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(Received for publication, June 21, 1995) From the
Cell-specific expression of the rat insulin II gene is in part
mediated through an element located in the 5`-flanking region. The
element, termed RIPE3b (-126 to -101), confers
The insulin gene is expressed exclusively in the Previous studies performed in our laboratory have identified several
important cis-elements, termed the rat insulin promoter
elements (RIPEs), The RIPE3a element contains an E-box sequence,
CANNTG. The E-boxes are recognized by a protein family carrying a basic
helix-loop-helix (bHLH) DNA-binding domain(14) . The E-boxes in
the rIns I (Nir and Far box) and rIns II genes
(RIPE3a) have been shown to bind islet-specific bHLH protein
complexes(11, 15, 16, 17) . Several
laboratories have cloned related bHLH proteins that bind to RIPE3a and
the corresponding region in the rIns I
gene(18, 19, 20, 21, 22) .
Most notably, our laboratory has recently cloned BETA2 (neuroD), the
tissue-specific component of bHLH-binding
complex(23, 24) . On the other hand, two specific 3b
protein-DNA complexes were observed by in vitro binding
assays(11) . The RIPE3b1 complex is islet-specific, whereas the
RIPE3b2 complex is expressed in all cell lines examined. A linker
substitution mutation in the RIPE3b element (mRIPE3b) not only
destroyed the ability of the element to form 3b1 and 3b2 complexes but
also abolished the RIPE3b activity in transfection
assays(11, 13) . The sequence of the 3b element shows
no obvious homology with any known consensus binding motif and thus is
possibly recognized by novel transcription factors. Here we report
the characterization of the RIPE3b2 complex and the cloning of the
first RIPE3b-binding protein. Our results show that RIPE3b2 complex is
composed of at least three polypeptides: p58, p62, and p110. By
screening a HIT cell (hamster insulinoma) cDNA library, we have
isolated a clone that binds specifically to RIPE3b. The encoded
protein, designated Rip-1, is the hamster homologue of the human and
mouse Smbp-2(25, 26) , which contains putative
helicase motifs and a transcription activation domain. Our data also
raise the possibility that Rip-1 might be a component of the
multi-subunit RIPE3b2-binding complex.
For in vivo expression of Rip-1, 2.5
Figure 1:
RIPE3b2 is a specific insulin
enhancer-binding complex. A, gel mobility shift assay with the
HIT cell nuclear extract and RIPE3b oligonucleotides. The probe
(RIPE3b) was labeled as described before(11) . Sequences of the
wild type and mutant RIPE3b oligonucleotides are shown in Fig. 6A (RIPE3b and m1,
respectively). RIPE7 is an oligonucleotide containing the sequence from
-305 to -281 of the rat insulin II gene promoter. All
competitors were used at 50-fold excess. B, methylation
interference analysis of the 3b2 complex. Methylation of the probe and
isolation of the binding complex was performed as
described(11) . The source of extracts is indicated at the top of each lane. Only the 3b2 complex was assessed
here. Free probe (F) was analyzed in parallel as a
control.
Figure 6:
RIPE3b2 but not the RIPE3b1 complex is
competed by the GFE. A, sequences of the wild type RIPE3b
element, GFE, and mutant RIPE3b (m1 and m2). The
direction of each CAGCC half-site is indicated by arrows. The
mutated nucleotides are shown in lowercase letters. B, gel mobility shift assay was performed with nuclear
extracts prepared from HIT and BHK cells. Competitors (as indicated at
the top of each lane) were used at 80-fold molar
excess relative to the probe RIPE3b. The positions of the 3b1 and 3b2
complexes are marked with solid arrows. The dashed arrow points to a
Figure 2:
Protein composition of the RIPE3b2
complex. UV cross-linking analyses were performed as described under
``Materials and Methods.'' Radioactive 3b2 complexes were
excised from the gel and analyzed on an SDS/8% polyacrylamide gel. Free
probe was also excised and analyzed in parallel (A and B, lane 1). Complexes formed with different nuclear
extracts were also compared (B). Complexes formed with
Figure 3:
Rip-1 is a hamster homologue of mouse and
human Smbp-2. A, comparison of the hamster Rip-1 with the
human and mouse Smbp-2. Deduced amino acid sequences are aligned such
that residues identical with and different from hamster Rip-1 are
indicated by dashed lines and uppercase letters,
respectively. Gaps were created to maximize the alignment and are
delineated by dots and lowercase letters. B,
schematic representation of the Rip-1 protein product. The cDNA encodes
a polypeptide of 989 amino acids with a calculated molecular mass of
108 kDa and several interesting features as described under
``Results.'' P and Q stand for the proline-
and glutamine-rich region. The GenBank accession number for the hamster
Rip-1 is L15625.
