(Received for publication, May 30, 1995)
From the
Vimentin, a cytoskeletal protein belonging to the intermediate filament protein family, exhibits a complex pattern of expression. In the case of the chicken vimentin gene, several regulatory elements within the 5` region of the gene have been characterized, including an enhancer activity between -160 and -320, which may contribute to the down-regulation of vimentin expression during myogenesis. In this study, sequences within this region were examined via transient transfections of various deletion constructs, and two distinct enhancer elements were found, one on either side of a previously described silencer element. These two enhancer elements also enhanced transcription when fused separately to the basal promoter region of the chicken vimentin gene. Gel mobility shift assays, UV cross-linking experiments, and DNase I protection studies indicate that these two enhancer elements and the silencer element all contain a common binding site for the previously described 95-kDa silencer element binding protein, suggesting that this regulatory protein can act as both an activator and a repressor.
The initiation of transcription is a key control point for eukaryotic gene expression, and regulation of this critical event is central to producing the correct tissue-specific and temporal pattern of gene expression. Regulation is primarily accomplished by gene-specific DNA-binding transcription factors, which either increase or decrease the rate of transcription initiation(1, 2) . The target sequences for these transcription factors can be either upstream or downstream of the transcription start site and are often quite far removed from the start site. Over the last few years, a number of enhancer and repressor elements, along with their corresponding DNA-binding transcription factors, have been well characterized(1) . As a result, the distinction between enhancers and repressors has become less definitive. For example, several studies have demonstrated how a single gene, by way of either alternative splicing or the use of alternative translational start sites, can encode for both an enhancer and a repressor protein(3) . In other cases particular transcription factors have been shown to either enhance or repress transcription depending on the absence or presence of a metabolic intermediate(4) , the concentration of the transcription factor itself(5, 6) , or the number of DNA-binding sites present(7) .
Vimentin belongs to the intermediate filament
protein (IFP) ()family, a related group of structural
proteins which are prominent components of the eukaryotic
cytoskeleton(8, 9) . IFPs can be subdivided into six
distinct types, based on sequence and site of synthesis. These include
keratins in epithelial cells, desmin in muscle cells, lamins in the
nucleus, neurofilaments in neurons, glial fibrillary acidic protein in
glial cells, and vimentin in cells of mesenchymal origin. IFPs are more
dynamic structures than previously thought(10) , and their
importance has been emphasized by the recent discovery that mutations
in specific keratin genes can give rise to genetic skin
diseases(11, 12, 13) . Among the different
IFP types, vimentin exhibits a complex pattern of expression (14, 15) and is often coexpressed with one of the
other IFPs, usually early in development. For example, vimentin is
coexpressed with desmin in the early stages of myogenesis but not in
the later stages(16) . Vimentin is also frequently expressed in
cultured cell lines, regardless of origin(17, 18) .
Regulation of vimentin gene expression is correspondingly complex,
involving multiple regulatory elements, including both enhancers and
silencers. In the case of the chicken vimentin gene, these regulatory
elements are all located upstream of a promoter region that encompasses
the first 160 bases upstream of the start site and provides a
constitutive level of activity(15, 19) . Several
regulatory elements upstream of this proximal promoter region, along
with associated DNA binding proteins, have been identified and
characterized. Three homologous silencer elements (SEs), denoted SE1,
SE2, and SE3, and an associated SE binding protein have been
identified(20, 21) . Approximately 1 kilobase upstream
of the most distal silencer element (SE3) is an antisilencer element,
which overrides the negative effect of the silencer element but shows
no independent enhancer activity(22) .
The SE closest to the transcription start site (SE1) lies in a region, between -160 and -320, that we have previously shown contains a tissue-specific enhancer of possible importance for the down-regulation of vimentin during myogenesis(23, 24) . In this report, we examine this upstream region in more detail and show that two homologous enhancer elements exist here, one on each side of SE1. Gel mobility shift assays, DNase I protection studies, and UV cross-linking experiments are used to argue that these two enhancer elements bind to the previously described SE binding protein, suggesting that this regulatory protein can both activate and repress transcription.
