (arrow) nucleus are indicated in a higher magnification in Figure 1B.


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)

Nano-gold TEM visualization of FIZ1's subcellular distribution in the P-35 mouse neural retina. A) Light micrograph (400X) of the adult mouse neural retina, illustrating the relative layers and their location: the Ganglion Cell Layer (GCL), whose neurons form the optic nerve; the Inner Nuclear Layer (INL), nuclei of bipolar, horizontal and amacrine cells; the Outer Nuclear Layer (ONL), containing photoreceptor nuclei; the photoreceptor Inner Segments (IS); and the photoreceptor Outer Segments (OS), the location of disk membranes and phototransduction proteins, including Rhodopsin. (Lowicryl embedded section, Toluidine blue stain.) A) and B) Rod nuclei display dense chromatin staining throughout most of their nuclear volume (arrowhead). Cones have dense chromatin staining in a much smaller proportion of their nuclear volume (arrow). C) Nano-gold labeling of FIZ1 in the central ONL (arrowhead). Rod photoreceptors dominate. D) FIZ1 labeling in rod (arrowhead) and cone (arrow) nuclei near the ONL/IS boundary. E) ONL, pre-adsorbed antigen control. F) FIZ1 labeling of the photoreceptor IS region. The outer limiting membrane at the ONL/IS interface is indicated (arrow). G) IS, pre-adsorbed antigen control. H) The OS region did not display label for FIZ1. Pigment granules from the Retinal Pigment Epithelium (RPE) are present (arrow). I) FIZ1 labeling of the GCL, nuclei are positive for FIZ1 (arrowhead). J) GCL, pre-adsorbed antigen control. K) The INL layer, nuclei are positive for FIZ1 (arrowhead). L) INL, pre-adsorbed antigen control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Nano-gold TEM visualization of FIZ1's subcellular distribution in the P-35 mouse neural retina. A) Light micrograph (400X) of the adult mouse neural retina, illustrating the relative layers and their location: the Ganglion Cell Layer (GCL), whose neurons form the optic nerve; the Inner Nuclear Layer (INL), nuclei of bipolar, horizontal and amacrine cells; the Outer Nuclear Layer (ONL), containing photoreceptor nuclei; the photoreceptor Inner Segments (IS); and the photoreceptor Outer Segments (OS), the location of disk membranes and phototransduction proteins, including Rhodopsin. (Lowicryl embedded section, Toluidine blue stain.) A) and B) Rod nuclei display dense chromatin staining throughout most of their nuclear volume (arrowhead). Cones have dense chromatin staining in a much smaller proportion of their nuclear volume (arrow). C) Nano-gold labeling of FIZ1 in the central ONL (arrowhead). Rod photoreceptors dominate. D) FIZ1 labeling in rod (arrowhead) and cone (arrow) nuclei near the ONL/IS boundary. E) ONL, pre-adsorbed antigen control. F) FIZ1 labeling of the photoreceptor IS region. The outer limiting membrane at the ONL/IS interface is indicated (arrow). G) IS, pre-adsorbed antigen control. H) The OS region did not display label for FIZ1. Pigment granules from the Retinal Pigment Epithelium (RPE) are present (arrow). I) FIZ1 labeling of the GCL, nuclei are positive for FIZ1 (arrowhead). J) GCL, pre-adsorbed antigen control. K) The INL layer, nuclei are positive for FIZ1 (arrowhead). L) INL, pre-adsorbed antigen control.
Mentions: Regions of the adult mouse neural retina are illustrated by the light micrographs in Figure 1A, B. The ganglion cell layer (GCL), Inner Nuclear Layer (INL), and the Outer Nuclear Layer (ONL) form the three major cell layers in the mature tissue. The photoreceptor Inner Segments (IS) and Outer Segments (OS, contain visual transduction proteins) are restricted to the outer-most layer of the neural retina. Note that rod photoreceptor nuclei in the ONL are extremely compact compared to neurons of the INL and GCL. Most of the photoreceptor nuclei represent rods, while about 3–5% are cones. Rod nuclear cross-sectional areas are almost entirely densely staining chromatin. Cone nuclei are localized on the outer-most edge of the ONL layer, viewed here just below the IS, and display a much smaller proportion of densely staining cross-sectional area in comparison to rod nuclei. Examples of a rod (arrowhead) and cone (arrow) nucleus are indicated in a higher magnification in Figure 1B.

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