(Received for publication, August 10, 1995)
From the
The human homeobox protein EVX1 (EVX1) is thought to play an important role during embryogenesis. In this study, the effect of EVX1 on gene transcription has been investigated in transfected mammalian cells. EVX1 expression represses transcription of a reporter gene directed by either cell-specific or viral promoter/enhancer sequences in a variety of mammalian cell lines and in a concentration-dependent manner. Transcriptional repression is independent of the presence of DNA-binding sites for EVX1 in all the promoters we tested. Furthermore, repression by EVX1 is evident also using a TATA-less minimal promoter in the reporter construct. A carboxyl-terminal proline/alanine-rich region of EVX1 seems to be responsible for the transcriptional repression activity, as suggested by transfection of EVX1 mutants. We speculate that the repressor function of EVX1 contributes to its proposed role in embryogenesis.
Homeobox genes code for transcription factors containing a
trihelical DNA-binding motif, termed homeodomain (HD) ()((1, 2, 3) , and references
cited therein). The HD is highly conserved with respect to structure
and function throughout evolution and is a common component of numerous
proteins that regulate transcription during development (for reviews,
see (4, 5, 6) ). The functions of HD proteins
range from activities in directing pattern formation to more restricted
roles in regulating specific cell fate(3, 7) . The
products of vertebrate homeobox-containing genes have been shown to
bind DNA in vitro(8, 9) and to regulate gene
transcription through specific target sequences in cell
culture(10, 11) . It is unlikely that HD-containing
proteins exert their different selective functions in vivo only through protein-DNA interactions, since they share similar
DNA-binding specificities in vitro(4, 5) .
Thus, it is presumed that although a single HD has the potential to
bind to many DNA-target sites it may be specifically recruited into a
functional complex at only a subset of those sites by selective
protein-protein
interactions(12, 13, 14, 15) .
Furthermore, in some cases the HD has been demonstrated to be
dispensable for activity in vivo(16, 17) .
Recently, the human homeobox genes EVX1 and EVX2 have been isolated and sequenced(18, 19) . These genes encode proteins containing a HD closely related to that encoded by the Drosophila even-skipped, which belongs to the pair-rule class of segmentation genes and is required for the proper development of the metameric body plan of the fruit fly(20, 21, 22) . Also the murine Evx 1 and Evx 2 genes have been cloned, and Evx 1 expression pattern during mouse embryogenesis has been studied(23) . During early embryogenesis, Evx 1 is expressed in a biphasic manner. From day 7 to 9 of development its expression emerges at the posterior end of the embryo within the primitive ectoderm and later in the mesoderm and neuroectoderm. From day 10 to 12.5, Evx 1 transcripts are restricted to specific cells within the neural tube and hindbrain, while no expression is detectable in a variety of adult tissues. Spyropoulos et al.(24) have recently shown that the targeted disruption of the Evx 1 gene in mice causes early postimplantation lethality of the conceptus.
Several authors have reported that the even-skipped gene product acts as a transcriptional repressor both in in vitro assays (25, 26, 27) and in cell transfection experiments(28, 29) . On the contrary, Jones et al.(30) showed that the mouse Evx 1 protein induces the expression of a reporter gene driven by the chicken Tenascin-C promoter. The same authors restricted the sequences that contributed to the activation to a segment containing a TRE known to bind transcription factors belonging to the AP-1 family. Currently, the role of the human EVX1 and EVX2 proteins in gene transcription is unknown.
The aim of our study was to investigate the transcriptional activity of EVX1 in transfected mammalian cells. Our data indicate that EVX1 expression strongly reduces, in a concentration-dependent manner, the basal and activated transcription of the reporter gene CAT directed by a variety of cell-specific and viral promoters in several different mammalian cell lines. EVX1 transcriptional repressor function is evident using both TATA-containing and TATA-less promoters. We also show that the repressor function of EVX1 is contained within a C-terminal region rich in alanine and proline residues and is independent of the presence of DNA-binding sites for EVX1.
