Download RBT1, a novel transcriptional co-activator, binds the second subunit

3478–3485 Nucleic Acids Research, 2000, Vol. 28, No. 18
© 2000 Oxford University Press
RBT1, a novel transcriptional co-activator, binds the
second subunit of Replication Protein A
John M. Cho, Daniel J. Song, Josee Bergeron, Naciba Benlimame, Marc S. Wold1 and
Moulay A. Alaoui-Jamali*
Departments of Medicine, Oncology and Pharmacology, Lady Davis Institute for Medical Research of the Jewish
General Hospital, McGill University, Montreal, QC H3T 1E2, Canada and 1Department of Biochemistry,
University of Iowa College of Medicine, 4403 Bowen Science Building, Iowa City, IA 52242-1109, USA
Received June 7, 2000; Revised and Accepted July 27, 2000
ABSTRACT
Replication Protein A (RPA) is required for DNA
recombination, repair and replication in all eukaryotes.
RPA participation in these pathways is mediated by
single-stranded DNA binding and protein interactions.
We herein identify a novel protein, Replication
Protein Binding Trans-Activator (RBT1), in a yeast
two-hybrid assay employing the second subunit of
human RPA (RPA32) as bait. RBT1–RPA32 binding
was confirmed by glutathione S-transferase pull-down
and co-immunoprecipitation. Fluorescence microscopy
indicates that green fluorescence protein-tagged
RBT1 is localized to the nucleus in vivo. RBT1 mRNA
expression, determined by semi-quantitative RT–PCR,
is significantly higher in cancer cell lines MCF-7, ZR-75,
SaOS-2 and H661, compared to the cell lines normal
non-immortalized human mammary epithelial cells
and normal non-immortalized human bronchial
epithelial cells. Further, yeast and mammalian onehybrid analysis shows that RBT1 is a strong transcriptional co-activator. Interestingly, mammalian
transactivation data is indicative of significant variance
between cell lines; the GAL4–RBT1 fusion protein
has significantly higher transcriptional activity in
human cancer cells compared to human normal
primary non-immortalized epithelial cells. We propose
that RBT1 is a novel transcriptional co-activator that
interacts with RPA, and has significantly higher
activity in transformed cells.
INTRODUCTION
Replication Protein A (RPA) is a ubiquitous and abundant
heterotrimeric protein composed of 70, 32 and 11 kDa subunits
and is required for DNA replication, repair and recombination
(1–6). In yeast, RPA is found to bind specific regulatory
sequences in the promoters of DNA repair and metabolism
genes (7) suggesting that RPA may have a role in the regulation
of transcription (8). Previous studies have demonstrated that
DDBJ/EMBL/GenBank accession no. AF192529
RPA function requires an ability to bind and destabilize
dsDNA and that its specific activity is mediated by protein
interaction and/or RPA phosphorylation (9). RPA–protein
interactions appear to be largely mediated by the large 70 kDa
subunit (RPA70). Of note, RPA70 has been reported to interact
with p53, GAL4, VP16, EBNA1, SV40 large T-antigen and
DNA polymerase α (9–14). RPA binding is also important for
the proper function of DNA repair proteins involved in damage
recognition and excision—interaction with Xeroderma
Pigmentosum Complementation Group F (XPF) stimulates its
5′ junction-specific endonuclease activity and interaction with
XPG targets this endonuclease to damaged DNA (15,16).
The possibility of interaction by the aforementioned repair
proteins with the second subunit of RPA (RPA32) has not been
clearly elucidated. However, interactions with some DNA
repair proteins appear to be mediated by RPA32, such as interaction with XPA and uracil-DNA glycosylase (UDG) (17,18).
A region of homology between XPA and UDG is suggestive of
a common binding motif to RPA32. Furthermore, some important
protein interactions, such as with RAD52, appear to involve all
three subunits of RPA (19).
The physiological role of RPA phosphorylation is less
clearly understood. RPA phosphorylation occurs on amino
acid residues proximal to the N-terminal of RPA32 resulting in
retardation in the electrophoretic mobility of this subunit (20–22).
Although RPA is phosphorylated throughout the S phase, in
response to radiation and during apoptosis, mutations of the
major phosphorylation sites do not produce a clear, detectable
phenotype in the model eukaryote, Saccharomyces cerevisiae,
nor do they affect DNA binding, SV40 DNA replication in
vitro or nucleotide excision repair (9,20,23–30). It is plausible
that phosphorylation, rather than being an absolute requirement
for physiological function, affects the conformation of RPA
modulating its affinity for DNA and its protein interactors, and
altering the balance between DNA replication and repair (9). It
is interesting to note that the interaction between RPA and p53
is disrupted by UV damage suggesting at least one mechanism
by which DNA replication and repair are co-ordinated with
checkpoint controls (31).