The deduced amino acid sequence of Rip-1
reveals several interesting features (Fig. 3B). As
indicated by Mizuta et al.(25) , the polypeptide has
putative helicase motifs in the N-terminal half, including a consensus
P-loop for ATP or GTP-binding sequence, GPPGTGKT, between amino acids
213 and 220(39) . Also in the N terminus is a region rich in
leucine residues. Leucine-rich regions have been implicated to be
protein-protein interaction domains in a few other
proteins(40, 41) . Finally, the hamster sequence also
reveals a proline- and glutamine-rich region in its C terminus, which
might be a potential activation domain as demonstrated in other
transcriptional regulatory proteins(42, 43) . Other
features not shown in Fig. 3B include a nuclear
localization signal KKKKK (amino acids 860-864) and several
putative protein kinase A and C phosphorylation sites.
To prove proper
expression of the transfected plasmid, an antibody was generated
against the recombinant Rip-1. As shown in Fig. 4, the
affinity-purified antibody recognized an endogenous protein of 110 kDa
in either the nuclear (lane 1) or the cytosolic extract (lane 2) prepared from HIT cells. The signal was specific,
because it was not detected by the preimmune serum (data not shown),
and the signal can be blocked by preincubating the Rip-1 antibody with
the recombinant glutathione S-transferase-Rip-1 fusion protein (Fig. 4, lanes 3 and 4).
Figure 4:
Immunoblot analysis of nuclear and
cytosolic extracts with the Rip-1 antibody. The analysis was conducted
with 110 µg of the nuclear extract (lanes 1 and 3) and 130 µg of the cytosolic extract (lanes 2 and 4) prepared from HIT cells. The specific signal (110
kDa) is indicated by an arrow. Lanes 1 and 2, blot incubated with the affinity-purified Rip-1 antibody. Lanes 3 and 4, same as lanes 1 and 2 except that the antibody was preblocked with the glutathione S-transferase-Rip-1 fusion protein for 30 min at room
temperature.
Upon introducing
the Rip-1 plasmid into COS cells, which have lower levels of the
endogenous protein, expression of a 110-kDa protein, as detected by
Rip-1-specific antibody, increased dramatically (Fig. 5B). As expression of Rip-1 increased, binding of
the RIPE3b2 complex was greatly enhanced (Fig. 5A,
compare lanes 1 and 2), whereas binding of other
complexes was not affected. The increased binding activity can be
further competed by unlabeled probe (Fig. 5, lane 3).
The results suggest that Rip-1 either is in the 3b2 complex or is
related to a component of the 3b2 complex.
Figure 5:
Overexpression of Rip-1 protein in cells
enhances the RIPE3b2 complex. 5 µg of the empty vector (pSG5, lanes 1) or vector carrying Rip-1 cDNA (lanes 2) were
introduced into COS cells by transient tranfection. 3 or 30 µg of
nuclear extracts were then used in gel mobility shift assay (A) or immuoblot analysis with Rip-1-specific antibody (B). The competitor indicated in A is 200-fold excess
of the unlabeled RIPE3b oligonucleotide.
