(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
-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.
The insulin gene is expressed exclusively in the -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) .
Previous studies performed in our laboratory have identified several
important cis-elements, termed the rat insulin promoter
elements (RIPEs), 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) .
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 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) .
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 -cell-specific complex related to 3b2 (see
``Discussion'').
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 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.
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) 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.
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
(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.
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].