In the present study, protein–protein interactors of RPA32
were screened by the yeast two-hybrid assay. We describe a novel
protein interactor of RPA32 which we refer to as Replication
*To whom correspondence should be addressed. Tel: +1 514 340 8222; Fax: +1 514 340 7576; Email: [email protected]
Nucleic Acids Research, 2000, Vol. 28, No. 18 3479
Protein Binding Trans-Activator 1 (RBT1). RBT1, a 196 amino
acid protein, is able to transactivate reporter genes in both
yeast and mammalian one-hybrid experiments.
MATERIALS AND METHODS
Plasmids
RPA32 nucleotide sequence was PCR amplified from cDNA
derived from cell line MCF-7 and cloned into the yeast twohybrid plasmids pBTM116 and pACT2 in frame to LexA (1–202)
and GAL4-TA, respectively. Similarly, both XPA and UDG
nucleotide coding sequences were PCR amplified from cDNA
derived from cell line normal non-immortalized human
mammary epithelial cells (NHMEC) and cloned in frame into
pBTM116 and pACT2.
RBT1, obtained from the yeast two-hybrid screen, was
subcloned from pACT2 into pBTM116. For purposes of
cloning truncation plasmids, RBT1 was PCR amplified from
IMAGE hEST clone, locus AI003615, and cloned into both
pBTM116 and pACT2. Plasmids RBT1-∆C16, RBT1-∆C36,
RBT1-∆C57 and RBT1-∆C79 refer to the amino acid deletion
of each RBT1 construct truncated from the C-terminal end.
Similarly, mammalian one-hybrid constructs of RBT1 were
cloned into pSG424 subsequent to PCR using the IMAGE hEST
clone as template. Plasmids RBT1-∆C16, RBT1-∆C36, RBT1∆C57 and RBT1-∆C84 refer to truncations from the C-terminal of
RBT1. The RBT1 clone obtained initially from the yeast twohybrid screen was also cloned into pSG424 by digestion with
BamHI and ligation; orientation was confirmed by restriction
digest, and this construct, referred to as RBT1-∆N22, has a
deletion of 22 amino acids from the RBT1 N-terminus. The
clone representing the entire open reading frame of RBT1
fused to GAL4 in pSG424 is referred to as RBT1. The
sequence for the 22 amino acids proximal to the C-terminus of
RBT1 was cloned from the PCR product generated using
primers RBT-5-TD (5′-CAACGAATTCTGTGCCCCAGGTTCTTGGGA-3′) and RBT-MAM-3-F (5′-CCAGAGTTGCATTCAGGGATCCAGG-3′) and is called RBT1-TD22.
The GAL4-LUC and GAL4-TLS (positive control) plasmids
were obtained from Dr Rongtuan Lin (Lady Davis Institute,
Montreal, Canada) (32). The full RBT1 coding sequence was
cloned in frame into pEGFP-C1 (Clontech, Palo Alto, CA) to
yield RBT1–GFP.
The RBT1 and RPA14 clones obtained from the yeast twohybrid screen were subcloned by digesting with SmaI and XhoI
and ligating into the respective sites in plasmid pGEX-4T-2 to
yield plasmids pRBT1-GST and pRPA14-GST, respectively.
RPA32 was cloned into Bluescript SK+ (Stratagene, La Jolla,
CA) for subsequent use as template for in vitro translation. The
hEST clone AI003615 was digested with EcoRI and BamHI,
ligated into Bluescript SK+ also for purposes of in vitro translation.
Primer sequences for semi-quantitative RT–PCR are: RBTSQ-5A (5′-TCCTCATGTCATCTTCGGTGG-3′), RBT-SQ3B (5′-GGTTTCCACCCTATTGCACG-3′), GAPDH-SQ-5A
(5′-CCATGGAGAAGGCTGGGG-3′) and GAPDH-SQ-3B
(5′-CAAAGTTGTCATGGATGACC-3′).