Figure 7:
RNA analysis of Rip-1. A,
Northern analysis of poly(A)
We have characterized an insulin enhancer-binding complex,
RIPE3b2, and demonstrated that it is composed of at least three
subunits: p58, p62, and p110. We have also isolated a RIPE3b-binding
protein, designated Rip-1, which turns out to be the hamster homologue
of the putative helicase Smbp-2 and the transcription factor GF-1. A
complex involving multiple subunits is not without precedents. The same
phenomenon has been observed for factors binding to the
interferon-stimulated response element and several others. The complex
residing on the interferon-stimulated response element is actually
composed of four polypeptides; interaction between the four subunits
greatly enhanced binding of the complex to the interferon-stimulated
response element(44) . Several lines of evidence suggest that
Rip-1 is involved in the 3b2 complex. First, Rip-1 was cloned by
binding to the RIPE3b element. Second, overexpression of Rip-1 in cells
greatly enhanced the 3b2 complex (Fig. 5A). Finally,
the formation of the endogenous 3b2 complex was competed by RIPE3b and
the GF-1-binding sequences that share little sequence similarity (Fig. 6). Because the Rip-1 antibody, although it functioned
well in immunoblot analysis, was unable to recognize the native protein
and super-shift or block the 3b2 complex, we are not able to make a
definitive conclusion at the present time. It is possible that the
identified clone, Rip-1, is either a component of or related to the 3b2
binding complex. It is noteworthy that the deduced amino acid
sequences of Rip-1 seems to possess both features of helicase and
transcription factor. The N-terminal half of Rip-1/Smbp-2 contains
putative helicase motifs (25) as well as multiple leucine-rich
domains. Sequence comparison shows a high degree of conservation
( What is the
function of Rip-1/GF-1/Smbp-1 in the insulin gene regulation? Because
Rip-1 is expressed about equally in HIT and BHK cells, it is not likely
that the protein plays a crucial role in determining tissue
specificity. Nevertheless, one cannot exclude the possibility that
through some kind of post-translational mechanism, the protein can only
function in certain cell types or in response to specific stimuli.
Evidence that suggests such a possibility may be seen in a gel mobility
shift assay (Fig. 6B), in which a complex closely
related to 3b2 (indicated by a dashed arrow) was observed in
insulin-producing HIT cells but not in the fibroblast BHK.
Interestingly, the activator GF-1, which has only the C-terminal
portion of human Smbp-2, activated transcription from JCV promoters
10-fold in glial cells(38) , whereas the full-length hamster
Rip-1 had at most 2-fold activation (data not shown) when tested on the
RIPE3b element in HIT cells. The same result was obtained with a
construct that expressed only the C-terminal half of Rip-1. It seems
that cellular environment and DNA context play important roles in
transcriptional activation. Recently, a family of transcription
factors with putative DNA helicase and ATPase motifs has emerged: to
name a few, the yeast protein SNF2/SWI2(45) , which is required
for the activation of many yeast genes; the protein hBRG1(46) ,
which is necessary for normal mitotic growth and transcription; and the
basic transcription factor TFIIH (47) . The cloned factor
Rip-1/Smbp-2/GF-1 represents another member of this family that
combines two functions in one protein: DNA helicase and transcriptional
activation. The two functional domains may perform cooperatively, as
Laurent and Carlson (45) have suggested for the yeast activator
SNF2/SWI2. That is, the protein may facilitate transcription by
altering chromatin structure, helping contact between activators and
the transcription apparatus, thereby enhancing transcription.
Alternatively, two domains can function independently on separate
occasions, depending on the composition of associated proteins. Whether
the activity of Rip-1/Smbp-2/GF-1 is mediated via the first or second
mechanism awaits detailed structure-function analysis.
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank(TM)/EMBL Data Bank with accession
number(s) L15625[GenBank].
Volume 270,
Number 37,
Issue of September 15, pp. 21503-21508, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CLONING OF A BINDING FACTOR WITH PUTATIVE HELICASE MOTIFS (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-cell-specific expression in conjunction with an adjacent element
RIPE3a (-110 to -86). Here we report the characterization
of one of the RIPE3b-binding complexes, 3b2. UV cross-linking analysis
demonstrated that it is composed of at least three polypeptides: p58,
p62, and p110. Furthermore, a cDNA was isolated via expression
screening for binding to RIPE3b. Sequence analysis reveals that the
encoded protein, designated Rip-1, possessed putative helicase motifs
and a potential transcription activation domain. Overexpression of
Rip-1 in cells greatly enhances the 3b2 binding complex, suggesting
that Rip-1 is involved in the binding of 3b2.
-cells of
pancreatic islets. The tissue specificity results in part from cell
type-specific transcription directed by the 5`-flanking region of the
insulin gene(1, 2, 3) . Unlike humans, who
have only one insulin gene, rodents (rats and mice) have two nonallelic
insulin genes, I and II (rIns I and rIns II) (for
review see (4) and (5) ). The rIns I and rIns II genes share homologous sequences not only in the
coding region but also in their promoters, suggesting that they are
controlled by similar regulatory mechanisms. In fact, similar cis-elements have been defined as a result of systematic
mutagenesis and in vitro binding analysis within the promoter
sequence(6, 7, 8, 9) . These
elements interact with ubiquitous and/or -cell-specific factors to
confer cell type-specific
expression(6, 10, 11, 12) . 
in the rIns II gene promoter.
