Download Mapping functional regions of the segment

© 7992 Oxford University Press
Nucleic Acids Research, Vol. 20, No. 10
2485-2492
Mapping functional regions of the segment-specific
transcription factor Krox-20
Christine Vesque and Patrick Charnay*
Laboratoire de Genetique Moleculaire, CNRS D 1302, Ecole Normale Superieure, 46 rue d'Ulm,
F-75230 Paris Cedex 05, France
Received February 7, 1992; Revised and Accepted April 10, 1992
ABSTRACT
Krox-20, a zinc finger transcription factor with similarity
to Sp1, is likely to play an important role in the
development of the vertebrate central nervous system.
A knowledge of its molecular properties will help to
understand its physiological functions. We have
therefore performed a structure-function analysis of the
protein to identify the regions involved in DNA-binding
and transcriptional activation. Our data suggest that
only the zinc fingers are required for high affinity,
specific DNA-binding. Transcriptional activation was
not affected by deletion of the C-terminal tail of the
protein. In contrast, deletion of the N-terminal half,
upstream of the zinc fingers, completely abolished
transactivation without affecting DNA-binding or
nuclear localization. Two transcriptional activation
domains were identified in this region. They cooperate
to establish full activity. They are rich in negativelycharged amino acids and are therefore may constitute
acidic activation domains. Comparative analysis of the
amino acid sequences of several zinc finger proteins
belonging to the Krox-20 subfamily indicates that they
contain acidic regions at similar locations within their
N-terminal region, suggesting that the functional
organization of these proteins has been conserved
during evolution.
INTRODUCTION
The mouse gene Krox-20 was originally identified as a member
of a subset of genes, termed immediate-early genes, whose
expression is induced following serum stimulation of NIH3T3
cells (1). These genes are activated very rapidly and transiently
at the transcriptional level by mechanisms that do not require
de novo protein synthesis and they are thought to mediate long
term cellular responses such as proliferation or differentiation.
Krox-20 encodes a protein with three C2H2-type zinc fingers
(1—5), which are similar to those of the transcription factor Spl
(6). As anticipated from the presence of the zinc fingers, the
Krox-20 protein was shown to bind to a specific DNA sequence
and to act as a transcription factor (7). It belongs to a small
* To whom correspondence should be addressed
subfamily of proteins that have very similar zinc fingers and
which recognize identical or very closely related GC-rich
sequences. So far, three other members have been identified:
Krox-24 (also known as Egr-1, Zif268, NGFI-A and TIS8
(8-12)), EGR-3 (13) and NGFI-C (14). Although these proteins
are very closely related within their putative DNA-binding
domains, they appear to be much less conserved elsewhere. The
Wilm's tumor gene product WT1 (15) recognizes a similar DNA
target, but it contains an additional zinc finger. The zinc fingers
of Spl are more distant from those of Krox-20 and indeed this
protein binds to a slightly different GC-rich sequence (16).
Recently, site-directed mutagenesis of the zinc fingers of Krox-20
allowed the identification of the amino acids responsible for the
difference in DNA binding specificity of Krox-20 and SP1 (17);
the establishment of the structure of the three zinc fingers of
Zif268/Krox-24 complexed with their target DNA sequence has
provided a framework for understanding how zinc fingers
recognize DNA (18).
Further interest for Krox-20 arose from the analysis of the
pattern of expression of the gene during development. During
mouse embryogenesis, Krox-20 is specifically expressed in two
non-adjacent rhombomeres of the developing hindbrain (19).
Rhombomeres have been shown to constitute anterior-posterior
metameric units within this region of the neural tube (20—22).
This pattern of expression, which appears before morphological
segmentation and partially overlaps with that of several
homeobox-containing genes (23), raises the possibility that
Krox-20 might play a role in the regulation of hindbrain
segmentation (19) and participate in the control of the expression
of some of the homeobox-containing genes (24). The role of
Krox-20 is likely to have been conserved throughout evolution,
since frog and zebra fish homologs of the gene have been cloned
and show segmented patterns of expression during hindbrain
development (25; D. Wilkinson, personal communication).
Consequently, it has become increasingly important to perform
a detailed analysis of Krox-20 molecular properties.
We present here the results of a structure-function analysis
designed to identify the role of the different regions of the protein.
A series of external and internal deletions in the Krox-20 cDNA
were built to analyze the involvement of the corresponding parts
2486 Nucleic Acids Research, Vol. 20, No. 10
of the protein in DNA-binding, nuclear localization and
transcription control. Our data suggested that only the zinc fingers
were required for high affinity, specific DNA recognition. Two
transcriptional activation domains were identified within the Nterminal half of Krox-20. Rich in negatively-charged amino acids,
they may be of the acidic type. Acidic regions are also observed
at similar positions in other members of the Krox-20 subfamily.
