Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St Johns, Newfoundland, Canada1
Author for correspondence: Laxminarayana R. Devireddy. Present address: Howard Hughes Medical Institute, University of Massachusetts Medical Center, 373 Plantation Street, Suite 309, Worcester, MA 01605, USA. Fax +1 508 856 5473. e-mail Laxminarayana.Devireddy{at}umassmed.edu
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Abstract |
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Introduction |
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Embryonal carcinoma (EC) cells are pluripotent in nature and can be differentiated with chemicals into several different cell lineages. For example, retinoic acid (RA) and DMSO induce neuron- and skeletal muscle cell-like morphologies, respectively. Thus, the EC cell system serves as a model system for studying neuron-specific gene expression. Previous studies from our laboratory have shown that the JCV promoter is active only in RA-differentiated and not in undifferentiated or DMSO-differentiated EC cells (Nakshatri et al., 1990 ; Kumar et al., 1993
). Nuclear extracts prepared from RA-differentiated cells protect nuclear factor-1 (NF-1)-like sequences in the JCV promoter (Nakshatri et al., 1990
; Kumar et al., 1993
) and disruption of these sites reduces the activity of the JCV promoter (Kumar et al., 1993
). Furthermore, a novel factor present in RA-differentiated EC cells seems to interact with these sites, as judged by the results of band-shift and UV cross-linking assays (Kumar, 1994
). However, the identity and nature of this protein remain unknown. Cloning and characterization of such factors may shed more light on neuron-specific expression of human JCV.
BAG-1 (Bcl-2-associated athano gene-1) is a novel Bcl-2-interacting protein that was first identified as a multifunctional molecule that binds to the anti-apoptotic protein Bcl-2 and promotes cell survival (Takayama et al., 1995 ). Since its discovery, BAG-1 has been shown to form complexes with several other proteins, including tyrosine kinase growth-factor receptors such as hepatocyte- and platelet-derived growth factors (Bardelli et al., 1996
), the serine/threonine protein kinase Raf-1 (Wang et al., 1996
; reviewed in Wang & Reed, 1998
) and the retinoic acid receptor (Liu et al., 1998
). Alternative translation of BAG-1 generates a series of proteins with variable lengths (Packham et al., 1997
). Longer forms of BAG-1, with unique amino-terminal domains, have also been reported to form complexes with steroid-hormone receptors (Packham et al., 1997
; Froesch et al., 1998
). Binding of BAG-1 to these molecules modulates their activity. The mechanism by which BAG-1 influences the activities of such diverse proteins can perhaps be attributed to its ability to bind heat-shock proteins directly, which in turn interact with multiple target proteins in cells. BAG-1 contains an amino-terminal region with similarity to ubiquitin and a central region that binds Bcl-2 (Takayama et al., 1995
). Its carboxy terminus is required for formation of complexes with Raf-1 and growth factor receptors.
It is well established that the expression of JCV early and late genes, encoding the tumour (T) antigen and capsid proteins, respectively, determines the narrow host range and specificity of this virus. Studies from our lab and other laboratories have demonstrated that the JC promoter/enhancer functions more efficiently in neuronal cells than in non-neuronal cells. The studies presented here attempt to identify the proteins that bind the JCV promoter/enhancer region. Here, we report the cloning of a novel protein by Southwestern screening of a P19 RA-differentiated cDNA library with a JCV NF-1 probe. Interestingly, this protein is identical to a Bcl-2-interacting protein, BAG-1. Furthermore, this protein is able to bind the NF-1 sequence and transactivate JCV promoters.
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Methods |
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Oligonucleotides.
The sequences of wild-type (WT) and mutant (mt) JCV NF-1 oligonucleotides used are: WT NF-1, 5' TGGCTGCCAGCCAA 3'; and mt NF-1, 5' gtaCTGCCAGaCcAGCA 3' (see Kumar et al., 1993 ). Lower-case letters indicate mutated bases.
Construction and screening of the cDNA library.