All cDNA clones were sequenced using a dye primer cycle
sequencing kit (Perkin-Elmer, Norwalk, CT) at Guelph Molecular
Supercentre (University of Guelph, Guelph, Canada) using a
DNA sequencer (Perkin-Elmer ABI 377).
cDNA library screening
The pLexA-RPA32 yeast two-hybrid plasmid construct was
transformed into yeast strain L40 [MATa trp1 leu2 his3
URA3::(lexAop)8-lacZ LYS2::(lexAop)4-HIS3 lys2 ura3 ade2
gal80 gal4] prior to library transformation. A human osteosarcoma cDNA library was obtained as a gift from Dr Karaplis
(Lady Davis Institute, Montreal, Canada) and amplified
according to the Clontech recommended protocol. All yeast
transformations were done using lithium acetate as previously
described (33).
Cell line and cell culture
Cell lines NHMEC, NHBEC (normal non-immortalized human
bronchial epithelial cells) and PrEC (normal non-immortalized
human prostate epithelial cell line) were maintained in medium
supplemented by the manufacturer, Biowhittaker (Walkersville,
MD). B2BN (normal bronchial epithelial cell line immortalized
with SV40 large T-antigen) obtained from Dr B. I. Gerwin
(NCI, Bethesda, MD), PC3 (human prostate adenocarcinoma
cell line), SaOS-2 (human osteosarcoma cell line), ZR-75,
MCF-7, MDA-231 and Hs578T (human breast adenocarcinoma
cell lines), H460, H661 and H322 (human non-small cell lung
cancer cell lines), HEK293 (human normal embryo kidney
cells), Hep3B (human hepatoblastoma cell line) and NIH3T3
(mouse fibroblasts) were cultured in American Tissue Culture
Collection (ATCC, Rockville, MD) recommended media
containing 5 or 10% fetal bovine serum (FBS) and supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin.
All cell lines were maintained in culture at 37°C in an atmosphere of 5% CO2.
Semi-quantitative RT–PCR
Cell lines were grown in appropriate media to 80% confluence,
collected and RNA was isolated using the High Pure RNA
Isolation Kit (Boehringer Mannheim, now Roche Diagnostics,
Laval, Canada). RNA from each sample (5 µg of total) was
used for synthesis of cDNA using the First Strand cDNA
Synthesis Kit (MBI Fermentas, Amherst, NY) where the
reverse transcriptase reaction was primed with the included
oligo (dT)18 primer. PCR was performed using 1 µl of a 1000-fold
dilution of each first strand cDNA reaction mix and included
primers for both RBT1 (RBT-SQ-5A and RBT-SQ-3B) and
GAPDH (GAPDH-SQ-5A and GAPDH-SQ-3B). [α-32P]dCTP
was added to each PCR reaction tube and 20 cycles performed
(94°C for 15 s, 55°C for 30 s, 72°C for 40 s). Aliquots of 2 µl from
each PCR reaction mix were loaded on a 6% acrylamide
sequencing gel, electrophoresed for 2 h and exposed to film. Band
intensity was quantitated using the software program, NIH Image.
Localization
To analyze the subcellular localization of RBT1, a green fluorescence protein (GFP) fusion-based method was employed.
MDA-231 cells were transfected using LipofectAMINE
reagent according to the manufacturer’s recommendations
(Gibco BRL, Rockville, MD) with an expression plasmid
coding for EGFP alone (pEGFP-C1) or for an EGFP–RBT1
(pRBT1-GFP) fusion protein. After 48 h, transfected cells
were washed with phosphate buffered saline (PBS) and fixed
3480 Nucleic Acids Research, 2000, Vol. 28, No. 18
in 3% paraformaldehyde/PBS. Subsequent to fixation, cells
were incubated for 1 h with PBS containing 2% BSA, 2%
normal goat serum and 0.2% gelatine at room temperature to
reduce non-specific binding of the RPA32 antibody. All
washing and incubations with both primary (RPA32) and
secondary (Texas Red conjugated) antibodies were done in
PBS containing 0.2% BSA. After labeling, the coverslips were
mounted in Airvol (Air Products and Chemicals Inc., Allentown,
PA) and photographed with a Zeiss Axiophot fluorescent
microscope. Monoclonal antibody against RPA32 was
purchased from Oncogene Research Products (Cambridge,
MA).
Transactivation assays
Yeast. L40 cells were transformed with plasmid(s) and assayed
for LacZ reporter activity by using the liquid culture β-galactosidase assay using o-nitrophenyl B-D-galactopyranoside
(ONPG) as substrate as described in Clontech Matchmaker
protocols. All experiments were done in triplicate.
