Queensland Institute of Medical Research and University of Queensland Joint Oncology Program, 300 Herston Road, Herston 4029, Brisbane, Australia1
Author for correspondence: Kenia Krauer. Fax +61 7 3362 0106. e-mail keniaK{at}qimr.edu.au
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Abstract |
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Introduction |
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Studies of viral gene expression initially established RBP as a transcriptional repressor of adenovirus polypeptide IX gene expression (Dou et al., 1994 ). So far, only a few mammalian cellular genes have been shown to be targetted by RBP; IL-6 (Kannabiran et al., 1997
; Plaisance et al., 1997
), CD23, CD21 and IL-1
(Krauer et al., 1998
). Kannabiran et al. (1997)
demonstrated that RBP repressed the activation of the IL-6 promoter by NF-
B and C-EBP and demonstrated that the location of the RBP site was critical in mediating repression. Plaisance et al. (1997)
showed that RBP partially blocked access of NF-
B to the promoter region and postulated that RBP was responsible for the low basal levels of IL-6. Our previous study identified an RBP-binding site, which overlapped a previously characterized NF-
B-binding site, in the -300 region of the IL-1
promoter (Krauer et al., 1998
). This study also demonstrated that RBP bound preferentially to the region over NF-
B, suggesting that RBP may be responsible for constitutive repression of the IL-1
gene.
The Drosophila homologue of RBP is suppressor of hairless, Su(H), which plays a functional role in the development of the peripheral nervous system in Drosophila (Furukawa et al., 1994 ). Studies have gone on to demonstrate that Su(H) participates specifically in the developmental Notch receptor signalling pathway (Artavanis-Tsakonas et al., 1995
). When co-expressed with Notch, Su(H) is sequestered in the cytoplasm. However, following Notch ligand binding, Su(H) is translocated to the nucleus where it can modulate gene expression. A number of proteins can interact with Su(H), including the different forms of Notch, which lead to transcriptional activation, and Hairless, which inhibits DNA binding (reviewed in Artavanis-Tsakonas et al., 1995
). The developmental role of Su(H) therefore appears to be determined by interaction with other proteins, which can then regulate gene expression. Given the crucial role that this protein plays in the Notch signalling pathway and that RBP-J
knock-out mice die at around 9 days of gestation (Oka et al., 1995
), Su(H)/RBP is likely to play an important role in cell development and differentiation.
The herpesvirus EpsteinBarr virus (EBV) utilizes RBP to mediate transcriptional regulation of both viral and cellular genes. The nuclear proteins EBNA-2 and the EBNA-3 gene family of proteins (EBNA-3, -4 and -6) have all been shown to interact with RBP (Krauer et al., 1996 ; Robertson et al., 1995
; Grossman et al., 1994
). The EBNA-2 protein is responsible for transcriptional activation of viral proteins, including TP-1, TP-2 and LMP-1, and a number of cellular genes including CD23 and CD21. Following EBNA-2 binding to RBP, which leads to masking of the RBP repression domain, the complex then targets promoters containing the RBP DNA consensus sequence. The EBNA-3 family of proteins, however, appear to regulate RBP-mediated gene expression by binding to RBP and preventing RBP from binding to DNA (Robertson et al., 1995
). Modulation of cellular gene expression has been shown following expression of the EBNA-3 gene family (Kienzle et al., 1996
; Silins & Sculley, 1994
). In addition, the EBNA-3 family proteins can repress EBNA-2-mediated transcriptional activation of the TP promoter (Le Roux et al., 1994
). Given the crucial roles that EBNA-2, -3, -4 and -6 play in EBV transformation of B cells, and given that they all bind RBP, it is likely that RBP is a key player in the transcriptional regulation of genes following EBV infection and hence immortalization of B cells.
Little is known about the RBP protein in human B cells. This study has examined the cellular localization of RBP in B-cell lines and showed that the intranuclear forms of RBP are altered following the expression of EBV genes, such that the majority of RBP becomes bound to chromatin.