Such a conservation suggests that these acidic regions are involved
in transcriptional activation like those of Krox-20.
MATERIALS AND METHODS
DNA constructions
The reporter plasmid p6E-tkCAT was constructed as described
previously (7). All the bacterial expression plasmids were derived
from pET-Krox-20 (7). The deletions were created by polymerase
chain reaction (PCR) amplification of a part of the cDNA and
subsequent substitution to a wild-type cDNA fragment in pETKrox-20 (17). In the case of the N-terminal deletions, the 5' PCR
primer included a Ndel restriction site for cloning into the vector
and an in frame initiator ATG codon. In the case of the C-terminal
deletions, the 3 ' PCR primer included an in frame stop codon
and a BamHI restriction site. The internal deletion A186—331
was obtained by creation of a BamHI restriction site at the position
corresponding to amino acid 331 by PCR amplification and
proline-rich
proline-rich
DH
subsequent deletion of the portion of the cDNA in between this
site and a natural BamHI site corresponding to position 186. The
PCR products were cloned and completely sequenced using a
Pharmacia sequencing kit. The Drosophila cells expression
constructs were derived from the corresponding pET constructs
by purification of a Ndel-BamHI fragment containing the Krox-20
coding sequence and subsequent cloning into the expression
vector pPacU + Ndel (generous gift of A.Courey and R.Tjian).
This places the Krox-20 cDNA under the control of the
Drosophila actin 5C promoter. In this case, the expected proteins
contain two additional amino acids (methionine and histidine) at
their N-termini as compared to the corresponding proteins
produced in bacteria.
Bacterial protein extracts and Western blotting
The expression system and protocols of Studier and collaborators
were used to produce the different proteins in E. coli (26, 27).
Protein extracts were prepared according to Kadonaga and
collaborators (6). For immunoblotting, 8 to 80 ng of bacterial
protein extract were separated by SDS-PAGE. Blotting and
immunodetection were carried out according to standard
procedures (28). 539A is a rabbit antiserum raised against a
Krox-20 fusion protein (M.Zerial, P.Chavrier, P.Charnay and
R.Bravo, unpublished result). The 539A antiserum and the
peroxydase conjugated anti-rabbit antibody (Jackson
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Figure 1. Schematic representation of the Krox-20 protein and of its deletion derivatives, and estimation of their DNA-binding, nuclear localization and transactivation
properties. Several features of the Krox-20 protein are represented: zinc fingers, acidic, basic and proline-rich regions. The positions of the extremities of the different
deletions are indicated along the wild-type sequence. The 'M' within Krox-20/2ER zinc fingers symbolize the double amino acid modification responsible for a
change in DNA-binding specificity (17). DNA-binding was estimated by gel retardation experiments (see Figure 2) after synthesis of the protein in E. coli. + + ,
proportion of complex similar to that formed by the wild-type Krox-20 protein within a 3-fold range; - , proportion at least 50-fold lower than the control; ND,
not determined. For deletions N238 and N298, the parentheses indicate that the relative concentration of the protein in the extract could not be precisely estimated.
Cellular localization was determined by indirect immunofluorescence with an antiserum directed against the N-terminal part of Krox-20 (see Figure 3): + , the protein
is mostly nuclear. Transcriptional activation was determined by transient cotransfection with a reporter CAT gene in SL2 cells (see Figures 4 and 5). + + , transactivates
the reporter gene like the wild-type protein within a 2-fold range; + , transactivates 4- to 6-fold less efficiently than the wild-type Krox-20; - , no transactivation detected.
Nucleic Acids Research, Vol. 20, No. 10 2487
Immunoresearch Laboratories) were diluted 2000- and 2500-fold
respectively.
Cell lines, DNA transfection and immunofiuorescence
Schneider 2 cells (29) were grown and transfected as described
(30, 31). Each plate received the following plasmids: 5 ng of
reporter DNA, 100 or 250 ng of expression plasmid, 5 /*g of
a control plasmid, pAdh-/3gal, which is capable of expressing
the E. coli lac Z gene under the control of the Drosophila Adh
promoter, and pUC19 DNA up to a total amount of plasmid DNA
of 20 fig. For immunofluorescence analysis, the cells were grown
on cover slides. Two days after transfection, they were washed
twice with PBS and fixed by incubation in PBS (Phosphate
Buffered Saline) containing 3.7% formaldehyde and 0.1 % NP40
for 15 min. After three washes in PBS containing 0.1% NP40,
the cells were preincubated for 30 min in PBS containing 1 %
Bovine Serum Albumin (BSA), before exposure for 1 hr to a
Pel3A
Krox-20
N106
N143
C418
C424
4186-331
B
Pet3a
Krox-20
N106
N143
C418
C424 A186-331
Figure 2. DNA-binding activity of the different Krox-20 derivatives synthesized
in E. coli. A, estimation of the relative amount of Krox-20 present in the extracts
by Western blotting using the 539A antiserum. For each derivative, three amounts
(100 /ig, 30 tig, 10 ^g) of extract were tested. Pet3A, control bacterial extract
containing no Krox-20 protein. The molecular weights of size markers are
indicated. B, determination of the DNA-binding activity by gel retardation with
an oligonucleotide including a consensus Krox-20 binding site. The amounts of
extract used for each derivative were normalized according to the results of the
Western blotting analysis. Two dilutions ( l x and 0.5x) were tested. F, free
oligonucleotide; C, complexed oligonucleotide.
