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Human Molecular Genetics, 2003, Vol. 12, No. 4 365-373
© 2003 Oxford University Press

Interaction of retinal bZIP transcription factor NRL with Flt3-interacting zinc-finger protein Fiz1: possible role of Fiz1 as a transcriptional repressor

Kenneth P. Mitton1,3, Prabodh K. Swain1,{dagger}, Hemant Khanna1, Mary Dowd3, Ingrid J. Apel1 and Anand Swaroop1,2,*

1Departments of Ophthalmology and Visual Sciences and 2Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA and 3Oakland University Eye Research Institute, Rochester, MI, USA

Received August 21, 2002; Revised December 3, 2002; Accepted December 11, 2002


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NRL (neural retina leucine zipper) is a basic motif leucine zipper transcription factor of the Maf-subfamily. Multiple phosphorylated isoforms of NRL are detected specifically in rod photoreceptors. NRL regulates the expression of several rod-specific genes, including rhodopsin and cGMP phosphodiesterase ß-subunit, in synergy with other transcription factors (e.g. the homeodomain protein CRX). Missense mutations in the human NRL gene are associated with autosomal dominant retinitis pigmentosa, whereas the loss of its function leads to rodless retina in Nrl-knockout mice that exhibit enhanced S-cone function. To further elucidate the molecular mechanism(s) underlying NRL-mediated transcriptional regulation, we used yeast two-hybrid screening to isolate NRL-interacting proteins in the retina and report the identification of Flt3-interacting zinc-finger protein, Fiz1. Interaction of Fiz1 and NRL-leucine zipper was validated by GST pulldown assays and co-immunoprecipitation from bovine retinal nuclear extracts. Fiz1 suppressed NRL- but not CRX-mediated transactivation of rhodopsin promoter activity in transiently transfected CV1 cells. The mRNA and the protein for both Fiz1 and its only other known interacting protein Flt3, a receptor tyrosine kinase, are expressed in the retina. Our results indicate potential cross-talk among signaling pathways in the retina and suggest that the function of NRL is modulated by its interaction with specific repressor proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mammalian retina consists of a diverse array of neurons that display a unique laminar organization (1). During retinal development, the differentiation of distinct neurons requires coordinated action of multiple signaling pathways involving extrinsic and intrinsic factors (2–4). Similar stringent control is needed to maintain the function of these terminally differentiated yet highly active cells. Molecular mechanisms that underlie the regulation of complex biological networks in developing and mature retina are now under intense scrutiny (4–7). Like other systems, tissue- or cell type-specific expression of genes in the retina and the response to extracellular environment is accomplished by the combinatorial and synergistic (or antagonistic) action of a limited number of transcription factors (7–12). A better understanding of retinal gene regulation, therefore, necessitates the delineation of protein–protein interactions that guide transcription of specific genes in retinal neurons.

Photoreceptors are unique neurons involved in the first stage of phototransduction (13). Rod and cone photoreceptors have characteristic morphology, express a specific G-protein coupled receptor (opsin) and exhibit distinctive spectral properties (13,14). Regulation of the rod-specific opsin gene, rhodopsin, provides an excellent paradigm to investigate cell-specific gene expression in developing and mature retina (15,16). Numerous studies have revealed two distinct cis-regulatory regions (rhodopsin proximal promoter region, RPPR, from -170 to +70 bp and rhodopsin enhancer region, RER, about 2 kb upstream) (16–21) and several DNA binding proteins that are implicated in transcriptional regulation of rhodopsin expression (20–25). RPPR is sufficient for rod-specific expression and includes several sequence elements that bind to proteins in vitro; these include Ret-1/PCE-1 (16,18), BAT-1 (19), eopsin-1 (22), Ret-4 (21) and NRE (NRL response element) (20).