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Methods |
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Transient transfection of dG75 and dG75 E346 cells.
dG75 and dG75 E346 cells were transfected by electroporation under conditions that have been described previously (Kienzle et al., 1996 ). Briefly, 3x106 cells (exponential-phase growth) were resuspended in 0·4 ml RPMI supplemented with 10% FCS and placed in a Gene pulser cuvette (Bio-Rad). Plasmid DNA (10 µg) was introduced into the cells by electroporation in a Bio-Rad Gene Pulser with 0·23 kV at 960 mF (time constant ~30 ms). The cells were incubated for 5 min at room temperature before transfer into 10 ml RPMI 1640 with 10% FCS and then incubated at 37 °C. Transfected cells were analysed for gene expression by Western blotting and immunofluorescence after 48 h incubation.
Construction of green fluorescent protein (GFP) expression plasmids and fluorescent microscopy
pEGFP-RBP-J.
The RBP-J cDNA was excised from pSG5-J
(a gift from Clare Sample, St Jude Childrens Research Hospital, Memphis, TN, USA) by restriction digestion with BamHI and BglII and subcloned into the BglII site of pEGFP-C1. This plasmid contains the enhanced GFP (EGFP) (Clontech) under the control of a CMV promoter. The resulting plasmid was sequenced to ensure correct reading frame and orientation of the RBP-J
cDNA.
pEGFP-RBP-2N.
The RBP-2N cDNA was excised from pACT-2N clone G4(3) (Young et al., 1997 ) by restriction digestion with XhoI and subcloned into the XhoI site of pEGFP-C1. The resulting plasmid was sequenced to ensure correct reading frame and orientation of the RBP-2N cDNA.
Fluorescent microscopy.
Expression of the GFPRBP-J and GFPRBP-2N fusions was determined by visualization under a fluorescent microscope. Transfected cells were harvested 48 h after transfection and placed on a slide in a minimal amount of medium, covered with a coverslip and then visualized. The percentage of fluorescent cells was analysed by FACScan analysis and the results are shown as means±SD.
Immunoblotting.
Protein extracts from 1x106 cells were electrophoresed on 7·5% SDSpolyacrylamide gels (Sambrook et al., 1989 ) and electrotransferred onto nitrocellulose filters (Hybond-ECL nitrocellulose; Amersham). The membrane was processed as described previously (Krauer et al., 1996
). Expression of the EBNA-1, -2, -3, -4 and -6 proteins was detected by incubation with human serum (MCr serum; Sculley et al., 1984
) diluted 1:200 in 5% Blotto in PBS, while expression of RBP was detected by addition of anti-RBP antibody (supplied by T. Honjo; Sakai et al., 1995
) diluted 1:1000. The proteins were visualized by using the ECL Western blotting detection system (Amersham).
Cell extracts and gel retardation assays.
Extracts were prepared by using a previously described method (Sambrook et al., 1989 ). Briefly, 2x107 cells were washed in PBS and resuspended in 1 ml cold buffer A [10 mM HEPESNaOH, pH 8, 50 mM NaCl, 500 mM sucrose, 1 mM EDTA, 0·25 mM EGTA, 0·6 mM spermidine hydrochloride, 0·5% (v/v) Triton X-100, 1 mM PMSF and 7 mM
-mercaptoethanol]. The cells were then homogenized in a Dounce homogenizer by using 15 strokes and transferred to a fresh tube. After centrifugation (650 g, 10 min, 4 °C), the supernatant (cytoplasmic extract) was collected. The pellet, containing the nuclei, was then resuspended in 300 µl buffer B [10 mM HEPESNaOH, pH 8, 400 mM NaCl, 25% (v/v) glycerol, 0·1 mM EDTA, 0·1 mM EGTA, 0·6 mM spermidine hydrochloride, 1 mM PMSF and 7 mM
-mercaptoethanol] and incubated on a rocking platform at 4 °C for 40 min. After centrifugation (1100 g, 10 min, 4 °C), the supernatant (nuclear extract) was collected. Gel retardation assays were performed using the Bandshift kit (Pharmacia) according to the manufacturers instructions. Briefly, the binding reactions were performed in 10 mM TrisHCl, pH 7·5, 50 mM NaCl, 0·5 mM DTT, 1·5 mM EDTA, 1 mM MgCl2, 4% glycerol and 0·5 µg poly(dIdC).poly(dIdC) in a volume of 15 µl. Competitors or antibodies were incubated with the nuclear protein extracts on ice prior to the addition of the radiolabelled probe. 32P-end-labelled, double-stranded oligonucleotide 5' TCTTCTAACGTGGGAAAATCCAGT 3' was used as the probe.
