From the Departments of Pediatrics and Cancer
Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263 and the § Department of Pediatrics, State University of New
York, Buffalo, New York 14222
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ABSTRACT |
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SCL is a basic domain helix-loop-helix (bHLH)
oncoprotein that is involved in T-cell acute lymphoblastic leukemia as
well as in normal hematopoiesis. Although it is believed that SCL
functions as a tissue-specific transcription factor, no molecular
mechanism has thus far been identified for this putative function. In
this report, we show that SCL interacts with p44, a subunit of the basal transcription factor TFIIH. The minimal region of SCL that interacts with p44 was mapped to a 101-amino acid sequence that includes, but is not limited to, the bHLH region; the SCL-binding site
of p44 is located in the carboxyl-terminal half of p44. This interaction was confirmed by glutathione S-transferase
fusion protein pull-down assays and a co-immunoprecipitation assay. As analyzed with a yeast two-hybrid system, p44 interacts specifically with SCL, but not with the other class A or B bHLH proteins tested. E2A
did not compete with p44 for SCL binding, as demonstrated by an
in vitro binding assay. These findings document a
previously unsuspected interaction between SCL and a subunit of
the basal transcription factor TFIIH, suggesting a potential means by
which SCL might modulate transcription.
Since its isolation from the multipotential DU528 stem
cell leukemia cell line, which carries a
t(1;14)(p32;q11) chromosome translocation (1-3), the SCL
(TCL5 or tal-1) gene has been studied in the
context of both normal and abnormal hematopoiesis. The observation that
the SCL locus is frequently disrupted in T-cell acute
lymphoblastic leukemia cells (4-6), resulting in aberrant SCL expression, has led to speculation that the gene product
of SCL is an oncoprotein. Indeed, recent transgenic mouse
models have confirmed that unscheduled SCL expression leads
to aggressive T-cell malignancies (7, 8). Moreover, despite its initial identification in leukemic cells, targeted disruption of the
SCL locus has demonstrated that SCL expression is
absolutely required for normal hematopoietic development (9-12).
SCL belongs to the basic domain helix-loop-helix
(bHLH)1 family of proteins
(13). Three principal classes of bHLH proteins have been identified
(14). Class A bHLH proteins include the E proteins E2A/ITF1
(immunoglobulin transcription
factor 1), E2-2/ITF2, and HEB (an E protein related to
E2A/ITF1 and E2-2/ITF2); these proteins are expressed ubiquitously. In
contrast, the expression of class B bHLH proteins, such as MyoD and
SCL, is restricted to specific organs or tissues. The myogenic (MyoD,
myogenin, myf5) bHLH proteins have been studied extensively (15) and
serve as a useful paradigm for the actions of tissue-restricted bHLH
proteins. MyoD forms a heterodimer with the ubiquitously expressed E2A
proteins (16), binds specific DNA sequence at the regulatory regions of
genes coding for muscle-specific proteins (such as muscle creatine kinase), and activates transcription of these genes (17). Although unproven, it is thought that SCL activates transcription of genes required for normal hematopoietic development in an analogous fashion
(18).
Several forms of the SCL protein have been found in mammalian cells
(19); the full-length form of SCL has a molecular mass variously
reported to be between 42 and 49 kDa, whereas an amino-terminally truncated form of 22-26 kDa is produced by translation of
alternatively spliced transcripts (20) in both normal and leukemic
cells. Although a transcription activation domain has been mapped to its amino terminus (21), unscheduled synthesis of an SCL protein that
lacks the transactivation domain leads to T-cell leukemia in transgenic
mice, indicating that this transactivation domain is not required for
leukemogenesis (8). Similar to many other bHLH proteins, SCL has been
shown to bind several E proteins, including E2-2/ITF2 (22), E12, and
E47 (two alternatively spliced forms of E2A/ITF1) (23). When bound to
E12, the SCL·E12 complex preferentially binds to a CAGATG nucleotide
sequence in vitro (24). Although SCL is suspected to
function as a sequence-specific transcription factor, no target genes
for SCL have thus far been convincingly identified, and any mechanism
by which SCL may control transcription remains unknown.
