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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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Comparison of FIZ1 recruitment and activated Pol-II association, at the Rhodopsin gene in P-3 and P-25 retina. ChIP was performed using antibodies specific for FIZ1 and the actively transcribing form of RNA Polymerase-II (Pol-II-S2). Protein binding is proportional to the amount of target DNA captured by ChIP, and is shown as copies of target DNA detected per 1000 cell equivalents of input DNA. Results were normalized for chromatin input and PCR efficiency. Error bars indicate standard deviation (n = 3). A) FIZ1: Q-PCR of two targets in the Rho proximal promoter (-711, -12), as illustrated in the diagram of the Rho gene. An untranslated region (Untr6), on chromosome-6, provided a reference for binding (negative control), and was not different comparing P-3 to P-25 neural retina. FIZ1 recruitment to the Rho promoter was significantly increased in P-25 neural retina compared to P-3 retina. B) Pol-II-S2: Q-PCR of a target inside intron-2 of the Rho gene (+3051), and the untranslated region (Untr6). Results confirm an increased amount of actively transcribing Pol-II in the transcriptional region of Rho in P-25 retina compared to P-3 retina.
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Figure 8: Comparison of FIZ1 recruitment and activated Pol-II association, at the Rhodopsin gene in P-3 and P-25 retina. ChIP was performed using antibodies specific for FIZ1 and the actively transcribing form of RNA Polymerase-II (Pol-II-S2). Protein binding is proportional to the amount of target DNA captured by ChIP, and is shown as copies of target DNA detected per 1000 cell equivalents of input DNA. Results were normalized for chromatin input and PCR efficiency. Error bars indicate standard deviation (n = 3). A) FIZ1: Q-PCR of two targets in the Rho proximal promoter (-711, -12), as illustrated in the diagram of the Rho gene. An untranslated region (Untr6), on chromosome-6, provided a reference for binding (negative control), and was not different comparing P-3 to P-25 neural retina. FIZ1 recruitment to the Rho promoter was significantly increased in P-25 neural retina compared to P-3 retina. B) Pol-II-S2: Q-PCR of a target inside intron-2 of the Rho gene (+3051), and the untranslated region (Untr6). Results confirm an increased amount of actively transcribing Pol-II in the transcriptional region of Rho in P-25 retina compared to P-3 retina.

Mentions: The relative quantity of FIZ1, associated with the regulatory protein complex on the Rho promoter, was significantly elevated (7-fold) in P-25 neural retina compared to P-3 neural retina (Fig. 8A). FIZ1 association with this regulatory complex was significantly higher than for the untranslated control region (Untr6) in both P-3 and P-25 neural retina. In this untranslated control region, there was no difference in the relative association of FIZ1 comparing P-3 to P-25 neural retina.


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)

Comparison of FIZ1 recruitment and activated Pol-II association, at the Rhodopsin gene in P-3 and P-25 retina. ChIP was performed using antibodies specific for FIZ1 and the actively transcribing form of RNA Polymerase-II (Pol-II-S2). Protein binding is proportional to the amount of target DNA captured by ChIP, and is shown as copies of target DNA detected per 1000 cell equivalents of input DNA. Results were normalized for chromatin input and PCR efficiency. Error bars indicate standard deviation (n = 3). A) FIZ1: Q-PCR of two targets in the Rho proximal promoter (-711, -12), as illustrated in the diagram of the Rho gene. An untranslated region (Untr6), on chromosome-6, provided a reference for binding (negative control), and was not different comparing P-3 to P-25 neural retina. FIZ1 recruitment to the Rho promoter was significantly increased in P-25 neural retina compared to P-3 retina. B) Pol-II-S2: Q-PCR of a target inside intron-2 of the Rho gene (+3051), and the untranslated region (Untr6). Results confirm an increased amount of actively transcribing Pol-II in the transcriptional region of Rho in P-25 retina compared to P-3 retina.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Comparison of FIZ1 recruitment and activated Pol-II association, at the Rhodopsin gene in P-3 and P-25 retina. ChIP was performed using antibodies specific for FIZ1 and the actively transcribing form of RNA Polymerase-II (Pol-II-S2). Protein binding is proportional to the amount of target DNA captured by ChIP, and is shown as copies of target DNA detected per 1000 cell equivalents of input DNA. Results were normalized for chromatin input and PCR efficiency. Error bars indicate standard deviation (n = 3). A) FIZ1: Q-PCR of two targets in the Rho proximal promoter (-711, -12), as illustrated in the diagram of the Rho gene. An untranslated region (Untr6), on chromosome-6, provided a reference for binding (negative control), and was not different comparing P-3 to P-25 neural retina. FIZ1 recruitment to the Rho promoter was significantly increased in P-25 neural retina compared to P-3 retina. B) Pol-II-S2: Q-PCR of a target inside intron-2 of the Rho gene (+3051), and the untranslated region (Untr6). Results confirm an increased amount of actively transcribing Pol-II in the transcriptional region of Rho in P-25 retina compared to P-3 retina.
Mentions: The relative quantity of FIZ1, associated with the regulatory protein complex on the Rho promoter, was significantly elevated (7-fold) in P-25 neural retina compared to P-3 neural retina (Fig. 8A). FIZ1 association with this regulatory complex was significantly higher than for the untranslated control region (Untr6) in both P-3 and P-25 neural retina. In this untranslated control region, there was no difference in the relative association of FIZ1 comparing P-3 to P-25 neural retina.

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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