General handling techniques for baculovirus expression system were performed essentially as described in (31) .
Mouse monoclonal antibodies to EVX1 were produced and characterized according to methods previously described(34, 35) . Determination of Ig classes was carried out by standard procedures utilizing the Mouse Typer Sub-Isotyping kit (Bio-Rad).
For immunoblot analysis, nuclear lysates of infected Sf9 cells were electrophoresed in 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes according to standard procedures. The filters were incubated with the supernatant of hybridoma cells, followed by an alkaline phosphatase-conjugated goat anti-mouse antibody from Sigma. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate were used as the substrates (Promega, Madison, WI) with a positive reaction resulting in purple color.
Immunohistochemistry was performed according to the streptavidin-biotin complex technique using a commercially available kit system (Dako, Glostrup, Denmark). Nuclei were counterstained for 5 min with Mayer's hematoxylin solution (Sigma).
General handling of plasmid DNA was performed according to previously described techniques(43) .
Figure 1:
EVX1 is
expressed in nuclei of transfected cells and is recognized by a
monoclonal antibody raised against the recombinant protein. A, left, SDS-polyacrylamide gel electrophoresis analysis of the
purified recombinant EVX1. 100 ng of the recombinant protein, purified
as detailed under ``Experimental Procedures,'' were loaded on
a 10% reducing acrylamide gel and, after electrophoresis, the gel was
silver-stained. Molecular mass markers are indicated on the left
side. Right, immunoreactivity of mAb B6-41 in
Western blot on nuclear lysates of Sf9 cells expressing HOXD4 and EVX1
proteins. Molecular mass markers are indicated on the right
side. B, immunohistochemical analysis of HeLa cells
transiently transfected with pRC-CMV vector (negative control)
utilizing mAb B6-41. Magnification is 500. C,
immunohistochemical analysis of HeLa cells transiently transfected with
pCMV-EVX1 vector utilizing mAb B6-41. Magnification is
500. D, immunofluorescence analysis of InR1-G9 cells
transiently transfected with pCMV-EVX1. Magnification is
500.
In order to determine whether mAb B6-41 is able to recognize the fully processed EVX1 expressed in human cells, HeLa cells were transiently transfected with the expression construct pCMV-EVX1 (see ``Experimental Procedures''). Both in immunofluorescence and immunohistochemistry assays nuclei of approximately 20% of HeLa cells showed a strong staining when reacted with mAb B6-41, while cells transiently transfected with pRC-CMV were negative (Fig. 1, B and C).
Figure 2: EVX1 represses basal and activated transcriptional activity of the rat proglucagon gene promoter in transiently transfected InR1-G9 cells. A, cells were transiently transfected in suspension using the DEAE-dextran technique (see ``Experimental Procedures'') with 10 µg of the reporter plasmid p[-1.1]GLU-CAT and 10 µg of the effector plasmids indicated. 30 h later cells were changed to serum-free medium with or without 20 µM forskolin. After a further 16 h, cells were harvested, and the CAT activity present in aliquots of cell extracts was measured, as detailed under ``Experimental Procedures.'' The results are the average (± S.E.) of five independent experiments (performed in duplicate). In the lower part of the panel, a representative autoradiogram of one experiment (performed in duplicate) is shown. B, cells were transfected with 10 µg of the reporter plasmid p[-1.1]GLU-CAT and 10 µg of the effector plasmids indicated. 30 h later cells were changed to serum-free medium with or without 160 nM phorbol 12-myristate 13-acetate. After a further 16 h, cells were harvested, and the CAT activity present in aliquots of cell extracts was measured, as detailed under ``Experimental Procedures.'' In the lower part of the panel, a representative autoradiogram of one experiment (performed in duplicate) is shown.