TFIIH is a multicomponent basal transcription factor complex that is
also known to function in certain DNA repair pathways (25). Nine
subunits have been identified within the TFIIH holoenzyme complex;
various enzymatic activities, including DNA repair, helicase (26), and
cyclin-dependent kinase (27) activities, have been identified. The p62, p52, p44, and p34 subunits are thought to constitute the "core" of the TFIIH transcription machinery (28). Although the p44 and p34 subunits have no defined enzymatic activity, their zinc finger structures suggest that they may be DNA-binding proteins (29) that might mediate interactions with soluble
transcription factors.
Here we report the identification of an unexpected interaction between
SCL and the p44 subunit of TFIIH. This observation provides a link
between SCL and the basal transcription machinery, suggesting that SCL
may exert its suspected transcription regulatory effects through an
interaction with TFIIH.
Yeast Two-hybrid Screen--
An SCL cDNA derived
from clone 67 (20) was subcloned into the pGBT9 two-hybrid vector
(CLONTECH) with the SCL coding sequence downstream
of the GAL4 DNA-binding domain. This vector, named pGBT9SCL,
was cotransformed into Saccharomyces cerevisiae YRG-2 yeast
cells (the relevant genotype of the YRG-2 strain is Mat In Vitro Transcription/Translation of SCL and E2-5--
An
SCL cDNA derived from clone 67 (20) was subcloned into
the pRC/CMV vector (Stratagene) and used as the DNA template for the
production of SCL protein. The E2-5 cDNA (30) was a gift from Dr. Adam Goldfarb. The in vitro
transcription/translation reactions were performed using the
TNT® Coupled Reticulocyte Lysate systems with T7 RNA
polymerase (Promega). The 35S-labeled proteins were
analyzed by 10% SDS-PAGE.
GST Fusion Protein Interaction Assay--
A full-length p44
cDNA was PCR-amplified using oligonucleotides
5'-GGGTGAATTCGGAGCCATGGATGAAGAACCTGAAAGAACTAAG-3'
and 5'-TAACGAATTCAATGTATACTACATGCTGG-3' as primers (the
EcoRI cloning sites are underlined, and the translation
start codon is indicated in boldface) and cloned into pGEX-1 Co-immunoprecipitation--
HEL and HL-60 cell lysates were
prepared as described (32) and incubated with rabbit polyclonal
antibodies directed against either the human TFIIH p62 subunit or human
cyclin D1 (Santa Cruz Biotechnology) and protein A-Sepharose (Amersham
Pharmacia Biotech) overnight at 4 °C. The precipitates were washed
three times with phosphate-buffered saline containing 0.1% Nonidet
P-40, dissolved and heat-denatured in 2× SDS loading buffer, and
resolved by 10% SDS-PAGE. The proteins were then transferred to a
HybondTM ECLTM nitrocellulose membrane
(Amersham Pharmacia Biotech) and detected with anti-SCL monoclonal
antibody BTL73 (a gift from Dr. Karen Pulford) (33), horseradish
peroxidase-conjugated anti-mouse IgG, and Western blot
Chemiluminescence Reagent Plus (NEN Life Science Products).
SCL and p44 Deletion Mutants--
Most of the SCL deletion
mutants were derived from pGBT9SCL using exonuclease III to
create nested deletion mutants (Erase-A-Base, Promega). Briefly, 5 µg
of pGBT9SCL was digested with PstI and BamHI, and the carboxyl-terminal deletion mutants were
generated with exonuclease III and S1 nuclease. For the amino-terminal
deletion mutants, a derivative clone of pGBT9SCL, which
lacks the EcoRI site located 3' to the SCL
cDNA, was first digested with EcoRI, and the
5'-overhangs were filled in with Klenow fragment of DNA polymerase I
and Construction of Additional GAL4-bHLH Expression
Vectors--
Partial cDNA sequences of HEB (amino acids 277-682;
GenBankTM accession number M80627), E2A/ITF1 (amino acids
349-582; GenBankTM accession number X52078), and E2-2/ITF2
(amino acids 210-622; GenBankTM accession number X52079)
isolated from the above yeast two-hybrid cDNA libraries were
subcloned into pGBT9. pGBT9NHLH1 (amino acids 8-133) was
derived from an NHLH1 cDNA clone (kind gift of Dr. Ilan
Kirsch) (34). An LYL1 cDNA encoding full-length
LYL1 was PCR-amplified using primers
5'-GTGGAATTCACCATGACTGAGAAGGC-3' and 5'-CCCTGAATTCTTCACTGGTCCTTCTT-3' and
cloned into pGBT9. The SCL cDNA was excised from
pGBT9SCL and subcloned into pGAD424 (CLONTECH). Full-length p44 cDNA was
PCR-amplified using the primers described above and subcloned into
pGBT9 and pGAD424.