A gt22A cDNA library from mRNA of P19 RA-differentiated cells was made by using a kit from GIBCO-BRL according to the manufacturers instructions. Screening of the cDNA expression library was performed by the Southwestern blot method of Vinson et al. (1988)
. Freshly prepared E. coli Y1090R- bacteria were infected with recombinant phage from the P19 RA
gt22A library at ~5x104 p.f.u. per 150 mm plate. The infection was performed by incubating at 37 °C for 30 min. Next, 7·5 ml 0·7% top agarose was mixed with the bacteria and the mixture was overlaid onto 1·5% T-Tyn (tryptone, yeast extract and NaCl) agar plates. After incubation at 42 °C for 4 h, IPTG-impregnated nitrocellulose filters were overlaid onto the plates and the plates were incubated for additional 6 h at 37 °C. The filters were then removed from the plates, air-dried and immersed in binding buffer (6 M guanidineHCl; 25 mM HEPES, pH 7·9; 3 mM MgCl2; 4 mM KCl and 1 mM DTT) for 5 min at 4 °C with gentle agitation. The binding buffer was then diluted with an equal volume of the same buffer but containing variable amounts of guanidineHCl and the filters were incubated in these diluted buffers for 5 min each. The filters were then washed with binding buffer without guanidineHCl. The filters were then blocked by incubating in binding buffer containing 5% non-fat dried milk powder for 30 min at 4 °C. After rinsing in binding buffer containing 0·25% milk powder, the filters were probed with 106 c.p.m./ml nick-translated JCV NF-1 oligonucleotide concatemer in binding buffer containing 0·25% milk powder and 10 µg/ml sonicated salmon sperm DNA. After 3 h hybridization at 4 °C, the filters were washed three times for 15 min each with binding buffer containing 0·25% milk powder. Finally, the filters were wrapped in Saran Wrap and exposed to X-ray film overnight at -70 °C. Positive plaques were identified and re-screened until they were homogeneously positive.
Sequence analysis.
The cDNA, which was cloned into pBluescript KS(+) (Stratagene), was sequenced by using a kit from United States Biochemical according to the manufacturers instructions. The sequence was compared with known sequences in GenBank by BLAST search.
Northern blot analysis.
Total RNA from undifferentiated (UD), RA- and DMSO-differentiated EC cells, U87 MG and HeLa cells was extracted by using a kit from Promega, according to the manufacturers instructions. Twenty µg total RNA was electrophoresed on a 1% formaldehydeagarose gel and transferred to nylon membrane (Gilman Sciences). The filter was blocked with Denhardts solution and probed with 106 c.p.m./ml nick-translated kNF-1 cDNA overnight at 60 °C. After hybridization, the filter was washed and exposed to X-ray film. The hybridized probe was stripped with 0·1xSSC and 0·1% SDS and reprobed with an actin probe as described above.
Gel-shift analysis.
The sequences of JCV NF-1 oligonucleotides were described by Kumar et al. (1993) . The probes were prepared by end-labelling the oligonucleotides in the presence of [
-32P]dCTP. The binding reactions were done in a volume of 35 µl in buffer containing 10 mM TrisHCl, pH 7·8; 1 mM EDTA; 5 mM DTT; 150 mM KCl; 12% glycerol; 10 µg poly(dI.dC), 10 ng labelled probe and 4 µl in vitro-translated BAG-1. The reaction mixture was incubated at room temperature for 30 min. The reaction products were resolved on a 4% non-denaturing PAGE gel. Gels were then dried and subjected to autoradiography.
Southwestern blot analysis.
Nuclear extracts from UD, RA- and DMSO-differentiated EC cells, HeLa and U87 MG cells were resolved on a 10% SDSPAGE gel and transferred to PVDF membrane in a semi-dry transfer apparatus (Bio-Rad) according to the manufacturers instructions. The filters were then blocked overnight at 4 °C with 5% milk powder in a binding buffer containing 25 mM HEPESNaOH, pH 7·9; 5 mM MgCl2; 0·5 mM DTT and 25 mM NaCl. After blocking, the filters were probed with 106 c.p.m./ml nick-translated JCV NF-1 oligonucleotide in binding buffer. The filters were then washed with binding buffer without milk powder, dried and subjected to autoradiography.
CAT assays.
CAT assays were performed as described previously (Nakshatri et al., 1990 ). The percentage of acetylation was quantified by liquid scintillation and normalized on the basis of
-galactosidase activity.
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Results |
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Analysis of DNA extracted from the two positive plaques yielded the same sequence, and only one plaque was characterized further. The positive recombinant phage encoding the JCV NF-1 region-binding protein was termed kNF-1 (Kumar, 1994 ). Analysis of kNF-1 revealed a ~1 kb DNA fragment (Fig. 1a
) with perfect sequence identity to BAG-1 (Fig. 1b
; Takayama et al., 1995
). The kNF-1 cDNA contains an open reading frame (ORF) of 229 amino acids with the potential to encode a 30 kDa protein. The ORF corresponds to sequences spanning the entire BAG-1 sequence. BAG-1 is a Bcl-2-interacting protein isolated from a mouse embryonic cDNA library in the yeast two-hybrid system (Takayama et al., 1995
). The sequence identity of kNF-1 to BAG-1 is likely to reflect the isolation of the same gene.