Neural retina leucine zipper (NRL) is a basic motif-leucine zipper (bZIP) protein of the Maf-subfamily (26) that was shown to bind to NRE and transactivate the rhodopsin promoter activity in cultured cells (20,23). Several differentially phosphorylated isoforms of NRL are expressed specifically in rods and not cones or other neurons (27). NRL is essential for rod differentiation and possibly acts as a molecular switch in photoreceptor cell fate determination since the deletion of Nrl in a knock-out mouse resulted in complete lack of rods but enhanced S-cones (28). The homeodomain protein CRX (cone rod homeobox) was identified as a Ret-4 and/or BAT-1 binding protein that transactivates the rhodopsin promoter as well (24). NRL and CRX physically interact (29) and function synergistically in rhodopsin promoter activity assays (24). NRL is also a key transcriptional regulator of rod-specific gene for cGMP phosphodiesterase-ß subunit (28,30). Mutations in NRL and CRX are associated with various retinopathies in humans, and a number of these mutations are reported to alter the transcriptional synergy between the two transcription factors (31–38).

To further identify protein interactions involving NRL, we performed yeast two-hybrid screening of a bovine retina library and discovered a zinc finger protein of unknown function, homologous to mouse Fiz1, which was previously identified as a Flt3 receptor tyrosine kinase (RTK)-interacting protein (39). We provide in vitro [glutathione S-transferase (GST)-pull-down assays] and in vivo (co-immunoprecipitation from bovine retinal nuclear extracts) evidence in support of NRL–Fiz1 interaction and demonstrate that Fiz1 can repress NRL- but not CRX-mediated transactivation of the rhodopsin promoter activity in transfected cells. We also show that both Fiz1 and Flt3 are expressed in the retina and propose a role for Fiz1 in mediating signal transduction in rods by modulating transcriptional activity of NRL.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NRL–Fiz1 interaction identified by yeast two-hybrid assay
To identify putative synergistic or antagonistic modulators of NRL's function in the retina, we used the yeast two-hybrid interaction assay as a screening strategy. The ‘bait’ construct including the N-terminal region of NRL (amino acid residues 1–142; Fig. 1A) (26) could transactivate reporter genes in the yeast two hybrid assay (data not shown) and was not used for isolation of Nrl-interacting clones. Screening of the bovine retina ‘prey’ cDNA library with the NRL–ZIP ‘bait’ (encoding NRL leucine zipper region, residues 171–231) (26) identified 26 His+ clones that also expressed the ß-galactosidase reporter gene. Partial sequence analysis of the double positive clones revealed five novel cDNAs; of these, two encoded a polypeptide with multiple C2H2 zinc finger motifs. Complete sequencing of these clones revealed a polypeptide of 390 amino acids that represented the bovine homolog (truncated bovine Fiz1 cDNA clone, called {Delta}bFiz) of the mouse Fiz1 protein of 500 amino acids (39) with over 85% identity in the corresponding regions (Fig. 1B). The {Delta}bFiz clone lacked the N-terminal region of the Fiz1 sequence. To confirm the specificity of interaction, bait strains containing pHybLexA–Laminin or pHybLexA–NRL–ZIP were transformed with Gal4AD–{Delta}bFiz (‘prey’). Yeast cells containing Gal4AD–{Delta}bFiz ‘prey’ grew well on minus-His medium and expressed lacZ in the presence of pHybLexA–NRL–ZIP ‘bait’ but not pHybLexA–Laminin (Fig. 1C).



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  		Figure 1. (A) Domain structure of the NRL protein showing the leucine zipper region that was used for yeast two-hybrid screening (amino acids 171–231). TA, transactivation domain; EH, extended homology domain; B, basic motif (DNA binding); ZIP, leucine zipper (dimerization). (B) Alignment of predicted human and mouse Fiz1 proteins with the truncated bovine Fiz clone obtained from two-hybrid screening ({Delta}bFiz). Black boxes indicate zinc finger motifs, clustered in four domains (I–IV). (C) Specificity of NRL–Fiz1 interaction: yeast two hybrid assay. Bait strains containing pHybLexA–Laminin or pHybLexA–NRL–ZIP were transformed with the Gal4AD–{Delta}bFiz ‘prey’ clone. Individual yeast transformants with LexA–NRL–ZIP and Gal4AD–{Delta}bFiz grew on minus-His medium containing 50 mM aminotriazole, but those with LexA–Laminin and Gal4AD–{Delta}bFiz did not grow (non-specific interaction control). Under these stringent conditions with the L40 strain, c-Fos-leucine zipper + c-Jun-leucine zipper (positive control) grew whereas Laminin + c-Jun-leucine zipper did not grow (negative control) (data not shown).

