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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 associated with photoreceptor gene promoters in photoreceptor neurons. Combined agarose gel images. A) Chromatin immunoprecipitation (ChIP) assays were performed using the anti-bFIZ1 antibody. Normal IgG and no chromatin (mock) samples served as negative controls, whereas input DNA (without IP) served as positive controls. ChIP DNA was analyzed using PCR amplicons corresponding to the proximal promoter regions of the indicated photoreceptor-specific genes (Sop, Mop, Rho, Pde6b and Rbp3) and a non-photoreceptor gene (Alb, liver gene). B) ChIP assays were performed using the anti-FIZ1-1 antibody, and retinas of the following mouse strains: Wild-type (C57BL/6J), and Rodless/Coneless (Rd1). ChIP DNA was analyzed by PCR for promoter regions of key photoreceptor-specific genes (Sop, Mop, Rho, Pde6b and Rbp3) and non-photoreceptor genes (mGluR6, bipolar cells).
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Figure 4: FIZ1 is associated with photoreceptor gene promoters in photoreceptor neurons. Combined agarose gel images. A) Chromatin immunoprecipitation (ChIP) assays were performed using the anti-bFIZ1 antibody. Normal IgG and no chromatin (mock) samples served as negative controls, whereas input DNA (without IP) served as positive controls. ChIP DNA was analyzed using PCR amplicons corresponding to the proximal promoter regions of the indicated photoreceptor-specific genes (Sop, Mop, Rho, Pde6b and Rbp3) and a non-photoreceptor gene (Alb, liver gene). B) ChIP assays were performed using the anti-FIZ1-1 antibody, and retinas of the following mouse strains: Wild-type (C57BL/6J), and Rodless/Coneless (Rd1). ChIP DNA was analyzed by PCR for promoter regions of key photoreceptor-specific genes (Sop, Mop, Rho, Pde6b and Rbp3) and non-photoreceptor genes (mGluR6, bipolar cells).

Mentions: To determine if FIZ1 is present in the regulatory protein complex on the chromatin of photoreceptor genes in neural retina, we performed chromatin immunoprecipitation (ChIP) from wild type mouse retina with an antibody specific to FIZ1 (anti-bFIZ1). Results show that FIZ1 is associated with the proximal promoter regions of several genes that are known targets of CRX and NRL (Fig. 4A). These included the Rho, Pde6b, M-Opsin, S-Opsin, and Rbp3 (Inter Retinoid Binding Protein) gene promoters. Control ChIP assays, minus antibody or minus input DNA, were negative. Additional negative controls with the pre-immune serum IgG further demonstrated the specificity of the positive results obtained with the FIZ1-antibody. Assays did not detect any association of FIZ1 on the liver specific Alb (Albumin) gene promoter.


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 associated with photoreceptor gene promoters in photoreceptor neurons. Combined agarose gel images. A) Chromatin immunoprecipitation (ChIP) assays were performed using the anti-bFIZ1 antibody. Normal IgG and no chromatin (mock) samples served as negative controls, whereas input DNA (without IP) served as positive controls. ChIP DNA was analyzed using PCR amplicons corresponding to the proximal promoter regions of the indicated photoreceptor-specific genes (Sop, Mop, Rho, Pde6b and Rbp3) and a non-photoreceptor gene (Alb, liver gene). B) ChIP assays were performed using the anti-FIZ1-1 antibody, and retinas of the following mouse strains: Wild-type (C57BL/6J), and Rodless/Coneless (Rd1). ChIP DNA was analyzed by PCR for promoter regions of key photoreceptor-specific genes (Sop, Mop, Rho, Pde6b and Rbp3) and non-photoreceptor genes (mGluR6, bipolar cells).
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Figure 4: FIZ1 is associated with photoreceptor gene promoters in photoreceptor neurons. Combined agarose gel images. A) Chromatin immunoprecipitation (ChIP) assays were performed using the anti-bFIZ1 antibody. Normal IgG and no chromatin (mock) samples served as negative controls, whereas input DNA (without IP) served as positive controls. ChIP DNA was analyzed using PCR amplicons corresponding to the proximal promoter regions of the indicated photoreceptor-specific genes (Sop, Mop, Rho, Pde6b and Rbp3) and a non-photoreceptor gene (Alb, liver gene). B) ChIP assays were performed using the anti-FIZ1-1 antibody, and retinas of the following mouse strains: Wild-type (C57BL/6J), and Rodless/Coneless (Rd1). ChIP DNA was analyzed by PCR for promoter regions of key photoreceptor-specific genes (Sop, Mop, Rho, Pde6b and Rbp3) and non-photoreceptor genes (mGluR6, bipolar cells).
Mentions: To determine if FIZ1 is present in the regulatory protein complex on the chromatin of photoreceptor genes in neural retina, we performed chromatin immunoprecipitation (ChIP) from wild type mouse retina with an antibody specific to FIZ1 (anti-bFIZ1). Results show that FIZ1 is associated with the proximal promoter regions of several genes that are known targets of CRX and NRL (Fig. 4A). These included the Rho, Pde6b, M-Opsin, S-Opsin, and Rbp3 (Inter Retinoid Binding Protein) gene promoters. Control ChIP assays, minus antibody or minus input DNA, were negative. Additional negative controls with the pre-immune serum IgG further demonstrated the specificity of the positive results obtained with the FIZ1-antibody. Assays did not detect any association of FIZ1 on the liver specific Alb (Albumin) gene promoter.

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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