Yeast Two-hybrid Interaction Assay--
Log-phase YRG-2 yeast
cells were made competent for transformation by suspension in TE buffer
(10 mM Tris-HCl and 1 mM EDTA, pH 7.5) and 100 mM lithium acetate, pH 7.5. One µg of plasmid DNA was
added to 120 µl of yeast competent cells before adding 600 µl of TE
buffer, 100 mM lithium acetate, and 40% polyethylene glycol 4000. The mixture was incubated at 30 °C for 30 min, mixed with 70 µl of Me2SO, and incubated for 15 min at
42 °C. The cells were plated in duplicate on -Leu/Trp and
-Leu/Trp/His SD plates. The plates were incubated for 5 days at
30 °C, and growth was monitored daily. The colonies grown on the
-Leu/Trp plate were transferred to a Whatman filter, and the cells
were disrupted in liquid nitrogen; Quantitative Assay of Yeast Two-hybrid Screen--
To help understand the mechanism of
SCL function during both normal hematopoiesis and leukemogenesis, we
searched for SCL-binding partners in eukaryotic cells using a yeast
two-hybrid system. Although the amino-terminally truncated 22-26-kDa
form of SCL had previously been used in a yeast two-hybrid screen (35), we used the full-length SCL protein as a bait to avoid bias against interactions that involved the amino-terminal portion of the SCL protein. Despite the fact that the full-length SCL protein contains an
activation domain that functions in mammalian cells (21), the GAL4
DNA-binding domain-SCL fusion protein (produced by pGBT9SCL) did not confer transcription activation activity in YRG-2 yeast cells
(Fig. 1). The libraries employed in these
screens were a human fetal brain cDNA library (Stratagene), a human
fetal liver cDNA library (CLONTECH), and a
human thymus cDNA library (CLONTECH). More than
106 cotransformants of each library were screened. Fifteen
independent clones of E2-2/ITF2 were isolated from the human fetal
brain cDNA library. One clone of DRG
(development-regulated GTP-binding
protein) was identified in the human fetal liver cDNA
library (36). We recovered three independent clones of E2A/ITF1, two
independent clones of HEB, one clone of DRG, and two identical clones
of p44 (a subunit of TFIIH) from the human thymus cDNA library.
Interestingly, although LMO1 (Lim
domain-only protein 1) and LMO2 have been shown to interact
with SCL (37, 38), we did not recover any clones encoding these
proteins from our screens.
Verification of a Specific SCL/p44 Interaction--
The specific
interaction between SCL and Tp44 was confirmed by cotransformation of
both pGBT9SCL and pGAD10Tp44 into YRG-2 cells to
reconstitute the GAL4 transcription activity (Fig. 1). To test whether
SCL will interact with full-length p44 as well as Tp44, a GAL4
transcription activation domain-p44 fusion protein was created by
cloning PCR-amplified full-length p44 into the pGAD424 vector; this
fusion protein was also found to specifically bind SCL in yeast cells
(Table I). An exchange-partner approach was attempted by fusing SCL to the GAL4 transcription activation domain
and p44 to the GAL4 DNA-binding domain and testing these constructs in
the yeast two-hybrid system. However, since we found that the GAL4
DNA-binding domain-p44 fusion protein conferred transcription activity
in YRG-2 yeast cells without an additional fusion
protein,2 this approach was
abandoned.