 
Interaction of NRL and Fiz1 by GST-pulldown assay
To further confirm the interaction, [35S]-labeled {Delta}bFiz protein was prepared by in vitro translation and incubated with glutathione-Sepharose beads containing GST (negative control), GST–{Delta}NRL (lacks N-terminal transactivation region but contains NRL residues 110–237, including EH, B and ZIP domains, as indicated in Figure 1A) or GST–NRL (29). GST–{Delta}NRL beads were included in the experiment to show the contribution of leucine zipper region in view of the fact that Fiz1 was identified as an interactor with NRL-ZIP ‘bait’. The [35S]-labeled {Delta}bFiz protein was retained on GST–{Delta}NRL and GST–NRL beads but not on the GST beads (Fig. 2A). The difference in 35S-{Delta}bFiz band intensity is not attributed to relative affinity for {Delta}NRL vs NRL since GST-fusion proteins showed varying efficiency of binding to glutathione-Sepharose beads. These results validate the yeast two-hybrid data showing {Delta}bFiz interaction with the NRL leucine zipper domain. To further establish the involvement of NRL–ZIP, reverse GST pull-down assays were carried out using full-length and two truncated forms of [35S]-labeled NRL protein (NRL1–237, NRL1–209 and NRL1–190) prepared by in vitro translation. The three labeled-NRL proteins showed the expected size by SDS–PAGE and could be immunoprecipitated with affinity-purified polyclonal antibody against NRL (27). Bacterially produced GST–{Delta}bFiz, bound to glutathione–Sepharose beads, was used for reverse pull-down experiments. In agreement with the results in Figure 2A, full-length 35S-NRL1–237 was retained on the GST–{Delta}bFiz beads (Fig. 2B). NRL1–209 missing part of the zipper domain showed reduced binding to GST–{Delta}bFiz and removal of the entire leucine zipper domain in NRL1–190 completely abolished any detectable binding to GST–{Delta}bFiz (Fig. 2B).



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  		Figure 2. Interaction of NRL–ZIP and Fiz1. (A) GST-pull-down assay. The [35S]-labeled {Delta}bFiz protein was prepared by in vitro translation and incubated with glutathione–Sepharose bound GST–NRL, GST–{Delta}NRL (minus transactivation domain) or GST alone (control). The leftmost lane shows 35S-bFiz input. The bFiz protein displayed binding to GST–{Delta}NRL and GST–NRL but not GST beads. (B) Reverse GST-pull-down assay. [35S]-labeled NRL was prepared by in vitro translation and incubated with GST–{Delta}bFiz bound to glutathione–Sepharose beads. Lanes from the left are: 35S-NRL1–237 (full length) translated input, protein bound to Sepharose–GST, protein bound to Sepharose–GST–{Delta}bFiz, immunoprecipitate of translated input protein with anti-NRL antibody. Experiments were repeated in a similar manner using 35S-NRL1–209and 35S-NRL1–190 as input.

 
Co-immunoprecipitation of NRL and Fiz1 from bovine retina nuclear extracts
To evaluate in vivo interaction of native Fiz1 and NRL proteins, co-immunoprecipitation experiments followed by immunoblotting were carried out with bovine retinal nuclear extracts using anti-bFiz or anti-NRL antibodies (Fig. 3). Immunoprecipitation with anti-bFiz antibody identified proteins that were immunoreactive to anti-NRL antibody (Fig. 3A). This protein pattern is similar to the one reported for multiple phosphorylated isoforms of NRL in the retina (27). The reverse co-immunoprecipitation experiments were carried out with anti-NRL antibody and the immunoprecipitated proteins were examined by immunoblot analysis using anti-bFiz antibody. Based on the molecular weight of the mouse Fiz1 protein (39), a protein of about 70 kDa corresponding to bovine Fiz1 was detected (Fig. 3B).