200-fold dilution of the 539A antiserum in PBS containing 0.5%
BSA and 0.1 % NP40. The cells were washed three times and
incubated for 30 min with a 100-fold dilution of the FITC antirabbit IgG antibodies (Biosys) in the same buffer. DNA was
stained by exposing the cells to a 0.5 /xg/ml solution of DAPI
(4',6-diamidimo2phenylindole 2HC1, Serva) in PBS.
Cellular extracts, transactivation and gel retardation assays
44 hours after transfection, half of the cells were resuspended
in 0.25 M Tris-HCl pH 7.8, to prepare the extracts for
chloramphenicol acetyl transferase (CAT) and /3-galactosidase
assays (32, 33). The amount of extracts used for the CAT assays
were normalized relative to the levels of /3-galactosidase. The
other half of the cells were used for the preparation of extracts
for gel retardation analysis. They were pelleted by centrifugation
and frozen in liquid nitrogen. The frozen cells were resuspended
in five volumes of extraction buffer (10 mM Hepes pH 7.9, 0.4
M NaCl, 0.1 mM EDTA, 0.5 mM DTT, 5% glycerol) and 0.5
mM PMSF were added. The gel retardation assays (34-36) were
performed as described (37) with the complementary
oligonucleotides 5'-CTCTGTACGCGGGGGCGGTTA-3' and
5'-CTCTAACCGCCCCCGCGTACA-3' containing a Krox-20
consensus binding site.
RESULTS
Specific DNA-binding
We have performed a detailed study of the regions of the Krox-20
protein responsible for transcriptional activation. It was based
on the analysis of a series of Krox-20 deletion derivatives. Such
a study required discrimination between regions involved directly
in transcriptional control and those necessary for specific DNAbinding or nuclear localization. Concerning DNA-binding, the
zinc fingers were shown previously to be involved in specific
DNA recognition (17). The question here was whether any region
of the protein was required in addition to the zinc fingers for
high affinity binding to the cognate sequence.
The DNA-binding activity and specificity of the Krox-20
protein were previously studied in bacterial extracts, using the
expression system of Studier and collaborators (7,17, 26, 27).
This approach allows an easy determination of the relative
affinities of different derivatives since the amount of Krox-20
protein present in the extract can be quantitated by Western
Figure 3. Nuclear localization of the N143 Krox-20 derivative. The expression
construct was transiently transfected into SL2 cells and the cellular localization
of the protein was studied by indirect immunofluorescence with the antiserum
539A directed against the N-terminal part of the protein. Three photographs were
taken from the same field: A, phase contrast; B, immunofluorescence with the
589A antibody; C, DAPI coloration of the DNA, indicating the positions of the
nuclei.
2488 Nucleic Acids Research, Vol. 20, No. 10
blotting. In order to evaluate the contributions of the different
regions of the Krox-20 molecule to DNA-binding, a series of
external and internal deletions were introduced into the Krox-20
cDNA. Figure 1 presents the schematic structure of the expected
proteins. It also illustrates some of the features of the Krox-20
protein that can be identified simply by examining the amino acid
sequence: i) the zinc fingers are located between positions 339
and 418; ii) they are immediately flanked, upstream and
downstream by two short basic sequences. The upstream basic
sequence is highly conserved between Krox-20, EGR-3 and
Krox-24 (8, 13), while the downstream one is not, although basic
amino acids are also observed at this position in the other
members of the subfamily; iii) two relatively acidic regions can
be identified between positions 23 and 67 (net charge - 8 ) , and
160 and 184 (net charge - 4 ) respectively; iv) finally, the protein
is very rich in proline residues, except in the zinc finger domain.
It contains a stretch of proline residues (positions 167-173) and
three regions where the content in proline is around or above
30% (positions 7 0 - 8 5 , 204-264 and 306-340). Several
deletions were selected to evaluate the contributions of these
different regions to DNA binding. In particular, deletions N331,
A186 —331 and C418, which eliminate the upstream or
downstream basic flanking regions respectively (Figure 1), were
designed to identify a possible role of these particular regions.