Mammalian. Mammalian cell transfections were performed
using the LipofectAMINE (Gibco BRL) reagent according to
the manufacturer’s recommendations; 0.36 µg of reporter
plasmid (p5XUPS-GAL4-LUC), 0.36 µg of a given RBT1pSG424 fusion plasmid or pSG424 empty vector or pGAL4TLS positive control and 0.08 µg pRL-TK (Renilla luciferase
reporter under the control of a TK promoter) internal control
plasmid for a total of 0.8 µg DNA in 12 well plates. Transfections
were done in triplicate and repeated 3–5 times. Twenty-four
hours after transfection, cells were washed with PBS without
Ca2+ or Mg2+ and harvested with Passive Lysis Buffer
(Promega, Madison, WI). Luciferase assays were performed
using reagents from the Dual-Luciferase Reporter Assay
System (Promega) and with an EG&G Berthold model Lumat
LB9507 luminometer. Relative luciferase activity was calculated
and reported as a ratio between firefly luciferase and Renilla
luciferase activity.
GST pull-down assay and in vitro translation
Glutathione S-transferase (GST), GST–RBT1 and GST–RPA14
were expressed and isolated from Escherichia coli DH5α
following a 3 h induction with 1 mM isopropyl-β-D-thiogalactopyranoside (Pharmacia) at 37°C according to the Pharmacia
recommended protocol; subsequent to lysis, GST fusion
proteins were incubated for 1 h with glutathione–Sepharose
beads (Pharmacia), the beads were washed three times with
PBS/1% Triton X-100 and once with PBS. Captured GST
fusion proteins were used for the GST pull-down assay.
RPA32 was in vitro translated in a reaction mixture
containing [35S]methionine (Amersham) using a TnT coupled
reticulocyte lysate system (Promega) according to the manufacturer’s instructions. The translated product was incubated
with the GST fusion proteins and GST alone and subsequently
washed. Binding was analyzed by 10–20% SDS–PAGE and
subsequent exposure to film. RBT1 was in vitro translated in a
similar manner to RPA32.
Protein extraction, immunoprecipitation
Mammalian cells were lysed with 10 vol of lysis buffer
[50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.1 mM EDTA,
0.5% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol,
0.2 mM phenylmethylsulfonyl fluoride, aprotinin (3 µg/ml),
pepstatin (1 µg/ml), leupeptin (1 µg/ml), 1 mM Na3VO4 and
10 mM NaF] and the supernatant was collected after centrifugation.
For immunoprecipitation, 400 µg of protein was mixed with
30 µl of Protein G-Sepharose and either anti-RPA32 mouse
monoclonal antibody, anti-RBT1 rabbit polyclonal antibody
(generated against purified GST–RBT1 protein at the McGill
Animal Resource Centre, Montreal, Canada) or non-specific
rabbit IgG antibody overnight at 4°C. Beads were pelleted,
boiled in SDS sample buffer and the immunoprecipitates were
subjected to 15% SDS–PAGE. Subsequent to transfer onto a
polyvinylidene difluoride membrane (Millipore, Bedford,
MA), the blot was incubated with anti-RPA32 monoclonal
antibody for 2 h at room temperature, washed and visualized
by enhanced chemiluminescence (Amersham).
RESULTS
A yeast two-hybrid screen of a human osteosarcoma GAL4
cDNA library using LexA-RPA32 as bait yielded 15 putative
positive colonies from a total of 400 000 colonies assayed.
Eight of the interactors were identified as the 14 kDa subunit of
RPA (RPA14) by PCR and/or cycle sequencing. RPA32–RPA14
binding represents the highest affinity of interaction in this
system based on the vigorous growth of the colonies on
dropout media lacking histidine (containing 25 mM 3-amino1,2,4-triazole) and strong β-gal activity; the isolation of
RPA14 suggested that RPA32 is faithfully translated in yeast
and that its conformation is not compromised.
RBT1 sequence
A putative positive identified as a probable RPA32 interacting
protein was sequenced and found to have no strong homology
to any other previously characterized protein and is subsequently
referred to as RBT1. The original clone obtained by yeast twohybrid was fully sequenced and used in a BLAST search of
human ESTs (expressed sequence tags). Sequence overlap was
used to pick AI003615 as representing the full coding
sequence of RBT1 and part of the 5′-UTR and 3′-UTR. The
full RBT1 sequence is derived from automated sequencing of
human EST clone locus AI003615. The nucleotide sequence of
clone AI003615 corresponds to the clone obtained by yeast
two-hybrid except that this latter clone is truncated at the N-terminal
and putatively reads from amino acid 22 (P-A-G-L-Q-S....).