Extraction of chromatin- and non-chromatin-bound nuclear extracts.
Nuclear proteins that are not associated with the chromatin can be extracted with 150 mM NaCl, while non-histone DNA-binding proteins (chromatin-bound) can be extracted with 500 mM NaCl (Busch et al., 1967 ). Nuclei were prepared as described above and were then fractionated by extraction in either nuclear extraction buffer B containing 150 mM NaCl or a buffer containing 500 mM NaCl.
DNase treatment of cell nuclei to release DNA-binding proteins.
Nuclei were prepared as described above and were then extracted with buffer B containing 150 mM NaCl, which led to the extraction of nuclear proteins that were not tightly associated with nuclear components. The nuclei were then treated for 15 min at room temperature with 150 U RNase-free DNase (Boehringer Mannheim) in buffer B containing 150 mM NaCl and supplemented with 5 mM MgCl2. The resulting extract contained DNA-binding proteins that were released following DNase treatment.
Co-immunoprecipitation.
Immunoprecipitations were performed according to a method modified from that of Harlow & Lane (1988) . Co-immunoprecipitations were prepared from nuclear extracts prepared with buffer B containing 500 mM NaCl (see above). The nuclear extracts were pre-cleared with protein-GSepharose (Pharmacia) for 1 h at 4 °C with constant rocking. An appropriate antibody (5 µg) was added to the supernatant and the mixture was incubated for 2 h at 4 °C with rocking. Protein-GSepharose (30 µl) was added and then incubated for 1 h at 4 °C with rocking. The samples were then centrifuged at 10000 g for 1 min, the supernatant was discarded and the pellet was washed twice with 1·5 ml nuclear lysis buffer. The pellet was resuspended in 40 µl SDSPAGE loading buffer, heated at 85 °C for 10 min and centrifuged at 10000 g for 5 min and 15 µl of the supernatant was electrophoresed on a 7·5 or 10% SDSpolyacrylamide gel.
Two-dimensional (2D) gel electrophoresis.
2D gel electrophoresis was performed with the Immobline Drystrip (pH 310·5) and ExcelGel SDS gradient (818%) according to the manufacturers instructions (Pharmacia). LCL cells (1x107 dG75 and CS-B95.8) were lysed in a buffer containing 9 M urea, 2% Triton X-100, 2% -mercaptoethanol, 1·4 µg/ml PMSF and 2% pharmalyte 310 (Bio-Rad). Sample solution (8 M urea, 2%
-mercaptoethanol, 0·5% Triton X-100 and 2% pharmalyte 310) was added to each lysate and then loaded onto the rehydrated Immobline Drystrip for isoelectric focussing. After the first dimension, the drystrip was placed on the ExcelGel SDS gradient (818%). After electrophoresis through the second dimension, the proteins were transferred onto nitrocellulose filters and subjected to Western blot analysis with the anti-RBP antibody as described above.
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Results |
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Discussion |
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The cellular location of RBP has been linked to the differentiation status of cells (Sakai et al., 1995 ). In the human B-cell lines dG75 and MUTU I, fractionation studies showed that RBP was predominantly located within the cell nucleus, with small amounts being observed in the cell cytoplasm (Figs 2
and 5
). RBP was absent from the membrane fractions, however (data not shown). Analysis of the intranuclear fractions demonstrated that RBP was distributed between free, nuclear forms and chromatin-bound forms, with a predominance in the chromatin-bound fraction (Fig. 5
). The B-cell lines used in this study show a germinal B-cell phenotype and are considered to be at the intermediate stage of B-cell differentiation (Cushley & Harnett, 1993
). RBP might therefore be expected to be distributed between the cytoplasmic and nuclear fractions.