As an additional confirmation of the SCL/p44 interaction, we generated
fusion proteins with both the full-length and truncated forms of p44
fused to GST for use in an in vitro "pull-down" binding assay. GST, GST-p44, and GST-Tp44 fusion proteins were produced in
E. coli and purified using glutathione-Sepharose beads. The glutathione-Sepharose beads, containing bound GST fusion proteins, were
then divided into two equal aliquots. One aliquot was resolved by 10%
SDS-PAGE (Fig. 2A). The second
equal aliquot of glutathione-Sepharose beads, containing bound GST
fusion proteins, was incubated with 5 µl of 35S-labeled
in vitro translated SCL protein. The GST fusion proteins were again precipitated by centrifugation of the glutathione-Sepharose beads, and coprecipitated SCL was identified by resolving the coprecipitated material using 10% SDS-PAGE (Fig. 2B). As
shown in Fig. 2B, GST-p44 and GST-Tp44 both precipitate the
in vitro translated SCL protein, whereas GST does not. Note
that since the amount of GST fusion proteins used for precipitation was
equal to that shown in Fig. 2A, slightly more GST protein
than either GST-p44 or GST-Tp44 was used for the coprecipitation.
To study the association of the native SCL and p44 proteins in
mammalian cells, we isolated cell lysates from HEL (a human erythroleukemia cell line) and HL-60 (a human myeloid leukemia cell
line) cells. HEL cells synthesize abundant amounts of SCL mRNA and protein, whereas HL-60 cells do not produce detectable levels of SCL mRNA or protein (39). Lysates from these
two cell lines were immunoprecipitated with antibodies directed against either human TFIIH subunit p62 or, as a negative control, human cyclin
D1. The immunoprecipitates were size-fractionated on an SDS-polyacrylamide gel and further analyzed by Western blotting with an
anti-human SCL monoclonal antibody (BTL73) (33). Fig. 3 shows that SCL was associated in
vivo with the TFIIH complex and could be co-immunoprecipitated
with an antibody that recognized the p62 subunit of TFIIH. These
findings demonstrate the existence of a complex containing both SCL and
the p62 subunit of TFIIH, potentially mediated through the interaction
between SCL and p44.
Mapping the Regions of SCL/p44 Interaction--
To define the
regions of SCL that bind p44, a series of SCL cDNA
deletion mutants fused to a GAL4 DNA-binding domain cassette were
produced. These SCL mutants were employed in the yeast two-hybrid system as described above. To determine if these deletion mutants could
still bind p44, expression of the HIS3 and lacZ
reporter genes was monitored by growth on histidine-deficient medium
and production of
A dramatic decrease in the SCL/p44 interaction was noted between the
C-terminal mutants C262 and C275, demonstrating a requirement for this
region. Interestingly, the principal difference between mutants C275
and C262 is a polyglycine tract consisting of 10 consecutive glycine
residues, the loss of which essentially eliminated SCL binding
activity, suggesting that this polyglycine tract is necessary for the
SCL/p44 interaction. Therefore, the minimal region of SCL that could
bind p44 was between amino acids 174 and 275, a region including, but
not limited to, the bHLH region. Of note, the interaction between p44
and full-length SCL produced twice as much
To determine which portion of the p44 protein binds SCL, p44 mutants
were used in a yeast two-hybrid assay (Fig.
5). Although p44 was originally isolated
as an N-terminally truncated form, full-length p44 also bound SCL, and
we wondered whether the amino terminus of p44 might also contain a
region that could bind SCL. A PstI site was utilized to
delete the C-terminal region of p44, and the N-terminus-only mutant
( Specificity of the SCL/p44 Interaction among bHLH
Proteins--
Since the p44-binding site of SCL encompasses its bHLH
region, it is easily conceivable that the bHLH and flanking regions of
other bHLH proteins could also bind p44. When several bHLH proteins
were tested in the yeast two-hybrid system, only SCL was shown to bind
p44 (Table I). In addition, we were able to use the yeast two-hybrid
system to demonstrate previously undescribed interactions between SCL
and NHLH1 (neural HLH protein 1, previously named NSCL) (34) as well as an SCL homodimeric interaction (Table I).