The RBT1 cDNA has an open reading frame of 196 amino
acids; the nucleotide and amino acid sequence is shown in
Figure 1A. In vitro translated RBT1 migration corresponded to
the theoretical protein size of ∼22 kDa and this corresponded
with western blot analysis using the generated polyclonal antiRBT1 antibody (data not shown).
Although RBT1 does not share significant homology with
previously characterized proteins, it does contain four motifs
with proline residues arranged in a manner similar to the core
consensus sequence for SH3 binding proteins or the PY motif
of WW domain-binding proteins (Fig. 1B). These motifs are
arranged pseudo-symmetrically with the first and fourth SH3like binding motifs sharing the common sequence P-P-E-P, and
the second and third P-P-Q. Also, a W-E-W motif is evident 19
amino acids from the N-terminal and 15 amino acids from the
C-terminal end of RBT1. In concert with the SH3-like binding
Nucleic Acids Research, 2000, Vol. 28, No. 18 3481
Figure 2. GST pull-down. Input of labeled RPA32 is shown in the first lane.
The fraction of labeled RPA32 pulled down by GST–RBT1, GST–RPA14 and
GST alone, respectively, are shown in the following lanes.
Figure 3. Co-immunoprecipitation. 293 kidney cell extracts were incubated
with anti-RPA32, anti-RBT1 and non-specific polyclonal IgG (negative control)
antibody, respectively; these were done in duplicate. Lanes labeled ‘None’
contain 100 µg of protein extract as a positive control for the anti-RPA32
antibody used to immunoblot this membrane.
Figure 1. Structure of RBT1 cDNA. (A) The nucleotide sequence of RBT1 cDNA
and the deduced amino acid sequence (GenBank accession no. AF192529).
(B) SH3-like consensus binding sites, where X tends to be alipathic (G, A, V,
L, I), the two prolines (P) are necessary for high affinity binding and the
scaffolding residue (p) is often proline.
motifs, RBT1 may be a protein with ‘mirrored symmetry’
possibly important for protein–protein interactions.
GST pull-down assay and co-immunoprecipitation
The binding of RPA32 to RBT1 was analyzed by performing a
GST pull-down assay. As shown in Figure 2, the GST–RBT1
and GST–RPA14 bind to RPA32 in vitro, whereas GST alone
does not bind. RPA32 pulled down by GST–RPA14 appears
significantly stronger than that of GST–RBT1; this was as
expected since the affinity of interaction between RPA32 and
RPA14 is extremely strong. Further, we verified that RPA32 had
specificity in this binding assay by including in one experiment
GST-tagged Basal Transcription Factor 3 (BTF3) and IKKγ
both of which did not pull down the in vitro translated RPA32
(data not shown).
Co-immunoprecipitation using anti-RBT1 antibody and
revealing with anti-RPA32 antibody also showed RBT1
binding to RPA32 (Fig. 3); the presence of RBT1 in the antiRPA32 immunoprecipitate was not demonstrated because of
significant background on the immunoblot. While the proportion of RPA32 brought down by the anti-RBT1 polyclonal
antibody is less than that of RPA14, it was able to bind more
RPA32 than the same amount of anti-XPA polyclonal antibody, a known interactor of RPA32 (data not shown). RPA32
binding to RPA14 has extremely high affinity, but this does not
preclude RPA32 binding to other proteins with moderate
affinity.
RBT1 mRNA size and semi-quantitative RT–PCR
A single transcript of ∼1.6 kb was detected by northern blot
analysis in cell line H322 (data not shown). Semi-quantitative
PCR and analysis of the clonal origins of the hESTs of RBT1
in GenBank suggest that RBT1 is expressed in various human
tissues, but with variance in abundance. We noted a high
frequency of EST matches from cancer cell lines and sought to
ascertain whether there was a difference in expression between
cancer and normal cell lines. Indeed, RBT1 expression levels
appear to be significantly higher in all cancer cell lines examined
by semi-quantitative RT–PCR in comparison to non-transformed
cell lines (Fig. 4). Levels of RBT1 mRNA appear to be at least
5–10 times more abundant in cancer cell lines MCF-7, ZR-75,
3482 Nucleic Acids Research, 2000, Vol. 28, No. 18
Figure 4. Semi-quantitative RT–PCR. RBT1 mRNA expression normalized
to GAPDH mRNA in cell lines NHMEC, MCF-7 and ZR-75, SaOS-2,
NHBEC and H661.
SaOS-2 and H661 as compared to both NHMEC and NHBEC
normalizing to GAPDH mRNA.