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  		Figure 3. Co-immunoprecipitation of NRL and Fiz1 from bovine retinal nuclear extracts. (A) Bovine retinal nuclear extract (300 µg) was incubated with anti-bFiz antibody and the immunoprecipitated proteins were analyzed by immunoblotting using anti-NRL antibody. Lanes are: 1, bovine retinal nuclear extract (50 µg); 2, immunoprecipitation with anti-bFiz; 3, immunoprecipitation using Protein A–Sepharose beads in the absence of anti-bFiz (negative control). The rabbit IgG band and multiple phosphorylated Nrl isoforms are indicated. A cross-reactive retinal protein of 45 kDa (p45) is also detected by the anti-Nrl antibody (27). (B) Bovine retinal nuclear extract (300 µg) was incubated with anti-NRL antibody and the immunoprecipitated proteins were analyzed by immunoblotting using anti-bFiz antibody. Lanes are: 1, prestained molecular weight markers; 2, bovine retinal nuclear extract (50 µg); 3, immunoprecipitation with anti-NRL; 4, immunoprecipitation using Protein A–Sepharose beads in the absence of anti-NRL (negative control). Anti-bFiz and anti-NRL antibodies were IgGs derived in rabbit and therefore the IgG heavy chain band was detected in both immunoblots.

 
Repression of NRL-mediated transactivation of the rhodopsin promoter activity by Fiz1
We had previously developed rhodopsin promoter activity assays using luciferase reporter gene in heterologous cell culture systems as a quantitative measure of NRL function and its synergy with CRX (20,23,24,33,34). To determine the physiological relevance of NRL–Fiz1 interaction, we co-transfected CV-1 cells with the rhodopsin promoter-luciferase reporter construct (p130–Luc) and the {Delta}bFiz mammalian expression construct (pDest26–{Delta}bFiz) in various combinations with the NRL (pED–NRL) and CRX (pcDNA–bCRX) expression constructs. In all instances, anti-bFiz antibody primarily detected a nuclear localization of the Fiz1 protein when pDest26–{Delta}bFiz was present (Fig. 4A). The immunostaining of transfected CV1 cells confirms that the expression construct produces {Delta}bFiz protein that is transported to the nucleus with or without NRL expression. In rhodopsin promoter activity assays, the {Delta}bFiz expression had no effect on luciferase reporter activity compared to control and with empty pED expression vector (data not shown). The expression of the {Delta}bFiz protein, however, repressed the NRL-mediated transactivation in a concentration-dependent manner (Fig. 4B, black bars). Under these conditions, the bFiz construct had no effect on the CRX-dependent transactivation (Fig. 4B, hatched bars); nevertheless, it reduced the synergy between CRX and NRL in rhodopsin promoter activity assays (Fig. 4B, open bars). Low levels of ubiquitously expressed Fiz1 are present in CV1 cells, probably keeping the rhodopsin promoter in repressed state, which is relieved by overexpression of transactivators NRL and/or CRX.



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  		Figure 4. (A) Immunolabeling with anti-bFiz antibody. CV1 cells were transfected with: a, pcDNA–bCRX; b, pED–NRL; c, pDest26–{Delta}bFiz; d, pDest26–{Delta}bFiz + pED–NRL; e, pDest26–{Delta}bFiz + pCDNA–bCRX; f, pDest26–{Delta}bFiz + pED–NRL + pcDNA–bCRX. (B) Repression of NRL-mediated transactivation of the rhodopsin promoter by Fiz1. Triplicate samples of CV-1 cells were co-transfected with expression constructs for NRL, CRX, {Delta}bFiz and a rhodopsin-promoter luciferase-reporter construct (p130-Luc). Increasing amounts of the construct expressing {Delta}bFiz (pDest26–{Delta}bFiz) repressed NRL-mediated transactivation of the rhodopsin promoter (black bars), but had no effect on CRX-mediated transactivation (hatched bars). bFiz expression also reduced the synergistic co-transactivation of the reporter construct by NRL and CRX (open bars; t-test; *P<0.05, **P<0.01, ***P<0.001). Error bars show the standard deviation, n=3.

 
Analysis and expression of the Fiz1 gene
In silico analysis of the genomic sequences using bioinformatics tools (www.ncbi.nlm.nih.gov; http://genes.mit.edu/GENSCAN.html) identified mammalian bFiz homologs and mapped the Fiz1 gene to mouse chromosome 7 and human chromosome 19. The Fiz1 gene spans about 7 kb of genomic sequence and consists of two exons (data not shown).