One element, RIPE3, located between -126 and -86, confers
-cell-specific expression when linked to a heterologous minimal
promoter in either orientation(13) ; thus it behaves as a cell
type-specific enhancer element. RIPE3 can be divided into two
subelements, a and b. Mutation in RIPE3a or RIPE3b in the context of
the whole promoter(-448) drastically reduced the promoter
activity by 25-fold. Element a and element b cooperate with each other
and give rise to full RIPE3 activity, whereas each element alone has
only marginal activity(13) . Therefore, it is very likely that
multiple protein factors are involved in cell type-specific control.
Indeed, it was later demonstrated by in vitro binding assays
that cell-specific as well as ubiquitous factors bind RIPE3a and RIPE3b (11) .
Nuclear Extract Preparation and Gel Mobility Shift
Assays
HIT-T15 M2.2.2(27) , a hamster insulinoma cell
line, and HeLa cells were cultured as described
previously(11) . BHK-21, a hamster kidney fibroblast cell line,
was grown in Dulbecco's modified Eagle's medium
supplemented with 15% horse serum, 2.5% fetal bovine serum, 100
units/ml penicillin, and 100 mg/ml streptomycin. Nuclear extracts were
prepared as described previously(11) . 
TC (28) and
TC (29) cells are transgenic mouse - and -cell
lines, respectively. Nuclear extracts of both cell lines are generous
gifts from Dr. Roland Stein (Vanderbilt University).
10
COS cells were
seeded to 60-mm dishes the day before transfection. 5 µg of either
pSG5 or pSGRip were introduced into cells by the calcium phosphate
method. Nuclear extract was then prepared from the dishes 40 h after
transfection using the method described by Attardi and Tjian (30) . Gel mobility shift assays were performed as described
previously (11) .In Situ UV Cross-linking
UV cross-linking
experiments were conducted essentially as described by Wu et
al.(31) . To prepare the probe, partially complementary
oligonucleotides corresponding to sequences from -111 to
-101 and -126 to -101 of the rIns II gene
promoter were synthesized. The partially double-stranded
oligonucleotide was then filled-in with Klenow enzyme (Promega) in the
presence of [-P]dCTP (ICN),
bromodeoxyuridine (Sigma), and other cold deoxynucleotides (Pharmacia
Biotech Inc.). The binding reaction was scaled up 5-fold and performed
as described before. After electrophoresis, the gel was covered with
plastic wrap and irradiated with 254 nm UV light at a distance of 3 cm
in the cold room for 1 h. The specific complexes were excised from the
gel following autoradiography. The gel pieces were minced, mixed with
20 ml of 2
SDS gel loading buffer, boiled, and loaded onto an
SDS-polyacrylamide gel. After electrophoresis, the gel was dried and
subjected to autoradiography.Library Screening
The HIT cDNA library was a gift
from Dr. Larry G. Moss (Department of Medicine, Tufts University). A
double-stranded oligonucleotide (RIPE3b) containing sequences from
-126 to -101 relative to the transcription start site of
the rat insulin II gene was concatenated, P-labeled using
a nick translation kit (Boehringer Mannheim), and used to screen the
library as described previously by Vinson et al.(32) and Singh et al.(33) with minor
modifications. Briefly, the nitrocellulose filters were soaked in 10
mM isopropyl-1-thio-
-D-galactopyranoside and air
dried. LB plates containing phage that had been grown for 4 h at 42
°C were overlaid with
isopropyl-1-thio--D-galactopyranoside filters and
subsequently incubated at 37 °C for 12 h. The filters were then
lifted and air-dried for 15 min at room temperature. The filters were
subjected to denaturation and renaturation as described in 1
binding buffer (20 mM Hepes, pH 7.9, 60 mM KCl, and 2
mM MgCl
) containing 6 M guanidine HCl.