Localization of RBT1
Cells transfected with GFP alone displayed diffuse green
fluorescence throughout the cells. On the other hand, a distinct
nuclear fluorescent pattern was observed in cells expressing
GFP–RBT1 (Fig. 5). RPA32 immunofluorescence overlapped
GFP–RBT1 demonstrating that the fluorescence is localized to
the nucleus. A putative nuclear localization sequence, K-R-K-H,
is also found near the N-terminal end of RBT1.
Yeast one-hybrid and two-hybrid data
Interactions between RPA32 and RPA14, RPA32 and XPA,
and RPA32 with UDG were confirmed by yeast two-hybrid
(Table 1 and Fig. 6). The protein product of pACT2-RBT1 did
not show interaction with the protein products of pBTM116,
pLexA-UDG or pLexA-RPA14 suggesting that it is not a
spurious positive interactor with RPA32.
RBT1 and RPA32 were shuffled into pBTM116 and pACT2,
respectively, to further verify the interaction. However, we
were unable to do so using pLexA-RBT1 because of extremely
strong transactivation of the yeast reporter genes by itself. This
observation suggested the possibility that RBT1 has characteristics of a transcriptional co-activator (Table 1 and Fig. 6).
Truncations of the RBT1 sequence suggest that the transactivation domain of RBT1 lies proximal to the C-terminal end
of the putative protein. Furthermore, RPA32 interacts with
RBT1-∆C79 which suggests that the binding domain lies
somewhere in the N-terminal half of RBT1; RBT1-∆C79 by
itself does not activate the HIS3 reporter gene and has minimal
β-gal expression.
Of note is that the fusion product of LexA-XPA alone transactivates both reporter genes approximately twice as strongly
as that shown by the XPA and RPA32 interaction in yeast. The
pLexA-XPA transformed yeast cells were plated on media
containing leucine, but this difference does not necessarily
explain the significant difference. In the context of GAL4
(BD), another group was able to show by yeast two-hybrid the
physiological interaction of XPA with RPA32; to perform the
yeast two-hybrid screen, XPA is not likely to have had significant
reporter gene activity in the context of GAL4 (BD) by itself.
Similarly, RBT1 transactivates much more strongly alone than
when co-transformed with RPA32—binding appears in these
cases to repress the strong transactivation by both XPA and
RBT1 possibly because the GAL4 transactivation domain is
weak relative to these genes or because there is conformational
interference between the transactivation domains of the interacting proteins.
The choice of DNA-binding domains in the yeast two-hybrid
may affect the conformation of the gene of interest and therefore
impact on observed interactions or physiological effects. It is,
however, unlikely that RBT1 is affected by the choice of LexA
or GAL4 DNA-binding domain since both such fusions gave
transcriptional promoting activities in yeast and mammalian
cells, respectively; we cloned into a yeast vector (pAS2-1),
RBT1 downstream from GAL4 (DB) and transformation into
yeast demonstrated transactivation by the protein product of
this construct (Table 1).
Mammalian one-hybrid
RBT1 in the context of the mammalian one-hybrid system
demonstrated strong trans-activity in support of our yeast data.
A clone representing the C-terminal 22 amino acids showed
extremely high luciferase activity suggesting that much of the
transactivation potential of this protein lies within this small
domain (Fig. 7A). The initial clone obtained via yeast two-hybrid
Figure 5. Nuclear localization of RBT1. (Top row) Diffuse expression of GFP in MDA231 cell line, RPA32 localization defining the nucleus and phase contrast
photograph of same cells. (Bottom row) Expression of RBT1–GFP and RPA32 localization to the nucleus and phase contrast photograph of same cells.
Nucleic Acids Research, 2000, Vol. 28, No. 18 3483
which lacks 22 amino acids from the putative N-terminal
(RBT1-FULL) also gives relatively high activity within the
context of the mammalian one-hybrid system.
Table 1. Yeast two-hybrid assays
DNA-binding domain
Activation domain
plasmid
Growth
(pBTM116)
(pACT2)
(colony formation)
RPA32
–
RPA32
–
RPA32
RPA14
++++
RPA14
RPA32
++++
XPA
++++
XPA
–
XPA
RPA32
++++
RPA32
XPA
+++
UDG
–
UDG
–
UDG
RPA32
+++
RPA32
UDG
+++
UDG
RBT1
–
RBT1
+++++
RBT1-∆C16
++++
RBT1-∆C36
+++
RBT1-∆C57
++
RBT1-∆C79
–
RBT1 (pAS2-1; strain
Y190)
+++++
RBT1
–
RBT1-∆C16
–
RBT1-∆C36
–
RBT1-∆C57
–
Figure 6. β-gal activity in yeast one-hybrid and two-hybrid assays. (Top
graph) Comparison of β-gal activity of RPA32 as bait: alone, with RBT1, with
UDG and with XPA. (Second graph) Comparison of β-gal activity of RBT1 in
the bait plasmid with RPA32 and RPA32 as bait with RPA14. (Third graph)
Comparison of β-gal activity of RPA32 in pACT2: alone, with UDG and with
XPA. Also shown are activities of UDG and XPA in the bait plasmid alone.