The SCL/p44 Interaction Is Not Disrupted by E2A--
Since SCL and
E2A form stable heterodimers (23, 40) and that interaction is mediated
through the bHLH domain, we wondered whether these E proteins might
interfere with SCL/p44 binding. Equal amounts of in vitro
translated SCL and E2A proteins were prebound by a 15-min incubation at
37 °C. The SCL·E2A complex was then incubated with the GST-p44
fusion protein and precipitated with glutathione-Sepharose beads as
described above. The coprecipitated material was washed extensively in
the presence of 0.1% Nonidet P-40. As shown in Fig.
6, the presence of in vitro
translated E2A did not prevent the interaction of SCL and p44.
Using a yeast two-hybrid system, we have isolated several
SCL-binding proteins, both expected and unexpected, from three
different human cDNA libraries. As expected, the most prevalent
SCL-binding partners recovered from these screens were E proteins,
including E2A/ITF1, E2-2/ITF2, and HEB (14). We also used the yeast
two-hybrid system to demonstrate previously unreported interactions
between SCL and NHLH1 as well as a homodimeric interaction of SCL with itself. A human form of the DRG protein was also isolated from a human
thymus cDNA library; this was not surprising given that mouse DRG
has been isolated from a mouse erythroleukemia cell line (MEL) (35)
using the 22-26-kDa form of SCL as the bait protein in a yeast
two-hybrid screen.
However, we were surprised by the isolation of the p44 subunit of TFIIH
from the human thymus cDNA library. Although interactions between
bHLH proteins (e.g. c-Myc and MyoD) and basal transcription machinery have been documented (41, 42), those studies involved the
interaction with TBP, the first basal transcription factor that is
recruited to an activated promoter (43). The interaction between a bHLH
protein and TFIIH, which is recruited to the transcription initiation
complex relatively late and functions in promoter clearance and
elongation of transcription (25), has not been previously reported.
Several functional domains have been mapped to portions of the SCL
protein; the amino-terminal region is proline-rich and has been shown
to contain a transcription activation domain (21), whereas the bHLH
domain is involved in both DNA binding and dimerization with other bHLH
proteins (44). The p44-binding region of SCL was mapped to a region
that includes the bHLH domain as well as amino acid residues
immediately N- and C-terminal to the bHLH domain. Although the bHLH
domain was included in the region essential for binding, it is possible
that the bHLH domain may only serve as an arm to link the N- and
C-terminal flanking regions together to form a binding domain for p44.
Support for this possibility is found in the observation that only SCL,
among several bHLH proteins tested, was able to bind p44 in the yeast
two-hybrid system, indicating that the bHLH structure alone is not
sufficient for this interaction. For example, LYL1 (a bHLH
protein named by virtue of its involvement in a chromosomal
translocation associated with lymphoblastic
leukemia) has a bHLH region that is quite similar (49/55
identical amino acids and four conservative substitutions) to SCL (1).
Despite this, an interaction between LYL1 and p44 was not detected with
this system, suggesting that the interaction between SCL and p44
requires amino acids proximal and distal to the bHLH region. Our
results also show that the C-terminal polyglycine tract is important
for the interaction; this is not surprising because glycine-rich
domains have been shown to mediate other protein/protein interactions
(45). However, the SCL C-terminal glycine tract itself is not
sufficient for the SCL/p44 interaction (see Fig. 4). The SCL-binding
region of p44 was mapped to the C-terminal region of p44. This region
is composed of 166 amino acids (from Lys-229 to Val-395), containing two putative functional motifs (29). One of these motifs, the TFIIIA-like zinc finger motif, was found to interact with DNA; the
second motif, whose function is unknown, is conserved in both p44 and
Ssl1, the yeast homologue of human p44. Although the SCL-binding domain
of p44 has not yet been clearly defined, the above two motifs are
potential targets for future studies.
It is not clear how the interaction between SCL and p44 might function
in a biological context. However, some insight may be gained through
the study of previously reported interactions between nonessential
transcription factors and elements of the basal transcription complex.