Differentiation of embryonic carcinoma cells leads to nuclear translocation of RBP (Sakai et al., 1995 ) and an increase in the chromatin-bound form of the protein. Given that RBP acts as a transcriptional repressor, these results suggest that nuclear translocation of RBP leads to decreased expression of cellular genes that control differentiation. An increase in nuclear RBP and a concomitant reduction in cytoplasmic RBP were observed in EBV-transformed B-cell lines (LCLs) in comparison to dG75 EBV-negative BL cells (Fig. 5
). In addition, a reduction in free forms of RBP and an increase in chromatin-bound RBP were also evident following expression of the EBV latent proteins (Fig. 5
). Expression of the EBV latent proteins led to a sequestering or recruitment of chromatin-bound forms of RBP in the nucleus. This resulted in the majority of cellular RBP existing either bound to chromatin (Fig. 5
, upper panel) or complexed to the EBV proteins, suggesting that RBP-mediated cellular gene expression may be partially under the control of EBV. This is not unexpected, as previous studies have suggested that approximately half of the cellular RBP is associated with the EBNAs in LCLs (Johannsen et al., 1996
). As RBP controls/regulates cell differentiation, the sequestering of free RBP by EBV latent proteins may lead to a blockage in further cellular differentiation. This would agree with an earlier finding that, following EBV infection, B cells do not differentiate further to become antibody-secreting plasma cells (Rickinson & Kieff, 1996
).
Do the two RBP isoforms (RBP-2N and RBP-J) expressed in B cells have different functions? This study makes a number of important observations with regard to functional differences between RBP-2N and RBP-J
. Firstly, RBP-J
and RBP-2N show different nuclear localization patterns (Fig. 2
). Secondly, the over-expression studies with EGFPRBP-2N and EGFPRBP-J
suggested either that B-cell lines show different sensitivities to RBP-2N and RBP-J
(Table 1
) or that RBP-2N and RBP-J
are processed differently within the cell. This suggests that RBP-2N and RBP-J
have different functions since, if their functions were interchangeable, cells would not display differential sensitivity to the two isoforms. That the EBNA-3 gene family of proteins was capable of increasing greatly the number of cells expressing EGFPRBP-2N, but not EGFPRBP-J
, supports the existence of functional differences between the two isoforms and suggests that RBP-2N interacts preferentially with the EBNA-3, -4 and -6 proteins (Fig. 4
). As our studies were performed on EGFP fusion proteins, in order to ensure that they were functionally active, gel-shift analysis was performed on nuclear extracts of DG75 cells transfected with EGFPRBP-J
(Fig. 3
). These results showed that the fusion protein was able to bind its DNA consensus sequence and that the complex could be supershifted with anti-RBP antibodies. As RBP-J
and RBP-2N have identical DNA-binding domains, it is logical to predict that both isoforms can bind their DNA binding sites. The finding of preferential association of the EBNA-3 family proteins with RBP-2N would also agree with the co-immunoprecipitation results, where the smaller, more predominant isoform, RBP-2N, was shown to associate more readily with EBNA-3 and -6 in comparison to EBNA-2. The first exon of RBP-J
encodes 19 amino acids, whereas the first exon of RBP-2N encodes only six amino acids. It is possible that the differences between the first exons could influence either the half-life of the proteins, post-translational modification or their three-dimensional structure, which could therefore affect interaction of these proteins with other proteins or DNA.
It remains to be determined whether the cellular genes regulated by EBV are the same as those that play a role in determining cellular differentiation status. However, from the results presented in this study, it is apparent that each of the RBP isoforms performs different functions and that EBV (or at least the EBNA-3, -4 and -6 gene products of EBV) target the RBP-2N isoform. This observation is supported by a previous study, which showed that the EBNA-3 gene family associated preferentially with the smaller RBP isoform in B-cell lines (Johannsen et al., 1996 ). In addition, this earlier study showed that the majority of EBNA-2 was associated with RBP, whereas approximately 20% of EBNA-6 was associated with RBP. As RBP-2N and RBP-J
show different nuclear localization patterns and differ in their ability to associate with the EBNAs, it is possible that one isoform may control cellular differentiation whereas the other may have a more generalized function in gene regulation.
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Acknowledgments |
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References |
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Received 4 May 1999;
accepted 10 August 1999.
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