Figure 1: Transient transfection assays of various chicken 5`-deletion constructs in mouse L cells. A, deletion constructs fused to the CAT reporter gene are identified by the number of upstream bases included, beginning at +42, relative to the transcription start site set as +1, and ending at the designated nucleotide. The CAT activity of each construct is expressed relative to that of pcV-320, which is set at 100%. Errorbars represent the S.E. B, relative position of several upstream regulatory elements in the chicken vimentin gene. A proximal promoter region with constitutive activity encompasses the first 160 bases upstream of the start site and includes several GC boxes and a CAAT box. A proximal enhancer region lies between -160 and -320 and includes the two enhancer elements, PEE1 and PEE2, identified by transient transfection. PEE1 and PEE2 flank the first of three silencer elements (SE1). The other two SEs are located upstream of this proximal enhancer region, between -462 and -486 (SE2) and between -567 and -607 (SE3).
Although both PEE1 and PEE2 enhanced transcription severalfold, the CAT activity of pcV-320, which contains both enhancers, was only about 2-3 times that of pcV-160. This is likely due to the silencer element that is located between PEE1 and PEE2. We have previously described three homologous silencer elements (SE1, SE2, and SE3), which are important in regulating expression of the vimentin gene(20, 21) . Their positions relative to PEE1, PEE2, and the basal promoter region are illustrated in Fig. 1B. One of these silencer elements, SE1, is located between -236 and -253 and has been shown to decrease the transcriptional activity of pcV-160 by about 75% when placed directly upstream of -160(21) . The repressive effect of SE1 was evident in the transfections described here, with construct pcV-283 showing a low level of CAT activity compared with pcV-200 (Fig. 1A). The single SE and the two enhancer elements are all present in construct pcV-320, with the net effect being a 2-3-fold increase over pcV-160.
To confirm the ability of PEE1 and PEE2 to independently enhance transcription, these two elements were separately cloned in front of the basal promoter region contained in pcV-160 and transiently transfected into mouse L-cells (Fig. 2). In these constructs, possible interactions between PEE1 and PEE2 or between SE1 and the enhancer elements were eliminated. When placed directly upstream of -160, both PEE1 and PEE2 continued to show enhancing activity of about 4- and 2-fold, respectively, providing additional evidence for their functional significance. PEE1 also appeared to be a stronger enhancer than PEE2, which correlates with the data shown in Fig. 1A, where PEE1 produced a larger increase in transcription than PEE2.
Figure 2: Activity of individual enhancer elements. Transient transfection assays in mouse L cells demonstrate independent enhancer activity for both PEE1 and PEE2. Each enhancer element was cloned directly upstream of the basal promoter region(-160). The CAT activity of each construct is expressed relative to that of pcV-160, which is set at 100%.
Figure 3: GMSA analysis. Crude nuclear extract (8 µg) prepared from HeLa cell cultures was incubated with 6 ng of radiolabeled DNA as described under ``Materials and Methods.'' A, nuclear protein binding to both PEE1 (22 bp) and PEE2 (19 bp). Lane 1, control reaction with no protein extract added. Either no competitor (lanes 2 and 7) or the following unlabeled competitor DNA was added. Lanes 3 and 8, PEE2 and PEE1, respectively; lanes 4 and 9, PEE1 and PEE2, respectively; lanes 5 and 10, SE3; lanes 6 and 11, nonspecific DNA. Both PEE1 and PEE2 bind a lower band (marked L), while PEE1 also binds an upper band (marked U). Reactions with unlabeled competitor DNA (lanes 3-6 and 8-11) include a 500-fold molar excess of competitor DNA. B, nuclear protein binding to both enhancer elements and all three SEs. Lane2 is a control with no protein extract added.
The observation that band L has nearly equal mobility with both PEE1 and PEE2 led us to investigate whether PEE1 and PEE2 were binding the same protein. Competition with the other enhancer element in excess (lanes 4 and 9) did indeed show effective cross-competition for band L, indicating that the same protein, or proteins with similar mobilities and DNA binding specificities, are binding to both PEE1 and PEE2. Band U was not affected by the addition of excess unlabeled PEE2 (lane 9). As an additional control, binding reactions with an excess of unlabeled SE3 were carried out for both PEE2 and PEE1 (lanes 5 and 10). Surprisingly, an excess of unlabeled SE3 abolished all binding to both PEE1 and PEE2, resulting in almost no detectable band L or band U. This suggests that this protein is binding to sequences common to PEE1, PEE2, and SE3.