Several proteins in addition to those of the basal transcription
complex have been shown to interact with TFIIH subunits. These proteins
include p53, which has been shown to interact in vitro with
the XPB subunit of TFIIH (46); this interaction is thought to modulate
either the nucleotide excision repair activity of TFIIH (47) or
p53-mediated apoptosis (48). Additionally, the transcription activation
domain of VP16 has been shown to bind the p62 subunit of TFIIH (49),
resulting in activation of transcription from an adenovirus type 2 major late promoter. With regard to interactions between bHLH proteins and basal transcription factors, the bHLH transcription factor c-Myc
has been shown to bind the basal transcription factor TBP (50).
Therefore, one hypothesis regarding the functional relevance of an
SCL/p44 interaction speculates that SCL, itself or as a heterodimer
with an E protein, interacts with p44, thus modulating the activity of
TFIIH and eventually the transcription elongation.
In summary, we have demonstrated a specific interaction, both in
vivo and in vitro, between the bHLH protein SCL and the
p44 subunit of the basal transcription factor TFIIH. Although this interaction was mapped to a region of SCL that encompassed, but was not
limited to, the bHLH domain, other bHLH proteins did not demonstrate
binding to p44. This novel interaction between a bHLH protein and TFIIH
suggests a potential mechanism by which the tissue-restricted bHLH
protein SCL might function during hematopoietic and vascular development.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
ura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3,112
gal4-542 gal80-538
LYS2::UASGAL1-TATAGAL1-HIS3
URA3::UASGAL4 17-mers(×3)-TATACYC1-lacZ) together with cDNA library plasmid DNA using Stratagene
reagents and protocols. The cDNA libraries were constructed in
either the pGAD10 (CLONTECH) or the pAD-GAL4
(Stratagene) vector, which contained the GAL4 activation domain fused
to random library cDNAs. Since the pGBT9 vector encodes
TRP1 and the pGAD10 or pAD-GAL4 vector encodes
LEU2, transformants were grown on -Leu/Trp/His synthetic dextrose (SD) plates to select for cotransformants that were able to
reconstitute GAL4 activity. Transformation efficiency was monitored by
plating cotransformants on a -Leu/Trp SD plate to ensure that ~106 independent clones were screened. Colonies that grew
on the -Leu/Trp/His plates were re-inoculated on a master plate and
tested for
-galactosidase activity. The pGBT9SCL plasmid
was then "dropped out" of the clones by differential growth on
-Leu and -Leu/Trp SD plates. Plasmid DNA was prepared from these
"drop out" clones and shuttled into Escherichia coli.
The plasmid DNA isolated from E. coli was sequenced. To
confirm the interaction between the library clone and SCL, the plasmid
DNA was cotransformed back into YRG-2 yeast cells together with
pGBT9SCL plasmid DNA, and the cotransformants were assayed
again for HIS expression and
-galactosidase activity.
(Amersham Pharmacia Biotech). A cDNA isolated from a human thymus
cDNA library that encodes an amino-terminally truncated p44 (Tp44)
was also subcloned into pGEX-1
. The coding sequence of both proteins
was confirmed to be in frame with the GST cassette, and the constructs
were transformed into E. coli BL21 cells. The binding assay
was performed as described previously (31), with minor modifications.
One ml of LB medium containing 100 mg/ml ampicillin and 1 mM isopropyl-
-D-thiogalactopyranoside was
added to 2 ml of overnight culture and incubated at 37 °C for 1 h. The cells were pelleted, resuspended in 150 µl of
phosphate-buffered saline, and disrupted by 30 s of sonication.
The bacterial cell lysate containing GST fusion proteins was incubated
at room temperature for 30 min with glutathione-Sepharose 4B beads
(Amersham Pharmacia Biotech). The beads were washed twice with
phosphate-buffered saline containing 1% Nonidet P-40. Half of the
beads were incubated with 1 ml of 0.5 M glutathione at room
temperature to release the GST fusion proteins (subsequently analyzed
by 10% SDS-PAGE); the other half were incubated with 5 µl of
in vitro translated SCL at 37 °C for 20 min. The beads
were then washed five times with phosphate-buffered saline containing
0.1% Nonidet P-40, boiled in 2× SDS loading buffer, and analyzed on a
10% SDS-polyacrylamide gel. After electrophoresis, the gel was soaked
in AutofluorTM (National Diagnostics, Inc.) for 1-2 h and
vacuum-dried before being exposed to Kodak XAR film.