The filters were then blocked in 5% milk and incubated with P-labeled, multimerized RIPE3b oligonucleotide in binding
buffer containing 0.25% milk and 50 µg/ml sheared, denatured calf
thymus DNA at a concentration of 1
10
cpm/ml. After
incubation at 4 °C for at least 4 h, the filters were washed three
times in the same buffer for 5 min each, then air-dried, and exposed to
x-ray film overnight.Plasmid Constructions
For nucleotide sequencing,
all Rip-1 cDNAs (full-length and truncated forms) were released from
the gt11 vectors by EcoRI digestion and were subsequently
cloned into the EcoRI site of the pGEM7fz(+) vector
(Promega). For in vivo expression of Rip-1 protein, the
longest cDNA (l3B6) was cloned into the EcoRI site of pSG5
(Stratagene). The resulting plasmid produced nearly full-length protein
in cells (except the three N-terminal amino acids).
Northern Analysis and RNase Protection
Assays
Total RNAs from hamster tissues and cell lines were
prepared with RNazol B (Biotecx, Houston, TX) or as described by
Sambrook et al.(34) using the guanidinium
thiocyanate extraction method. Poly(A) RNA was
isolated from total RNA using standard conditions(34) .
Northern blots were prepared as described (35) using 12.5
µg of poly(A)
RNA and probing with a
P-labeled, N-terminal EcoRI-EcoRV
fragment of the Rip-1 cDNA. RNase protection assays were performed as
described by Wu et al.(36) using an in vitro synthesized,
P-labeled antisense RNA probe
corresponding to a C-terminal NheI-EcoR I fragment of
the Rip-1 cDNA. The [
P]UTP-labeled RNA probe (1
10 cpm/reaction) was incubated with 40 µg of
total RNA isolated from various hamster tissues overnight at 45 °C
in 80% formamide buffer (80% formamide, 40 mM Pipes, pH 6.7,
0.4 M NaCl, and 1 mM EDTA). The hybridized mixture
was digested with RNase A (40 µg/ml) and RNase T1 (75 units/ml,
Boehringer Mannheim) at 30 °C for 1 h and then was treated with
proteinase K (150 µg/ml). After phenol/chloroform (1:1) extraction,
the reaction was precipitated with ethanol and run on a 6%
polyacrylamide sequencing gel.Generation and Purification of Antibody
To
generate antibodies against the cloned factor, the cDNA from one of the
initial clones, 3B17, which encodes amino acids 535-857, was
cloned into the vector pGEX-3X in frame with the glutathione S-transferase coding sequence and expressed as a fusion
protein in Escherichia coli essentially as
described(37) . Following purification, the fusion protein was
run on an SDS/10% polyacrylamide gel. The gel piece containing the
fusion protein was excised, mashed, mixed with adjuvant, and injected
into rabbits. The sera (immune and preimmune) obtained from the rabbits
were either clarified through a protein G-Sepharose column (Pharmacia
Biotech Inc.) or affinity-purified as described below. To affinity
purify the antibody, the antiserum was first passed through an E.
coli protein-Sepharose column made with crude E. coli extracts coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech
Inc.). The flow-through was collected and affinity-purified through a
second column that contained purified glutathione S-transferase Rip coupled to Sepharose beads.
Immunoblot Analysis
Protein extracts were first
separated on an SDS-polyacrylamide gel and then transferred to the
Immobilon-P membrane (Millipore) using the Bio-Rad electroblotting
system. The membrane was blocked in 1% milk in TBST (20 mM
Tris
HCl, pH 7.5, 136 mM NaCl, and 0.05% Tween-20) for 30
min at room temperature. The affinity-purified antibody was diluted
250-fold and incubated with the membrane for 1 h at room temperature.
The specific signal was detected by ECL chemiluminescence (Amersham
Corp.) following the manufacturer's instructions.
Two Specific Complexes Interact with RIPE3b
We
have shown previously that at least two specific protein complexes
interact with the RIPE3b element: 3b1 and 3b2 (Fig. 1A); 3b1 is islet-specific, whereas 3b2 is
ubiquitously expressed(11) . The binding site for the 3b2
complex was further analyzed here by methylation interference assay: it
contacted the RIPE3b element at -107G of the bottom strand (Fig. 1B), which overlapped but was distinct from those
recognized by 3b1 (Gs at -107, -108, -111, and
-114). Mutation in the region from -118 to -111
affected not only 3b1 but also 3b2 binding (Fig. 1A, lane 4), suggesting that nucleotides immediately upstream of
-107 are required for binding 3b2. No interference was seen with
methylations in the top strand. The experiment was also repeated with
nonislet extracts HeLa and BHK. Each extract showed an identical
pattern of interaction (Fig. 1B).