To further investigate the possibility that these elements are all binding the same protein, another GMSA, comparing all three SEs plus PEE1 and PEE2, was performed. As shown in Fig. 3B, all three SEs bound a protein to produce a band of equal mobility to band L observed with PEE1 and PEE2. We had previously reported that all three SEs bind the 95-kDa SE binding protein in GMSAs or Southwestern blots(21) , suggesting that the SE binding protein is present in band L in Fig. 3A. SE3 also produced a complex with the same mobility as band U observed with PEE1 (Fig. 3B, lane 6), explaining why unlabeled SE3 was an effective competitor for both bands (Fig. 3A, lane 10).
Figure 4: UV cross-linking of protein-DNA complexes. The lower bands from the GMSA (Fig. 3B) were UV-cross-linked as described under ``Materials and Methods'' and separated by 8% SDS-PAGE. Lane 1, control reaction using free DNA from the GMSA.
Figure 5: SDS-PAGE of affinity-purified extract. A sample of the 0.6 M KCl fraction from the SE1-DNA affinity column was separated by 8% SDS-PAGE. The positions of molecular mass markers are indicated on the right.
Figure 6: DNase I footprints and sequence comparison of PEE1, PEE2, SE1, and SE3. A, 20 fmol of each DNA element was incubated with 5 µl of affinity-purified HeLa nuclear extract and digested as described under ``Materials and Methods.'' Verticalbars to the right of each gelimage mark protected regions. The leftlane on each gel is a Maxam and Gilbert cleavage reaction used to identify bases in the DNase I cleavage pattern. B, underlined bases correspond to the protected areas of the DNase I footprints. Boldface bases in SE1 and SE3 correspond to areas protected in previous DNase I footprints using less purified nuclear extracts(21) . The boxedarea encloses a homologous region of each element. The human element is a matching sequence from the regulatory region of the human vimentin gene.
Figure 7: GMSAs with affinity-purified fractions. A, 0.5 µl of affinity-purified protein was used in the GMSA. Lanes1 and 6 are control reactions with no protein. Lanes3-5 and 8-10 include an excess of the following unlabeled competitor DNA. Lanes3 and 9, unlabeled SE; lanes4 and 8, unlabeled PEE; lanes5 and 10, unlabeled nonspecific DNA. B, lanes marked -66 were incubated with a DNA affinity-purified fraction containing the 95-kDa protein but missing the 66-kDa protein. Lanes marked Both were incubated with both extracts.
Previous studies of the chicken vimentin gene have demonstrated that several upstream regulatory elements, both positive and negative, function in an integrated but as yet incompletely understood fashion to regulate gene expression(15) . In the case of the human vimentin gene, positive and negative elements have also been identified that appear to correspond to the regulatory elements in the chicken vimentin gene, although not necessarily in the same number or absolute position (28, 29) .
We
have previously described a region between 160 and 320 bases upstream
of the transcription start site in the chicken vimentin gene, which
contains a tissue-specific enhancer of possible importance for the
down-regulation of vimentin expression during
myogenesis(23, 24) . Here we have used transient
transfection assays with various deletion constructs (Fig. 1A) to show that two separate enhancer elements,
PEE1 and PEE2, exist in this region, one on each side of a previously
characterized SE(21) . Transient transfection assays with each
of these enhancer elements fused separately to the basal promoter
region(-160) support the functional importance of these two
enhancer elements (Fig. 2). When binding to nuclear proteins was
tested via GMSA, both PEE1 and PEE2 produced a protein-DNA complex with
equal mobility (Fig. 3A, band L), and the
addition of both specific and nonspecific competitor DNA confirmed that
this complex is the result of a sequence-specific interaction.
Unlabeled SE3 was also an effective competitor in this experiment (Fig. 3A, lanes 5 and 10), and a
second GMSA confirmed that both enhancer elements and all three
silencer elements produce a complex with equal mobility (Fig. 3B). UV cross-linking of these complexes suggests
that the 95-kDa SE binding protein is binding to all of these elements (Fig. 4). Efforts to purify the SE binding protein using a
SE1/DNA affinity column were under way at the same time as these
experiments, and a nearly pure fraction containing the SE
binding protein was available for use in DNase I protection studies.
This purified fraction contains only two major components when stained
with either Coomassie Blue (Fig. 5) or silver (data not shown):
the SE binding protein around 95 kDa and another protein around 66 kDa.