Note the strong transactivation of XPA by itself. (Bottom graph) Comparison
of β-gal activity of full-length RBT1 with truncations of RBT1 from the putative
C-terminal domain. Also included are RBT1–RPA32 and RPA32–RBT1 β-gal
activities as comparative reference.
relative luciferase activity is significantly higher in cancer cell
lines in comparison to non-transformed lines. RBT1-FULL
showed highest activity in MCF-7 cells. These results are not a
function of transfection efficiency since the data are reported
as ratios of firefly to Renilla luciferase activity and the raw
data was indicative of transfection efficacy.
RBT1-∆C79
–
RBT1
RPA32
++++
DISCUSSION
RPA14
RBT1
–
RPA32
RBT1
+++
RPA32
RBT1-∆C16
+++
RPA32
RBT1-∆C36
+++
RPA32
RBT1-∆C57
+++
RPA32
RBT1-∆C79
+++
RPA plays a pivotal role in DNA metabolism. Its physiological
role is associated with DNA destabilization, binding and
recognition; it is not surprising then that it has numerous
functional interactions with different proteins involved in one
or more aspects of DNA replication, repair or recombination.
While conventional biochemical methods have described the
major interactors of RPA, our library screen suggests that there
may be other physiologically relevant interactions that remain
uncharacterized. RBT1 may be one of these.
We attempted to determine the protein interactors of RBT1
by shuffling the clone obtained in the screen into the bait
plasmid and subsequently screening our library, but were
unable because of the extremely high trans-activity produced
by the LexA–RBT1 fusion protein alone. We therefore examined
whether RBT1 has a functional transactivation domain. Indeed,
several proteins have been serendipitously characterized as
transcriptional co-activators prior to the initial yeast twohybrid screen for protein–protein interactors; proteins such as
Growth on histidine deficient media containing 25 mM 3-amino-1,2,4-triazole
determined qualitatively by vigour of colony formation. Where two proteins
are assayed for interaction, the selection media is also deficient for tryptophan,
leucine and histidine. Where a protein is assayed for activation of the HIS3
reporter gene by itself, the media is deficient for either tryptophan or leucine
and histidine. Plus signs refer to how quickly and the vigour with which the
resultant colonies grow.
The activity of RBT1 was examined in several cell lines,
both cancerous and non-transformed, and compared to the
activity produced by GAL4-TLS. As can be seen in Figure 7B,
3484 Nucleic Acids Research, 2000, Vol. 28, No. 18
Figure 7. Relative luciferase activity of RBT1 in mammalian one-hybrid
assay. (A) Activity in MCF-7 cell line. Comparison of relative luciferase activity
of full-length RBT1, the C-terminal 22 amino acids of RBT1 (RBT1-TD), the
initial clone obtained via yeast two-hybrid which lacks 22 amino acids from
the putative N-terminal (RBT1-∆N22) and RBT1 truncations (RBT1-∆C16,
RBT1-∆C36, RBT1-∆C57 and RBT1-∆C84). (B) Activity of RBT1 in cancerous
and non-transformed cell lines in comparison to TLS. Transactivation by
RBT1 in cell line MCF-7 is greater than the scale provided but is the same as
that shown in Figure 7A.
BRCA1 and Npw38 were found to contain transcriptionpromoting activities in yeast and were subsequently found to
have similar activities in mammalian cells (34,35).
RBT1 does not have significant homology to any previously
characterized protein. In silico analysis (TBLASTN) using the
RBT1 amino acid sequence did not show any EST homologs in
other species with the exceptions of human and mouse.
However, we note the presence of SH3-like (or WW domain-like)
consensus binding sites arranged in mirrored symmetry. Interestingly, the PY motif is found in the activation domains of
many transcriptions factors, including c-Jun, AP-2 and NF-E2
(36). One hypothesis for the relevance of the PY motif
suggests that transcription stimulation is partially conferred by
interaction with WW domain-containing proteins which themselves contain strong transactivation domains. The significance of the proline rich sites in RBT1, which are similar to the
PY motif, remains to be determined, but they may be important
for interaction with enhancers or repressors of its transactivation
function.