Figure 4: EVX1 represses the transcriptional activity of several cell-specific and viral promoter/enhancer sequences in several different transiently transfected cells. Indicated cells were transiently transfected (detailed under ``Experimental Procedures'') with 10 µg of the reporter plasmids indicated and 10 µg of the pCMV-EVX1 expression plasmid. 46 h later, cells were harvested, and the CAT activity present in aliquots of cell extracts was measured as detailed under ``Experimental Procedures.'' Results represent the average (± S.E.) of three independent experiments performed in duplicate.
Figure 6: EVX1 represses the transcriptional activity of a minimal TATA-less promoter in transiently transfected InR1-G9 and NIH-3T3 cells. Cells were transiently transfected in suspension using the DEAE-dextran technique with 10 µg of the reporter construct and 10 µg of effector plasmids, as indicated. 46 h after the transfection, cells were harvested, and the CAT activity present in aliquots of cell lysates was assayed, as detailed under ``Experimental Procedures.'' Results represent the average (± S.E.) of three independent experiments (performed in duplicate). In the lower part of the figure, a representative autoradiogram of one experiment (performed in duplicate) for each cell line is shown.
Next we investigated whether transcriptional repression is exerted by EVX1 at any concentration of the transfected expression vector. As shown in Fig. 3, repression by EVX1 is a concentration-dependent phenomenon in a range of transfected pCMV-EVX1 vector from 0.1 to 8 µg.
Figure 3: EVX1 functions as a transcriptional repressor in a concentration-dependent manner. Subconfluent InR1-G9 cells were transfected in suspension using the DEAE-dextran technique with 10 µg of the reporter plasmid p[-1.1]GLU-CAT and different amounts (as indicated) of pCMV-EVX1. 46 h after transfection, cells were harvested, and aliquots of lysates were assayed for CAT activity as detailed under ``Experimental Procedures.'' The results are the average (± S.E.) of three independent experiments performed in duplicate.
It has been reported that the mouse Evx 1 activates the chicken Tenascin-C promoter in transfected
cells by interacting with a TRE(30) . The rat proglucagon gene
promoter contains binding sites for transcription factors belonging to
the AP1 and CREB families and is regulated by protein kinase A and
protein kinase C activators in pancreatic cell
lines(36, 37, 38) . The transcriptional
activation of p[-1.1]GLU-CAT by forskolin (Fig. 2A) and phorbol 12-myristate 13-acetate (Fig. 2B) was approximately 9O% reduced by
cotransfection of pCMV-EVX1 in comparison with pRC-CMV alone. Similar
results were obtained using -TC1 insulinoma cells (not shown).
To investigate whether the transcriptional repression is restricted to the specific promoter we used in the above described experiments, other reporter plasmids containing mammalian and viral promoter/enhancer regions were tested in different mammalian cell lines. The cotransfection of pCMV-EVX1 with pRSV-CAT, pCAT-promoter, and p7B2-I-CAT, containing 524 bp of the 3` LTR of the Rous sarcoma virus (39) , 202 bp of the SV40 promoter(51) , and 1.5 kilobases of the 5`-flanking region plus 1.5 kilobases of the first intron of the human 7B2 gene (which encodes a neuroendocrine molecular chaperone expressed in insulinoma and glucagonoma cells)(52) , respectively, decreased the transcriptional activity of the reporter by 50-90% in different cell lines (Fig. 4, and data not shown). These results indicate that the transfected EVX1 exerts its transcriptional repressor activity in cells either expressing or not expressing the endogenous EVXI and that this effect is not promoter-specific.
The observed repression of CAT activity by EVX1 corresponds to a decrease in CAT mRNA, as measured either by RNase protection analysis or by RT-PCR experiments (Fig. 5). It is noteworthy that the CAT transcript is strongly reduced in EVX1-transfected cells 24 h after transfection and that the reduction persists after 46 h (Fig. 5A). This finding suggests that the observed EVX1 effect on CAT activity predominantly depends on the transcription rate of CAT mRNA. This finding is supported by the RT-PCR experiments depicted in Fig. 5B.