-thiol deoxyribonucleotides before digestion with
NotI. The deletion mutants were sequentially generated with exonuclease III and S1 nuclease. Mutant N100 was produced by utilizing the ApaI restriction site within the SCL
cDNA, whereas mutants N174, N186, C262, and C275 were PCR-amplified
DNA fragments that were subcloned into pGBT9. The amino-terminal p44
deletion mutant was constructed by subcloning the carboxyl-terminal
portion of the p44 cDNA derived from the human thymus cDNA
library (see "Results") into pGBT9, whereas the carboxyl-terminal
deletion mutant was generated by PstI digestion of
pGBT9p44 to delete the 3'-portion of the full-length p44
cDNA, followed by religation of the plasmid.
-galactosidase activity was
detected with Z buffer (60 mM
Na2HPO4·7H2O, 40 mM
NaH2PO4·H2O, 10 mM
KCl, and 1 mM MgSO4·7H2O)
containing 0.27%
-mercaptoethanol and 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside.
-Galactosidase--
Five replicate
colonies were picked from -Leu/Trp SD plates containing
cotransformants and inoculated in -Leu/Trp SD medium, which was
incubated at 30 °C with shaking until the culture reached mid-log
phase. The cells were pelleted, resuspended in Z buffer, and disrupted
with liquid nitrogen. One-hundred-µl samples were mixed with 700 µl
of Z buffer containing 0.27%
-mercaptoethanol before 160 µl of 4 mg/ml o-nitrophenyl
-D-galactopyranoside in Z
buffer was added. The reaction was incubated at 30 °C until a yellow
color developed. Four-hundred µl of 1 M
Na2CO3 was added to stop the reaction, and the
incubation time was recorded. The absorbance of the supernatant was
measured at 420 nm and used to calculate the
-galactosidase units.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
In vivo interaction between SCL and
p44. The yeast two-hybrid system shows the specific interaction
between SCL and Tp44. The interaction was demonstrated by both the
growth on -Leu/Trp/His SD plates and positive (+++) -galactosidase
activity. Each pair represents a two-hybrid interaction. The first
plasmid listed produces a GBD fusion protein; the second plasmid
represents a GAD fusion protein. Growth on -Leu/Trp SD plates
indicates successful cotransformation; an interaction between the pair
allows for growth on -Leu/Trp/His SD plates. SIL (51), lamin C, and
p53 are proteins structurally dissimilar from SCL used as negative
controls. No interaction was found between SCL and GAD alone, lamin C
and Tp44, p53 and Tp44, SIL and Tp44, and GBD alone and Tp44.
Protein/protein interactions between bHLH proteins and p44 in a yeast
two-hybrid system
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Fig. 2.
In vitro interaction between SCL and
p44. A, GST fusion proteins produced in E. coli BL21 cells. The cell lysates were resolved by 10% SDS-PAGE,
either before ( ) or after (+) purification with glutathione-Sepharose
4B beads, and stained with Coomassie Brilliant Blue. B,
coprecipitation of in vitro translated SCL with GST-p44 and
GST-Tp44. In vitro translated 35S-labeled SCL
(input *SCL lane) was incubated with GST fusion proteins and
precipitated by centrifugation with glutathione-Sepharose beads as
described under "Materials and Methods." Coprecipitated proteins
were resolved by 10% SDS-PAGE. The position of the in vitro
translated SCL protein is indicated with two arrows.
Molecular mass markers are shown on the left. Bands of 39 and 43 kDa,
representing the in vitro translated SCL proteins, are seen
in the GST-p44 and GST-Tp44 lanes, but not in the
GST lane.
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Fig. 3.
In vivo association of SCL and
TFIIH. HEL and HL-60 cell lysates were immunoprecipitated with
either rabbit anti-human cyclin D1 or rabbit anti-human TFIIH p62, and
the precipitates were analyzed with a monoclonal antibody against human
SCL (BTL73), as demonstrated by Western blot assay. The
co-immunoprecipitated SCL protein is indicated with an
arrow. Molecular mass markers are indicated on
the left. The band above the SCL protein is due to cross-reactivity of
the secondary antibodies (anti-mouse IgG) with the rabbit polyclonal
antisera used in the immunoprecipitations.
