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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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Co-immunoprecipitation of native FIZ1 and CRX from bovine retina. Immunoblots of Co-IP assays. A) Bovine retinal nuclear extract (300 μg protein) was incubated with anti-CRX antibody or pre-immune serum, and the immunoprecipitated proteins were analyzed with anti-bFIZ1 antibody. A mock sample (no extract) was included. B) The reverse Co-IP, using the anti-bFIZ1 antibody for precipitation and the anti-CRX antibody for detection.
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Figure 3: Co-immunoprecipitation of native FIZ1 and CRX from bovine retina. Immunoblots of Co-IP assays. A) Bovine retinal nuclear extract (300 μg protein) was incubated with anti-CRX antibody or pre-immune serum, and the immunoprecipitated proteins were analyzed with anti-bFIZ1 antibody. A mock sample (no extract) was included. B) The reverse Co-IP, using the anti-bFIZ1 antibody for precipitation and the anti-CRX antibody for detection.

Mentions: To investigate if native FIZ1 complexes with CRX in neural retina, Co-IP experiments were carried out with bovine retina nuclear extract. Co-IP with the CRX antibody identified a band corresponding to FIZ1 (Fig. 3A). The full size gel format was used to separate FIZ1 from the heavy IgG band on immunoblots. Co-IP with pre-immune serum served as a negative control and identified the IgG heavy chain of the precipitating antibody. The reverse experiment was also performed, as Co-IP with the FIZ1 antibody, which identified a band corresponding to CRX on mini-gel immunoblots (Fig. 3B).


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)

Co-immunoprecipitation of native FIZ1 and CRX from bovine retina. Immunoblots of Co-IP assays. A) Bovine retinal nuclear extract (300 μg protein) was incubated with anti-CRX antibody or pre-immune serum, and the immunoprecipitated proteins were analyzed with anti-bFIZ1 antibody. A mock sample (no extract) was included. B) The reverse Co-IP, using the anti-bFIZ1 antibody for precipitation and the anti-CRX antibody for detection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2571102&req=5

Figure 3: Co-immunoprecipitation of native FIZ1 and CRX from bovine retina. Immunoblots of Co-IP assays. A) Bovine retinal nuclear extract (300 μg protein) was incubated with anti-CRX antibody or pre-immune serum, and the immunoprecipitated proteins were analyzed with anti-bFIZ1 antibody. A mock sample (no extract) was included. B) The reverse Co-IP, using the anti-bFIZ1 antibody for precipitation and the anti-CRX antibody for detection.
Mentions: To investigate if native FIZ1 complexes with CRX in neural retina, Co-IP experiments were carried out with bovine retina nuclear extract. Co-IP with the CRX antibody identified a band corresponding to FIZ1 (Fig. 3A). The full size gel format was used to separate FIZ1 from the heavy IgG band on immunoblots. Co-IP with pre-immune serum served as a negative control and identified the IgG heavy chain of the precipitating antibody. The reverse experiment was also performed, as Co-IP with the FIZ1 antibody, which identified a band corresponding to CRX on mini-gel immunoblots (Fig. 3B).

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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