Figure 5:
RNase protection and RT-PCR analysis of
the CAT transcript in transfected InR1-G9 cells. A,
subconfluent cultures of InR1-G9 cells were transiently transfected
using the calcium phosphate technique with p[-1.1]GLU-CAT
together with either pRC-CMV or pCMV-EVX1. Total RNA was prepared at
different times, and 10 µg RNA were subjected to RNase protection
analysis as indicated under ``Experimental Procedures.'' Lane 1, RNA CAT probe; lane 2, negative control (Escherichia coli RNA CAT); lane 3,
positive control (E. coli RNA CAT
); lanes
4 and 6, RNA from pRC-CMV transfected cells, 24 and 46 h
after transfection, respectively; lanes 5 and 7, RNA
from pCMV-EVX1-transfected cells, 24 and 46 h after transfection,
respectively. Arrows point the RNA probe and the protected CAT
fragment, respectively. Expression of
-actin is shown in the lower part of the panel as an internal control. The numbers to the left indicate the length of the
fragments in nucleotides. B, cell transfection and RNA
isolation were performed as in panel A. 5 ng of RNA were
subjected to RT-PCR as described under ``Experimental
Procedures.'' RT-PCR products were electrophoresed, and agarose
gels were stained with ethidium bromide and photographed. The bands
corresponding to the CAT transcript were densitometrically scanned, and
the relative absorbances (expressed in arbitrary units) of two
independent experiments were 23.1 (lane 1, pRC-CMV transfected
cells) and 2.5 (lane 2, pCMV-EVX1 transfected
cells).
In each transfection experiment, both the expression and the correct transport to the nucleus of the EVX1 protein were checked by immunofluorescence using mAb B6-41 (Fig. 1D, and data not shown).
Figure 7:
The
deletion mutant EVX1 lacking the C terminus loses the ability to
repress transcription in transfected cells. A, a schematic
representation of EVX1, the deletion mutants, HOXC6, and the chimeric
construct HOXC6/EVX1. Details on the construction of the mutant
expression vectors are reported under ``Experimental
Procedures.'' B, immunohistochemical analysis of
transiently transfected InR1-G9 cells with pCMV-
EVX1 utilizing mAb
B6-41. Magnification is
250. C, InR1-G9 was
transiently transfected with 10 µg of the reporter plasmid
p[-1.1]GLU-CAT and 10 µg of the effector plasmids
indicated. 46 h after transfection, cells were harvested, and the CAT
activity present in aliquots of cell lysates was assayed as detailed
under ``Experimental Procedures.'' Bars represent
the average (± S.E.) of three independent experiments performed
in duplicate.
Our findings indicate that a human HD protein, EVX1, is a potent transcriptional repressor and that this effect is independent of the presence of its specific DNA-binding sites in the promoter. Here we show that EVX1 represses basal and activated transcription directed by TATA-containing and TATA-less promoters. Furthermore, the carboxyl-terminal region of the protein seems to contain the ``repressor domain(s).''
It has been shown in Drosophila that Eve binds DNA (55) and functions as a sequence-specific transcriptional repressor in transfected cells (28, 29) and in transcriptionally competent extracts (25) . However, Han and Manley(29) , using Eve deletion mutants, observed a significant correlation between strong repression and weak binding to DNA. Moreover, TenHarmsel et al.(27) , in addition to a repressor mechanism based on Eve binding to its DNA sites, described also a second mechanism that does not require binding to DNA. These authors suggested that Eve might interact with the general transcription factors to inhibit their function. Thus, the finding that EVX1, which is homologous to Eve, represses reporter gene transcription directed by promoters lacking any obvious binding site for the protein is not totally unexpected. Furthermore, other HD proteins seem to behave similarly. Msx-1, a murine HD protein, inhibits transcription with a mechanism very similar to that we describe for EVX1(54) . Also SCIP, a member of the POU class of HD proteins, actively represses transcription of myelin-specific genes independently of the presence of its specific DNA binding sites on the promoter(56) . Much evidence indicates that the function of HD proteins in transcriptional regulation may not be limited to their interaction with specific DNA-binding sites. Although HD proteins share similar DNA-binding properties in vitro, they present different functional specificities in vivo, and this is likely due to protein-protein interactions(4, 5, 14, 57, 58) . Furthermore, several authors have shown that proteins lacking all or part of their HD may retain some aspects of function(16, 17, 54, 59) . Therefore, all of this experimental evidence indicates that the simple interaction between the HD and its DNA-binding site can be dispensable for some transcriptional properties of homeobox-containing proteins.