Consistent with the previous study in mouse (39), we detected a ubiquitously expressed FIZ1 transcript of 2.7 kb in all human tissues examined (data not shown). Northern analysis of mouse retinal mRNAs from various developmental stages also detected a 2.7 kb transcript, corresponding to Fiz1, as early as embryonic day (E) 14.5 (Fig. 5A). Similarly, a protein of about 70 kDa (consistent with the mouse Fiz1 study) (39) was detected in immunoblots of human, bovine and mouse retinal protein extracts using affinity-purified rabbit IgG against E. coli-expressed GST–{Delta}bFiz (Fig. 5B). An identical band was also observed in nuclear extracts of bovine retina. In the previous study (39) varying levels of Fiz1 were detected in various cell types both in the nucleus and the cytoplasm. Using affinity-purified anti-bFiz antibody, we show that Fiz1 is present throughout the retina but at varied levels and with more intense labeling in the ganglion cell layer (Fig. 5C). As previously reported (39), immunocytochemistry did not show heavy nuclear staining relative to the cytoplasm even though we detected a strong Fiz1 band in the retinal nuclear extract upon immunoblotting.



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  		Figure 5. Expression of Fiz1. (A) Northern blot of mouse retinal mRNA. A 2.7 kb transcript was detected with a probe to the 3' non-coding region of mouse Fiz1 mRNA. The same blot was also probed for glyceraldehyde-3-phosphate dehydrogenase. Retinal RNA samples from embryonic day 14.5 to postnatal day 10.5 were used for northern analysis; however, only four time points are shown. Lanes are: 1, E14.5; 2, P0.5; P 4.5; P10.5. (B) Immunoblot analysis. The protein extracts from human, bovine and mouse retina (100 µg) and bovine retinal nuclear extract (60 µg) were analyzed by SDS-PAGE, and the immunoblot was probed with affinity-purified anti-bFiz antibody. Lanes are: 1, bovine retina; 2, bovine retinal nuclear extract; 3, human retina; 4, mouse retina; 5, no extract control. (C) Indirect immunofluorescence of adult mouse retinal sections using anti-bFiz antibody. FITC-labeled goat anti-rabbit IgG was used as secondary antibody. Control sections were processed without the primary antibody. GCL, Ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments of photoreceptors; OS, outer segments of photoreceptors.

 
Expression of Flt3–RTK in the retina
Fiz1 was originally identified as a novel interacting protein in a two-hybrid screen using the cytoplasmic domain of the Flt3-RTK (39). We, therefore, determined the expression of Flt3 in the retina. A 300 bp PCR product corresponding to FLT3-RTK mRNA could be amplified both from QuickcloneTM human retinal cDNA (Clontech) and a human retinal cDNA library (26) (Fig. 6A). Using a purified polyclonal antibody to the extracellular domain, Flt3 immunoreactivity was primarily detected in the photoreceptor inner segments and the plexiform layers of the mouse retina (Fig. 6B).



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  		Figure 6. Expression of Flt3–RTK in the retina. (A) PCR amplification of Flt3-RTK product. Flt3-RTK primers that spanned intron 1 were used and the amplified product was validated by sequencing. Lanes are: 1, 100 bp ladder; 2, PCR from QuickcloneTM human retinal cDNA; 3, PCR from a human retinal library; 4, no template negative control. (B) Indirect immunofluorescence of a mouse retinal section near the optic nerve exit using anti-Flt3 antibody. Texas-red conjugated goat anti-rabbit IgG was used as secondary antibody. Control sections were processed without the primary antibody. GCL, Ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments of photoreceptors; OS, outer segments of photoreceptors.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Photoreceptors are unique neurons that continuously renew their outer segments with highly regulated periodicity (40). It is, therefore, expected that the expression of phototransduction genes (e.g. rhodopsin) is stringently controlled by positive and negative regulatory transcription factors. To date, NRL and CRX are the two transcription factors which have been shown to synergistically activate the expression of several photoreceptor-specific genes (20,23,24,29,41,42). Of these, NRL is specifically expressed in rods (27) and is essential for rod differentiation (28). To better elucidate the transcriptional regulatory networks in the photoreceptors, it is imperative to identify proteins that display functionally relevant interactions with NRL. Here, we report the identification of Fiz1 as an NRL-interacting protein that is expressed in the retina and can modulate the function of NRL in cultured cells.