-cell-specific complex related to 3b2 (see
``Discussion'').
The RIPE3b2 Complex Is Composed of at Least Three
Polypeptides
To further characterize the RIPE3b2 complex, we
performed in situ UV cross-linking analysis. A gel mobility
shift assay was carried out with the HIT nuclear extract and a
radiolabeled, bromouridine-substituted RIPE3b oligonucleotide. The gel
was then irradiated with UV light, and the RIPE3b2 complex was excised.
The protein components of the complex were analyzed by
SDS-polyacrylamide gel electrophoresis and subsequently visualized by
autoradiography (Fig. 2). The analysis revealed that the RIPE3b2
complex contained at least three proteins that differed in their sizes:
p58 (58 kDa), p62 (62 kDa), and p110 (110 kDa) (Fig. 2A). The same components were detected in the 3b2
complex isolated from BHK, HeLa, TC, and TC cells (Fig. 2B), which was in agreement with the ubiquitous
nature of the 3b2 complex.
TC
and HeLa cell nuclear extracts were reassayed using a different UV
light source (transilluminator, model TM-3, UVP Inc.) with a longer
exposure time (C). Arrows indicate positions of three
polypeptides with sizes of 110, 62, and 58 kDa,
respectively.
Isolation of cDNA Clones That Bind RIPE3b
Previous
studies using linker-scanning mutagenesis(8) , transient
transfection experiments(13) , and in vitro protein
binding assays (11) demonstrated that the RIPE3b element,
spanning from -126 to -101 relative to the transcription
start site, is important for expression of the rat insulin II gene. To
study the molecular mechanism underlying the transcriptional
regulation, we set out to clone gene(s) coding for the RIPE3b binding
protein(s). A HIT cDNA expression library was screened by using a
concatenated, double-stranded RIPE3b oligonucleotide as a probe. Two
clones, 3B17 and
3B22, were isolated in the initial
screening. Both recombinant phages showed a stronger binding preference
to the wild type RIPE3b than to a mutated binding site. Partial
sequencing and restriction mapping of the two cDNAs demonstrated that
they were overlapping clones. A fragment derived from the 5`-end of the
3B17 clone was then used as a probe to rescreen the library. As a
result, a 3.3-kb cDNA clone (
3B6) was isolated. Sequence analysis
of the cDNAs revealed that the clone we had, designated Rip-1, was the
hamster homologue of the human and mouse Smbp-2, a protein that binds
to the immunoglobulin m chain switch (Sm)
region(25, 26) . Fig. 3A shows the
alignment of deduced amino acid sequences from the three species. The
open reading frame of hamster Rip-1 encodes a protein with a calculated
molecular mass of 108 kDa and is 86.6 and 77.4% homologous to its mouse
and human counterparts, respectively. It was also once cloned as the
human glial factor-1 (GF-1), a transcription factor that binds and
activates promoters of the human neurotropic virus JCV in glial
cells(38) . As pointed out by Fukita et
al.(26) , human GF-1 is identical to and is an incomplete
version of human Smbp-2.
Overexpression of Rip-1 Protein Enhances RIPE3b2
Binding
In the process of proving the DNA binding specificity of
Rip-1, we encountered the same problem that Fukita et al.(26) had experienced, that is, the overexpressed protein
has very weak binding affinity in electrophoretic mobility shift
assays. As a result, it is impossible to perform binding assays with
materials prepared from in vitro translation or bacterial
expression system. Because several DNA-binding proteins had been shown
to require accessory factors to bind stably to DNA in vitro,
an alternative approach was employed to test the possibility. A plasmid
that expressed Rip-1 (without the first three amino acids) under the
control of SV40 enhancer was introduced into COS cells by transient
transfection. Nuclear extracts were then prepared and examined by in vitro gel mobility shift assay.
GF-1-binding Element Competes with RIPE3b for Forming the
RIPE3b2 but Not the RIPE3b1 Complex
It has been shown previously
that using P-labeled RIPE3b oligonucleotide as a probe and
HIT cell nuclear extract as the protein source, two specific binding
complexes, 3b1 and 3b2, are observed. Because overexpression of Rip-1 in vivo greatly enhanced 3b2 binding, we were curious as to
whether sequences recognized by the human homologue, GF-1, could
compete for 3b2 binding. To this end, excess amount of the GF-1-binding
oligonucleotide (GFE) was used as a competitor in the gel shift assay.