The minimal transactivation domain at the C-terminal end of
RBT1 has no significant homology to previously described
domains that impart transcription-promoting activities.
However, this domain is not particularly acidic suggesting that
it is not a spurious trans-activator as can often be the case with
acidic amino acid regions. Furthermore, the relative luciferase
activity by this domain as well as for full-length RBT1 is at
least comparable and often much higher than that observed
with our positive control for transactivation, Translocated in
Liposarcoma (TLS). TLS was previously described within the
context of the mammalian one-hybrid system to have transactivity similar to levels observed with GAL4 fused to VP16
(37). We used TLS as a positive control, and while transactivity was similar in most cell lines, this was not the case in
MCF-7 and Hs578T cells where RBT1 had significantly
stronger activity. This suggests the possibility that, at least in
some cellular contexts, RBT1 trans-activity is affected by more
than one component or pathway of transcription.
While some random amino acid sequences can produce nonphysiological trans-activity in the context of one-hybrid
systems, the extremely high level of activity observed for
RBT1 in cancer cells indicate that it is likely that the activity
observed is physiologically relevant. We suggest that the short,
minimal transactivation domain of RBT1, as well as the fulllength RBT1, is functionally strong. Mutational analysis of this
region will allow us to determine which amino acids are essential
for basal transcriptional activity. While binding of co-factors
may be necessary for optimal RBT1 trans-activity, the strong
transactivation data obtained using the C-terminal of RBT1
suggests that it may itself interact with the basal transcription
machinery.
The observed RBT1 transactivation, within the context of
the mammalian one-hybrid system, is high in cancer cell lines
in comparison to non-transformed cell lines; transcript level is
correlated to this upregulation and this was found to be well
correlated to protein levels—we were able to detect RBT1
protein in extracts from cancer cell lines but not from normal
cell lines (data not shown). We suggest the possibility that
RBT1 may play a role in the regulation of cell proliferation or
may contribute to the maintenance or promotion of oncogenesis.
Overexpression of the GAL4–RBT1 construct does not show
similar levels of luciferase reporter gene activity in cancerous
and non-transformed cell lines suggesting that a co-factor
present in abundance in cancerous cells is necessary for
optimal transactivation and/or that an inhibitor is not present or
is non-functional. Since cancer cell lines usually proliferate
with greater vigour, they likely have an abundance of proteins
involved in replication and would be conducive to strong
RBT1 transactivation.
One candidate for inhibition of RBT1 transactivation is p53.
Preliminary data in this laboratory show significant inhibition
of RBT1 transactivation by wild-type (wt) p53. Expression of wt
p53 in several p53 null cells [SaOS-2, Hep3B and NIH-3T3(null)]
results in a >50% decrease in transactivation by RBT1 (data
not shown). While the mechanism of this inhibition is yet to be
Nucleic Acids Research, 2000, Vol. 28, No. 18 3485
clearly defined, it is plausible that binding of p53 by RPA is
correlated to a reduction in the affinity of binding between
RBT1 and RPA or that inhibition of RPA by p53 leads to a
concomitant inhibition of RBT1. It is also possible that p53
binds RBT1. These possibilities are currently being investigated in conjunction with the determination of the specificity
of the inhibition of RBT1 by wt p53 versus p53 mutant forms.
RBT1 is a novel cDNA encoding a nuclear protein capable
of binding RPA32 within the context of the yeast two-hybrid
system, as well as GST pull-down and co-immunoprecipitation.
We plan to address other significant questions such as: whether
the phosphorylation state of RPA32 affects the affinity of
binding to RBT1, whether RBT1 binds to the other subunits of
RPA and in which aspects of cellular function including DNA
metabolism and DNA repair does RBT1 play a role.
ACKNOWLEDGEMENTS
We thank Dr A. Karaplis, Dr R. Lin, Dr H. Kwon, Dr C. DeLuca,
Dr M. Katabi, Dr A. Wong, Dr M. Whiteway and Dr J. Hiscott
for materials and technical assistance. This work was
supported by a grant from the Canadian Institutes of Health
Research and in part by the Canadian Breast Cancer Initiative
of the National Cancer Institute of Canada (M.A.A.J.). J.M.C.
is supported by a studentship from the Canadian Institutes of
Health Research, and M.A.A.J. is a senior scientist of the
‘Fonds de Recherches en Sante du Quebec’.
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