DNase I protection studies of PEE1, PEE2, SE1, and SE3 confirm that all
of these elements have a region or regions protected against DNase I
cleavage when incubated with this purified fraction (Fig. 6A). Furthermore, a sequence comparison reveals a
homologous eight-base region where all four elements show protein
binding (Fig. 6B, box), providing a common
basis for DNA-protein interaction. The two SEs show an additional area
of protection, and a comparison of these DNase I footprints (underlined bases) with previously described footprints using
less purified extracts (highlighted bases) shows good
correlation for all protected areas(21) .
Studies on regulation of the human vimentin gene support the relevance of these results. A computer search of the human vimentin 5` end identified one sequence that exactly matches the eight-base sequence in PEE1 (Fig. 6B). Interestingly, this matching human sequence lies immediately upstream of a 19-bp negative element(29, 30, 31) , which was clearly protected in DNase I footprints(29) . We have previously shown using Southwestern blots and UV cross-linking that this 19-bp negative element binds the same 95-kDa protein as the chicken SEs(21) .
These results suggest that the same protein can bind both an enhancer and a silencer element, thereby activating or repressing transcription. In recent years, several instances of such dual regulation by the same transcription factor or related transcription factors, have been demonstrated. In the case of the Wilm's tumor gene product WT1, separate domains of WT1 mediate activation and repression, but the same DNA binding site is utilized in both cases. Whether WT1 activates or represses transcription depends on the number of binding sites and their position within the gene. In cases where related transcription factors with opposite effects have been described, the two proteins can be derived from the same gene, either as a result of alternative splicing (3, 32, 33, 34) or multiple translational start sites(35) , or from separate genes(36, 37) . Although two proteins of noticeably different molecular weights often result, this is not always the case. The gene for the POU domain nuclear protein I-POU, for example, can be alternatively spliced to create twin of I-POU, which activates transcription instead of repressing it like I-POU and differs from I-POU by only two amino acids(34) . An example involving two proteins from separate genes is found in the interferon regulatory system, where the regulatory factors IRF-1 and IRF-2 possess similar molecular weights and DNA binding specificities but produce opposite effects on the transcription of the interferon gene and interferon-inducible genes(36, 37) . IRF-1 and IRF-2 are derived from separate but related genes.
In our case, the 95-kDa SE binding protein is clearly interacting with both the positive and negative regulatory elements, as demonstrated by GMSAs, UV cross-linking experiments, and DNA footprints. It is unlikely that the opposing effects depend on binding site position or number, as with WT1, since these elements always increase (PEE1 and PEE2) or decrease (SE1, SE2, SE3) transcription when placed independently in front of the CAT reporter gene. A more promising hypothesis is that the SE binding protein is producing opposing effects due to differences in the binding site itself. To test this hypothesis, work is currently under way to construct a series of mutations that can convert the silencer element to an enhancer of transcription and vice versa.
Another possibility is that additional proteins, acting in concert with the SE binding protein, control whether these DNA elements act in a positive or negative manner. The 66-kDa protein, which copurified with the 95-kDa SE binding protein on the DNA affinity column, is one candidate, and preliminary results do indicate that it is essential for binding of the SE binding protein. A GMSA using a partially purified fraction of the SE binding protein that did not contain the 66-kDa protein produced no binding (Fig. 7B, lanes labeled -66). Subsequent addition of the 66-kDa fraction restored binding (lanes labeled Both). Interestingly, the 66-kDa protein was never detected in the UV cross-linking experiments, perhaps because it only contacts the 95-kDa protein and not the DNA itself. It is also possible that the 66-kDa protein and the SE binding protein are binding as a dimer, but the nature of the contacts between the 66-kDa protein and the DNA does not permit UV cross-linking under our conditions. In this case the 66-kDa protein might still be responsible for part of the DNase I protection pattern, and the eventual purification of this protein should allow us to examine more detailed and informative footprints. In addition to the 66-kDa protein, other proteins that play a role in gene regulation could be removed during purification. Since the functional assay used during purification was binding to SE1, proteins that do not directly bind to the DNA, but that nevertheless are important components in vivo, could be missed. The successful purification of the 95-kDa SE binding protein and production of antibodies will eventually allow us to immunoprecipitate intact complexes from labeled extracts. Analysis of these complexes will help us unravel how these regulatory elements and factors interact in vivo to activate or repress transcription of the vimentin gene.
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