-galactosidase, respectively (Fig.
4). No obvious change in the SCL/p44
interaction was detected when the N-terminal 100 amino acids were
deleted (Fig. 4). However, both the N156 and N174 (which closely
approximates the naturally occurring 22-26-kDa isoform of SCL) mutants
produced decreased level of
-galactosidase activity and less robust
growth on histidine-deficient medium, suggesting a decreased ability to
interact with p44. The SCL/p44 interaction was completely absent in the
N186 mutant, which retained the entire bHLH domain, demonstrating that
the bHLH domain was not sufficient to mediate the interaction. The
region between amino acids 100 and 186 was necessary for full binding
activity, whereas the region between amino acids 174 and 186 highlighted residues that were absolutely required for the SCL/p44
interaction.
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Fig. 4.
Mapping of regions of SCL that bind p44.
A series of SCL deletion mutants fused to GBD were tested in the yeast
two-hybrid system. SCL amino acid (aa) residues are
indicated; the solid black bars represent the bHLH domain.
The interaction between SCL mutants and p44 was quantitated by
observing the colony growth on -Leu/Trp/His SD plates and
-galactosidase assay. Interaction between SCL and ITF2 was used as a
positive control. +++, colony growth to full size 2 days after
transformation; ++, colony growth to full size 3 days after
transformation; +, colony growth to full size 4 days after
transformation; -, no colony growth 5 days after transformation.
-galactosidase as did the
SCL/ITF2 interaction, indicating that the interaction between SCL and
p44 is of similar magnitude to that seen with a well known SCL-binding
protein; the SCL/ITF2 interaction showed the strongest binding for SCL among the E proteins (data not shown).
p44) was tested in the yeast two-hybrid system. The lack of
interaction with this mutant demonstrates that the SCL-binding region
of p44 is located at the carboxyl terminus (Fig. 5).
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Fig. 5.
Carboxyl terminus of p44 binds SCL. p44
and its truncated mutants were fused to GAD and tested in the yeast
two-hybrid system with GBD-SCL. p44 represents the
carboxyl-terminally truncated mutant, whereas Tp44 is the
amino-terminally truncated mutant. The interaction was demonstrated by
the colony growth on the -Leu/Trp/His SD plate; growth on the
-Leu/Trp SD plate was used to monitor the transformation efficiency.
GBD/SCL and GAD/ITF2 interactions were used as positive controls.
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Fig. 6.
E2A does not interfere with SCL/p44
interaction. In vitro translated E2A
(*E2-5), SCL (*SCL), or SCL and E2A (*SCL + *E2-5)) were incubated with GST or GST-p44, and interactions were
detected by coprecipitation as described in the legend to Fig.
2B. The positions of SCL are indicated with two
arrows.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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ACKNOWLEDGEMENTS |
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We thank David S. Chervinsky for excellent technical assistance, Ellen Greco for art work, and the Roswell Park Cancer Institute Biopolymer Facility for oligonucleotide synthesis. We are also grateful to Dr. Ilan Kirsch for the NHLH1 cDNA, Dr. Adam Goldfarb for the E2-5 cDNA, and Dr. Karen Pulford for BTL73 monoclonal antibodies.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA16056-21 and CA67177-01 and by the Leukemia Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Scholar of the Leukemia Society of America. To whom correspondence should be addressed: Dept. of Pediatrics, Roswell Park Cancer Inst., Buffalo, NY 14263. Tel.: 716-845-4598; Fax: 716-845-4502; E-mail: paplan{at}sc3101.med.buffalo.edu.
The abbreviations used are: bHLH, basic domain helix-loop-helix; SD, synthetic dextrose; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PCR, polymerase chain reaction; Tp44, truncated p44; GAD, GAL4 transcription activation domain; GBD, GAL4 DNA-binding domain.
2 X.-F. Zhao and P. D. Aplan, unpublished data.
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REFERENCES |
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