Limits...
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.

Show MeSH

Related in: MedlinePlus

FIZ1 alters the CRX-mediated activation of two Opsin promoters. Two CRX-activated proximal promoters were used from the photoreceptor genes: M-Opsin and S-Opsin. Firefly luciferase activity was normalized to Renilla-luciferase activity (pRL-CMV). Fold Activation is relative to empty expression vectors. A) the M-Opsin promoter luciferase-reporter construct, Mop250-Luc, or B) the S-Opsin promoter luciferase-reporter construct, Sop552-Luc. Triplicate wells of CV1 cells were co-transfected with expression vectors for CRX, FIZ1, and NRL in the combinations indicated. CRX could activate both test promoters, relative to empty vectors. NRL or FIZ1 alone had no activation effect on either promoter. NRL combined with CRX did not alter the activation potential from that of CRX alone. FIZ1 combined with CRX significantly altered the CRX-mediated activation of both test promoters, when compared to CRX alone. Bars represent ± SD (n = 3). Statistical comparisons are described in results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2571102&req=5

Figure 7: FIZ1 alters the CRX-mediated activation of two Opsin promoters. Two CRX-activated proximal promoters were used from the photoreceptor genes: M-Opsin and S-Opsin. Firefly luciferase activity was normalized to Renilla-luciferase activity (pRL-CMV). Fold Activation is relative to empty expression vectors. A) the M-Opsin promoter luciferase-reporter construct, Mop250-Luc, or B) the S-Opsin promoter luciferase-reporter construct, Sop552-Luc. Triplicate wells of CV1 cells were co-transfected with expression vectors for CRX, FIZ1, and NRL in the combinations indicated. CRX could activate both test promoters, relative to empty vectors. NRL or FIZ1 alone had no activation effect on either promoter. NRL combined with CRX did not alter the activation potential from that of CRX alone. FIZ1 combined with CRX significantly altered the CRX-mediated activation of both test promoters, when compared to CRX alone. Bars represent ± SD (n = 3). Statistical comparisons are described in results.

Mentions: To determine if FIZ1 interaction with CRX could have any effect upon CRX-mediated activation of a test promoter, we employed a co-transfection luciferase-reporter assay system. Two test promoters were selected that represent photoreceptor-specific promoters that are capable of activation by CRX alone [9]. CV-1 cells were transfected with M- or S-Opsin reporter plasmids (firefly-luciferase) and expression plasmids for NRL, CRX and FIZ1. FIZ1 significantly altered the CRX-mediated activation of both test promoters. For the M-Opsin promoter, CRX alone caused a 2.5-fold activation relative to empty vectors (p < 0.01, Tukey Kramer post analysis) (Fig. 7A). Neither FIZ1 nor NRL alone had significant effect compared to empty vectors. FIZ1 combined with CRX increased the activation of the M-Opsin promoter, to 6-fold, relative to empty vectors (p < 0.01). The effect was to more than double CRX's activation potential.


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 alters the CRX-mediated activation of two Opsin promoters. Two CRX-activated proximal promoters were used from the photoreceptor genes: M-Opsin and S-Opsin. Firefly luciferase activity was normalized to Renilla-luciferase activity (pRL-CMV). Fold Activation is relative to empty expression vectors. A) the M-Opsin promoter luciferase-reporter construct, Mop250-Luc, or B) the S-Opsin promoter luciferase-reporter construct, Sop552-Luc. Triplicate wells of CV1 cells were co-transfected with expression vectors for CRX, FIZ1, and NRL in the combinations indicated. CRX could activate both test promoters, relative to empty vectors. NRL or FIZ1 alone had no activation effect on either promoter. NRL combined with CRX did not alter the activation potential from that of CRX alone. FIZ1 combined with CRX significantly altered the CRX-mediated activation of both test promoters, when compared to CRX alone. Bars represent ± SD (n = 3). Statistical comparisons are described in results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: FIZ1 alters the CRX-mediated activation of two Opsin promoters. Two CRX-activated proximal promoters were used from the photoreceptor genes: M-Opsin and S-Opsin. Firefly luciferase activity was normalized to Renilla-luciferase activity (pRL-CMV). Fold Activation is relative to empty expression vectors. A) the M-Opsin promoter luciferase-reporter construct, Mop250-Luc, or B) the S-Opsin promoter luciferase-reporter construct, Sop552-Luc. Triplicate wells of CV1 cells were co-transfected with expression vectors for CRX, FIZ1, and NRL in the combinations indicated. CRX could activate both test promoters, relative to empty vectors. NRL or FIZ1 alone had no activation effect on either promoter. NRL combined with CRX did not alter the activation potential from that of CRX alone. FIZ1 combined with CRX significantly altered the CRX-mediated activation of both test promoters, when compared to CRX alone. Bars represent ± SD (n = 3). Statistical comparisons are described in results.
Mentions: To determine if FIZ1 interaction with CRX could have any effect upon CRX-mediated activation of a test promoter, we employed a co-transfection luciferase-reporter assay system. Two test promoters were selected that represent photoreceptor-specific promoters that are capable of activation by CRX alone [9]. CV-1 cells were transfected with M- or S-Opsin reporter plasmids (firefly-luciferase) and expression plasmids for NRL, CRX and FIZ1. FIZ1 significantly altered the CRX-mediated activation of both test promoters. For the M-Opsin promoter, CRX alone caused a 2.5-fold activation relative to empty vectors (p < 0.01, Tukey Kramer post analysis) (Fig. 7A). Neither FIZ1 nor NRL alone had significant effect compared to empty vectors. FIZ1 combined with CRX increased the activation of the M-Opsin promoter, to 6-fold, relative to empty vectors (p < 0.01). The effect was to more than double CRX's activation potential.

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