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Transcriptional activity of kNF-1 cDNA in HeLa cells
To examine the transcriptional activity of the kNF-1 protein, the kNF-1 cDNA was cloned into a eukaryotic expression vector, pRc/CMV. Non-neuronal HeLa cells that do not support JCV transcription were employed (Tada et al., 1989 ). Reporter plasmids with the CAT gene under the control of the JCV early (JCVE) or late (JCVL) promoters were transfected into HeLa cells alone or with pCMV-kNF-1. A basal level of activity from the JCVE and JCVL promoters was detected in HeLa cells (Fig. 4a
). Co-transfection of kNF-1 cDNA resulted in 6-fold and 5-fold activation of the JCVE and JCVL promoters, respectively (Fig. 4a
). Co-transfection of pRc/CMV alone had no effect (data not shown). The RIIE expression plasmid, containing only one JCV 98 bp repeat, was also transactivated, by 2·5-fold (Fig. 4a
). The BK virus early promoter was not transactivated by kNF-1 cDNA (Fig. 4a
). A JCV enhancer/promoter containing mutations in the NF-1 sites was not transactivated by kNF-1, suggesting that the integrity of these sites is necessary for kNF-1-mediated transactivation (Fig. 4b
). In summary, these results suggest that kNF-1 specifically transactivated JCV promoters.
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Discussion |
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JCV infection occurs during childhood in the majority of the population, but only during immunosuppression does this virus become pathogenic and cause PML. The virus replicates only in neuronal cells, but the mechanism that determines neuron-specific expression of virus genes is not completely understood. It has been postulated that a regulatory pathway that includes participation of neuronal and non-neuronal transcription factors plays a role in the neuron-specific regulation of JCV (reviewed in Raj & Khalili, 1995 ). The functional redundancy of promoter elements and the requirement for multiple cellular transcription factors further support the hypothesis of multiple cis and trans determinants of JCV neurotropism.
The data presented here led to the identification of a novel transcription factor, BAG-1, that bound specifically to the JCV NF-1 sequences and up-regulated transcription of the JCV early and late promoters. Recent studies indicate that BAG-1 and its longer isoforms BAG-1M and BAG-1L influence a wide variety of cellular phenotypes through their interaction with Hsc70/Hsp70, including resistance to apoptosis, promotion of cell proliferation, enhancement of tumour cell migration and alteration of transcriptional activity of steroid-hormone receptors (Takayama et al., 1995 ; Wang et al., 1996
; Bardelli et al., 1996
; Liu et al., 1998
; Froesch et al., 1998
). The ability of the BAG-1 family of proteins to modulate the chaperone activity of Hsc70/Hsp70 may have different consequences for the various proteins whose functions are controlled by conformational change. For example, BAG-1 proteins have been reported to bind Bcl-2, Raf-1, androgen receptor and PDGF receptor and enhance their function. Conversely, interaction of BAG-1 with retinoic acid receptor, glucocorticoid receptor and Siah-1 seems to inhibit their activity (Takayama et al., 1995
; Wang et al., 1996
; Bardelli et al., 1996
; Liu et al., 1998
; Kullmann et al., 1998
; Matsuzawa et al., 1998
; Froesch et al., 1998
). The Bcl-2 protein, which has an anti-apoptotic function, recruits cytosolic proteins to internal membranes, including Raf-1 and BAG-1 (Wang et al., 1996
; Takayama et al., 1998
). Bcl-2 also negatively regulates p53 transcriptional activity (Froesch et al., 1999
), possibly by sequestering transcription factors necessary for p53 transcriptional activity. Analogously, BAG-1 may modulate JCV expression by interacting with transcription factors.
It is intriguing that a ubiquitously expressed, anti-apoptotic and chaperone regulator BAG-1 activates the expression of JCV. Many distinct promoter elements, including NF-1, contribute to the tropism of the JCV promoter (Nakshatri et al., 1990 ; Kumar et al., 1993
). The restriction in expression of individual transcription factors to glial cells, including Tst-1, NF-1 and Sp1, may confer neuron-specific expression to JCV (Nakshatri et al., 1990
; Henson et al., 1992
; Wegner et al., 1993
). Therefore, the relative abundance of transcription factors, rather than a single determining factor, may account for the neurotropism of JCV.
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Acknowledgments |
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Footnotes |
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b This work is dedicated to the fond memory of Dr Mary M. Pater, who passed away on 2 November 1994.
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References |
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Received 3 August 1999;
accepted 21 October 1999.