The sequence recognized by GF-1 in the JCV promoter shows little
similarity when compared with the RIPE3b element, except that both have
two CAGCC motifs in either a direct repeat or a palindrome (Fig. 6A). As shown in Fig. 6B, wild
type RIPE3b oligonucleotide competed with both 3b1 and 3b2 complexes (lane 2), whereas GFE only competed for the binding of 3b2
complex but had little effect on the 3b1 complex (lane 3).
Both complexes cannot be competed by m2, a RIPE3b mutant
oligonucleotide (Fig. 6B, lane 4). The same
results were observed with the BHK nuclear extract (Fig. 6B, lanes 5-8). Taken together,
the results clearly indicated that the RIPE3b2 complex consists of a
DNA binding activity that recognizes both RIPE3b and GFE sequences and
that it is distinct from the RIPE3b1 complex. This again demonstrates
the specific relationship between Rip-1 and the RIPE3b2 complex.
Expression of Rip-1 in Insulin-producing and
Non-insulin-producing Cells and Tissues
Although it was shown
that the human and mouse counterparts of Rip-1 were widely expressed,
we were curious about the expression level related to insulin
production. To assess the size of the Rip-1 message we probed Northern
blots containing BHK poly (A) RNA with a 0.8-kb
fragment corresponding to the 5`-end of the cDNA and detected a single
band of 3.7 kb (Fig. 7A). RNase protection analysis was
used to compare the expression level of Rip-1 in insulin-producing HIT
cells and non-insulin-producing BHK cells (Fig. 7B).
P-labeled antisense RNA corresponding to the 201
C-terminal nucleotides plus 80 nucleotides from the cloning vector was
synthesized in vitro and used as a probe for hybridization.
The results demonstrated similar levels of Rip-1 expression in these
cell lines. To determine the tissue distribution of Rip-1 RNA, total
RNA was prepared from various hamster tissues and was also examined by
RNase protection assays. The result revealed the highest amount of
expression in brain and testis, moderate expression in heart, spleen,
and kidney, and low level of expression in other tissues. No signal was
detected using the negative control yeast RNA.
RNA prepared from BHK
cells. 12.5 µg of poly(A)
RNA was run on a 0.8%
agarose/formaldehyde gel and transferred to nylon membrane. The blot
was probed with a N-terminal 0.8-kb fragment of Rip-1 cDNA. The signal
detected is about 3.7 kb in size. B, RNase protection assays
of total RNA prepared from various hamster tissues. The RNA probe was
prepared by in vitro transcription as described under
``Materials and Methods.'' Marker (M) was pBR322 cut
with HpaII. nt, nucleotides; yRNA, yeast
RNA.
85%) in these regions, suggesting that they carry important
functions. Contrary to the N terminus, the C terminus is more diverse (Fig. 3A). It contains a single-stranded DNA-binding
domain mapped by Fukita et al.(26) in the human
Smbp-2 (corresponding to a region in Rip-1 of amino acids
637-783) and a proline- and glutamine-rich domain, which could be
the putative activation domain in GF-1. The low degree of homology in
the single-stranded DNA-binding domain may explain why there is no
apparent sequence similarity except a string of Gs among the elements
recognized by Rip-1/Smbp-2/GF-1. Although there are CAGCC motifs in the
elements recognized by Rip-1 and GF-1, they are not present in the Sm
region. It is more likely that the protein recognizes the structure
rather than the sequence of DNA. Alternatively, there is an additional
DNA-binding domain that has yet to be identified. Additionally,
differences in the context of response elements may mediate diverse
functions such as recombination (in the case of Smbp-2) and
transcriptional activation (in the case of GF-1). It is possible that
by interacting with different factors the same protein can recognize
DNA elements with slightly different sequences and thus results in
different cellular responses (transcriptional activation versus recombination). The presence of multiple leucine-rich regions, the
putative protein-protein interaction domains (Refs. 40 and 41 and
references therein), seems to lend support to the hypothesis. Whether
the CAGCC motif is required specifically for transcriptional activation
(as in the case of Rip-1/GF-1) remained to be tested.
)
We thank Dr. Larry Moss for providing the HIT cDNA
library and Christina Chang for help with GenBank search and sequence
analysis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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