NRL–Fiz1 interaction was first identified by yeast two-hybrid screening of a bovine retinal library using human NRL–ZIP as bait, suggesting the interspecies conservation of important functional residues. This was confirmed by in vitro GST- pull-down experiments that also show the involvement of the leucine zipper domain of NRL in mediating the interaction. More importantly, NRL and Fiz1 could be co-immunoprecipitated from bovine retinal nuclear extracts, providing strong evidence for the interaction of native and full-length NRL and Fiz1 in vivo. Since NRL is expressed only in rod photoreceptors in the retina the co-immunoprecipitation studies indirectly support the immunolocalization studies, indicating the presence of Fiz1 in rods.

Fiz1 is a zinc-finger protein of currently unknown function, recently discovered in a yeast two-hybrid screen with the cytoplasmic domain of the Flt3-RTK as bait (39). It contains 11 zinc fingers of the C2H2 class, grouped in four clusters (see Fig. 1B). The bovine clone, {Delta}bFiz, that was isolated in our NRL interaction screen includes seven zinc fingers (clusters II–IV), suggesting that this region of Fiz1 is sufficient for its association with NRL and mediates nuclear localization as well as transcriptional repression of rhodopsin promoter activity observed in co-transfection experiments. The specificity of this interaction is further reflected by the lack of {Delta}bFiz effect on CRX-mediated transactivation of the rhodopsin promoter.

Repression is a significant component of transcriptional regulatory networks, providing an additional level of control and complexity (43–46). Repressor proteins can modulate gene expression by multiple distinct mechanisms and regulate diverse physiological processes, e.g. cell fate determination and dosage compensation (44,45). Zinc-finger proteins are involved in both activation and repression of gene expression. For example, KLF-1, Sp1 and GATA are able to target the SWI/SNF chromatin remodeling complex to the ß-globin promoter via their zinc fingers (47). A zinc finger protein Mok2 has been implicated in repressing the expression of the retinal protein IRBP (interphotoreceptor retinoid binding protein) (48). Recently, Wang et al. (49) have identified Baf (barrier to autointegration factor), a widely expressed protein involved in mitosis and retroviral integration, as a CRX-interacting protein in the retina. Baf is shown to repress the transactivation function of CRX but not of NRL. We hypothesize that Fiz1 (also a ubiquitously expressed protein) has a similar function in modulating NRL but not CRX function in the retina. Together, Baf and Fiz1 may modulate the transcriptional activation properties of NRL and CRX and provide additional level of control of photoreceptor differentiation and function. As a multi-zinc finger protein, Fiz1 may possess DNA binding activity and/or may sequester NRL from binding to NRE or associate with NRE-bound NRL, thereby preventing the recruitment of other activator proteins to retinal gene promoters. The recruitment of Fiz1 to the promoter region may also directly suppress transcriptional initiation. We propose that transcriptional repression (partial or complete) by Fiz1 may be used as a mechanism to attain an appropriate level of gene expression in different cell types. Our studies, therefore, suggest a novel, and as yet unrecognized, function of Fiz1 and open new possibilities for future investigations.

Flt3 (also called Flk-2) stimulates the proliferation of stem cells and differentiation of lymphoid progenitors (50–53). Though expressed in developing and mature central nervous system, the biological function of Flt3 in neurons is poorly understood. Activation of Flt3 with its ligand is shown to inhibit the proliferation of neural progenitor cells and promote neuron survival in synergy with NGF (54). This is the first report demonstrating Flt3 expression in mammalian retina. Possible role of RTKs in retinal cell fate determination has previously been proposed (55). Since RTKs participate in mediating the response to extracellular signals, it will be of interest to examine Flt3 function in the retina and decipher if and how Flt3 signaling pathways might affect photoreceptor differentiation.