A comparative analysis of the amino acid sequences of EVX1 and Eve revealed that, apart from the highly conserved HD, the two proteins show an alanine-/proline-rich region present at the C-terminal end of both proteins in a position very close to the HD (data not shown). Although glutamine/alanine/proline richness is not a universal feature of active repressors, these domains have been found not only in Eve, but also in other well characterized transcriptional repressors such as engrailed, Krüppel, Wilms tumor gene product, and Msx-1(53, 54) . Furthermore, the Dr1 protein, which also contains a glutamine/alanine-rich region, has been shown to interact with TFIID and to inhibit transcription(60) . Indeed, we have shown that EVX1 protein, while still able to bind DNA with high affinity, loses its ability to repress transcription when the alanine- and proline-rich carboxyl-terminal portion of the protein is removed. Furthermore, this region confers transcriptional inhibitory potential to the HD protein HOXC6 which is per se unable to affect transcription of the reporter plasmids used.
In recent years there
has been an explosion of information regarding the role of
sequence-specific DNA binding proteins in the selective activation of
eukaryotic promoters. On the contrary, less is known about how DNA
binding proteins repress transcription (for reviews, see (53) and (61) -63). However, it is reasonable to
believe that selective repression is an important mechanism of
transcriptional control and that it could be widely used during
development. Our results seem to rule out most of the repression
mechanisms proposed for the eukaryotic gene transcriptional
regulation(53, 61, 62, 63) . First,
EVX1 does not compete with an activator protein for binding to the same
DNA sequence, as it can inhibit promoters lacking its binding site.
Second, a mechanism by which EVX1 would interact with a specific
activator protein, preventing its contact with the transcription
machinery, is made unlikely by the generality of the phenomenon we
observed. A mechanism that must be taken into account when repression
is observed, particularly in transient transfection assays, is termed
``squelching''(64) . In this case, transcriptional
repression paradoxically results from the overexpression of an
activating protein. Such repression requires neither specific DNA
binding sites nor an intact DNA binding domain in the overexpressed
protein and appears to result from the sequestration of other
transcription factors with which the activator naturally interacts. Our
data are not consistent with this explanation since (i) transcriptional
activation was never observed under any circumstances we used,
regardless of the promoter, the cell type or the concentration of the
transfected EVX1 expression vector; (ii) the amount of the expression
plasmid required for repression is within the range that is normally
used to measure transcriptional activity in transient transfection
assays(54) ; and (iii) preliminary results obtained in pools of
HeLa cells stably transfected with pCMV-EVX1, which express relatively
low levels of the protein in comparison to the transient transfectants,
indicate that EVX1 represses transcription of transiently transfected
reporter constructs. ()On the basis of our data, we can
speculate that EVX1 functionally interferes with a general
transcription factor. However, this interference cannot be due to a
displacement of the factor caused by the binding of EVX1 to the TATA
sequence, which could in principle mimic homeobox protein binding
sites, because of its action on TATA-less promoters. The results seem
then to indicate a mechanism by which EVX1, as other repressor proteins (60, 65, 66, 67) , interferes with a
general transcription factor at an early stage of the assembly of the
transcription initiation complex, thereby preventing its further
assembly.
The potential for EVX1 to repress transcription through protein-protein rather than protein-DNA interactions may be an important feature of its proposed role as a regulator of embryogenesis.