In summary, the interaction of NRL with Fiz1 introduces a new dimension to understanding the transcriptional hierarchy in retinal gene regulation. Our studies implicate Fiz1 as a putative transcriptional repressor of NRL function in photoreceptors and provide new insights into combinatorial control and cross-talk between extrinsic and intrinsic signaling pathways. Since mutations in NRL and its target genes lead to retinopathies it is reasonable to propose that sequence variations in the human FIZ1 gene may either cause or modify clinical manifestations in the retina.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Yeast two-hybrid screening
The details of yeast two-hybrid screening have been described (29). The pHybLexA–NRL–ZIP ‘bait’ construct, used in this study, encodes the NRL–ZIP (amino acids 171–231) fused in frame with the LexA protein and did not display autologous activation of the reporter genes lacZ or HIS3 upon transformation in the yeast L40 strain [MATa his3 {Delta}200 trp1-901 leu2-3112 ade2 LYS2::(4lexAop-HIS3) URA3::(8lexAop-lacZ) GAL4]. A bovine retina cDNA library (in pACTII prey vector with Gal4-activation domain) (a gift of Dr C.-H. Sung) was used to isolate interacting clones. Other constructs were obtained as part of the ‘Hybrid Hunter System’ (Invitrogen). Yeast strain L40 was sequentially transformed with the bait and the prey (retinal library) and double transformants (>2.5x106) were selected for possible interactions per manufacturer's protocol (Invitrogen) (29). Interacting retinal clones were further evaluated by retransformation of L40 yeast containing plasmids pHybLexA–Laminin (expressing LexA–Laminin fusion protein) or pHybLexA–NRL–ZIP (expressing LexA–NRL–ZIP). Clones that activated His and lacZ reporter genes with LexA–NRL–ZIP but not LexA–Laminin were considered putative interactors.

NRL and Fiz1 constructs
The partial bovine Fiz1 cDNA ({Delta}bFiz) was sub-cloned into Gateway entry vector, pENT-1A (Invitrogen). The following destination constructs were generated by in vitro enzyme-mediated transfer using the manufacturer's protocol; for GST–{Delta}bFiz fusion protein in E. coli (pDest15–{Delta}bFiz), in vitro translation (pDest17–{Delta}bFiz), and transient expression in mammalian cells (pDest26–{Delta}bFiz). The full-length human NRL and its truncated isoforms were cloned in pcDNA3.1 and used for in vitro translation (29).

Recombinant GST–{Delta}bFiz fusion protein
The GST–{Delta}bFiz fusion protein was expressed in BL21-SI E. coli by inducing the culture with 50 mM NaCl for 3 h at 30°C. The sonicated cell extracts were centrifuged at 12000g for 30 min at 4°C and GST–{Delta}bFiz was purified by binding to glutathione–Sepharose (Amersham-Pharmacia Biotech) for 4 h at 4°C. The bound protein was eluted with reduced glutathione and dialyzed against three changes of 10 mM HEPES, pH 7.4. The purified GST–{Delta}bFiz protein was used to produce polyclonal antibodies in rabbits and subsequently non-specific immunoglobulins were removed by affinity purification using {Delta}bFiz (Invitrogen). GST–{Delta}bFiz bound to glutathione–Sepharose beads was also used for pull-down assays.

In vitro translation and GST-pull-down assay
The bFiz cDNA in pDest17–{Delta}bFiz construct was translated in vitro in the presence of [35S]methionine using T7-TNTTM Quick Coupled Transcription/Translation System (Promega). Pull-down assays are essentially as described (29). For the reverse pull-down assays, NRL cDNA in pcDNA3.1 (27,29) was used for producing [35S]-labeled protein by in vitro translation and GST–{Delta}bFiz–Sepharose beads were used.

Co-immunoprecipitation from bovine retinal nuclear extracts
The nuclear extract from bovine retina was prepared according to published procedure (56). Briefly, 4 g of bovine retina were homogenized in 10 ml of cold phosphate buffered saline (PBS) containing complete protease inhibitor cocktail (Amersham Pharmacia Biotech) using Dounce tissue grinder with five strokes of the B pestle. Tissue was homogenized again by five strokes after adding 0.5% NP-40. The suspension was incubated on ice for 10 min and centrifuged at 16 000g for 5 min at 4°C. The nuclear pellet was suspended in 1/10th volume of PBS, sonicated at 50% duty cycle for 5 min, and centrifuged at 16 000g for 25 min at 4°C. The supernatant (nuclear extract) was stored at -70°C as aliquots.

For immunoprecipitation, the bovine retinal nuclear extract (300 µg) was incubated with anti-Nrl or anti-Fiz1 polyclonal antibodies overnight at 4°C with gentle shaking. The immunoprecipitate was collected by incubation with Protein-A-Sepharose conjugate (Amersham Pharmacia Biotech) at room temperature for 2 h with gentle shaking. After washing three times with PBS containing 1% Triton X-100, the immunoprecipitate was subjected to SDS–PAGE, followed by immunoblotting with appropriate antibodies. The membrane was incubated with the secondary antibody, then washed three times with PBS and developed either using the enhanced chemiluminescence kit (Pierce) or tetramethylbenzidine as substrate (57). For the latter, the membrane was incubated in 10 ml solution [containing di-oictylsulfosuccinate (4 mg/ml final concentration), tetramethyl benzidine (prepared in methanol, 0.1 mg/ml final concentration) and 100 µl of 30% hydrogen peroxide] until green precipitate was formed. The membrane was then washed and fixed by adding 10% methanol.

Rhodopsin promoter activity assays
To assess the functional relevance of NRL–Fiz1 interaction, we performed rhodopsin promoter activity assays using luciferase reporter gene in transiently transfected monkey kidney cell line CV1 (American Type Culture Collection) in the presence or absence of NRL and CRX, as described (20,23,24,33,34). CV1 cells have been used for rhodopsin promoter activity assays since these cells completely lack NRL and CRX, the two known transactivators of the reporter construct. Briefly, cells grown in 24-well plates were transfected at 50–75% confluency using Lipofectamine-plusTM (Invitrogen). The following expression constructs were used: pED–NRL for NRL (34); pcDNA–bCRX for CRX (34); and pDest26–{Delta}bFiz for {Delta}bFiz. All co-transfections included pCMV–lacZ vector and the rhodopsin proximal-promoter luciferase-reporter construct, pBR130–Luc (34). Luciferase expression was measured using the luminescence-based assay system (Promega Corporation, Madison, WI, USA) with duplicate assays per sample. ß-Galactosidase activity was measured with Galacto-Light chemiluminesent assay system (Tropix Inc., Bedford, MA, USA). Fold activations in relative light units (luciferase/ß-galactosidase) were calculated relative to control transfections containing empty pED vector and the pBR130-Luc reporter construct. Triplicate transfections were used for each test condition.

Immunolocalization of Fiz1 and Flt3 in mouse retina
For Fiz1 localization in retina, mouse eyes were obtained after perfusion with 4% paraformaldehyde. Frozen sections were obtained from eyes embedded in OCT (Tissue-Tek, Miles Inc.). Sections were blocked for non-specific protein binding with 20% goat serum in PBS (23°C, 1 h) and then incubated with the primary antibody (affinity purified rabbit anti-bFiz) overnight at 4°C at 1/500 dilution in PBS containing 2% goat serum (Sigma). After washing with PBS+0.2% Triton X-100 (23°C, 3x10 min), the sections were incubated with goat anti-rabbit IgG-FITC conjugated secondary antibody (1 µg/ml, Sigma) in PBS+2% goat serum. Control sections were processed simultaneously without the primary antibody.

Purified rabbit polyclonal IgG against the extracellular domain of the mature mouse Flt3 protein (Upstate Biotechnology) was used at a concentration of 10 µg/ml in PBS+2% goat serum (4°C, overnight). This antibody works well with methanol fixation (Upstate Biotechnology); however, this provided poor retinal morphology. Frozen sections of mouse retina were, therefore, post-fixed a series of times (4% paraformaldehyde in PBS, 23°C) with 1–2 min fixation optimal for both preservation of morphology and surface epitopes. Sections were then washed in PBS and blocked for 1 h in PBS+20% goat serum (23°C). Goat anti-rabbit IgG-Texas red conjugated secondary antibody was used at 1/500 dilution. Control sections were processed simultaneously without the primary antibody.


   ACKNOWLEDGEMENTS
 
The authors thank Drs. Ching-Hwa Sung, Donald J. Zack and Alan J. Mears for reagents and constructive discussions and Ms Sharyn Ferrara for assistance. This research was supported by grants from the National Institutes of Health (EY11115, EY07003), the Foundation Fighting Blindness, and Research to Prevent Blindness.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Ophthalmology and Visual Sciences, W. K. Kellogg Eye Center, University of Michigan, 1000 Wall Street, Ann Arbor, MI 48105, USA. Tel: +1 7347633731; Fax: +1 7346470228; Email: swaroop{at}umich.edu Back

{dagger} Present address: P. K. Swain, National Brain Research Center, Gurgaon, India. Back


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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