The selected Krox-20 derivatives were expressed in E. coli
and the amount of Krox-20 protein in the extracts was estimated
by Western blotting with a rabbit polyclonal antiserum, 539A,
directed against the N-terminal half of the wild-type protein
(Figure 2A and data not shown). The polyclonal nature of the
antiserum may have led to a slight underestimation of the amount
of some of the N-terminal deletion mutants. However, this did
not compromise our conclusions. The proteins N298 and N331
were not recognized at all by the antiserum, preventing estimation
of their relative concentrations. The extracts were then used to
evaluate the affinity of the different proteins for a consensus
Krox-20 nucleotide target sequence by a gel retardation assay
(34-36). In these conditions, formation of the complex between
the Krox-20 derivatives and the oligonucleotide gave rise to major
retarded bands (Figure 2B). In addition, in some cases, lower
shifted bands were also observed. They were likely to correspond
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Figure 4. Transactivation by the different Krox-20 derivatives. SL2 cells were cotransfected with Krox-20 derivatives expression plasmids, CAT reporter plasmids
and a normalization plasmid, pAdh-/3gal. A, structure of the reporter plasmids. ptkCAT contains the CAT gene (open box) driven by the HSV tk promoter (closed
box) and followed by the SV40 polyadenylation signal (striped box). p6E-tkCAT was obtained by insertion of six Krox-20 binding sites of the E-type (7) into the
BamHI site. B, estimation by gel retardation assay of the Krox-20 DNA-binding activity present in the cell extracts. pPacU-Ndel (Pac) is the expression vector.
C and F refer to the positions of complexed and free oligonucleotide respectively . The arrow indicates a retarded band corresponding to an endogenous binding
factor. C, transactivation assay. CAT assays were performed after normalization of the amounts of extracts according to /3-galactosidase activity. 'C' corresponds
to the unreacted substrate [ I4 C] chloramphenicol and '3-AC to the major acetylated form (in position 3).
Nucleic Acids Research, Vol. 20, No. 10 2489
to complexes involving partially degraded Krox-20 protein. The
proportion of complexed versus free oligonucleotide obtained with
each of the Krox-20 derivatives was determined and taken as an
estimate of the relative affinity of the protein for the target
sequence. The outcome of these experiments is summarized in
Figure 1: none of the deletions tested significantly affected the
binding activity. In contrast, a double amino acid change in finger
2 (construction Krox-20/2ER) has been shown previously to
change the recognition specificity and to abolish binding to the
oligonucleotide (Figure 1 and reference 17), which illustrates the
involvement of the zinc fingers in DNA recognition. The Cterminal endpoint of deletions A186-331 abutted the zinc fingers,
eliminating the basic region located upstream. The deletion C-418
eliminated the entire C-terminal part of the protein, including
the basic region located immediately downstream to the zinc
fingers. The fact that these deleted proteins bound efficiently to
the cognate sequence (Figure 1 and 2) indicated that the basic
regions were not required simultaneously for DNA-binding. In
fact, our data suggest that the DNA-binding domain consists of
only the zinc fingers and they are consistent with the results of
Pavletich and Pabo indicating that a similar region of
Krox-24/Zif268 binds efficiently to the target sequence (18).
Nuclear localization
We have previously studied transactivation by Krox-20 by
transient transfection into Drosophila Schneider line 2 (SL2) cells
(7). These cells were selected because they are devoid of Spl
and Krox-20 endogenous activities that might have interfered in
the assay (31) and since they allow a high level of transactivation
by Krox-20 (7). We have used the same system to evaluate the
transcriptional properties of the different Krox-20 derivatives.
An essential point in the analysis was to determine whether the
mutations affected nuclear localization. Therefore, the cellular
localization of the Krox-20 derivatives that were recognized by
the 539A antibody was determined by immunofluorescence after
transient expression in SL2 cells. The coding sequences of the
different mutants were placed under the control of the Drosophila
actin 5C promoter as described previously (7). Representative
results for the deletion mutant N143 are shown in Figure 3.
Examination of the same field under phase contrast, immunofluorescence and DAPI staining, which labels the DNA, indicated
that the protein was found localized preferentially within the
nucleus. The background fluorescence observed in the cytoplasm
of all cells was also obtained with preimmune serum or
untransfected cells (data not shown). The same pattern was
observed for all the other deletion mutants tested (data not shown
and Figure 1). Although the N-terminal external deletions N-298
and N-331 could not be tested in this assay, because they were
not recognized by the antiserum, the combination of the data
obtained with the other external deletions and with the internal
deletion A186—331 indicated that any region outside of the zinc
fingers, including the two flanking basic regions, could be omitted
without affecting nuclear localization.
plasmids are represented in Figure 4A: ptkCAT includes the
herpes simplex virus thymidine kinase (tk) gene promoter driving
the chloramphenicol acetyl transferase (CAT) gene; it contains
no Krox-20 binding site; p6E-tkCAT was derived from ptkCAT
by insertion of six Krox-20 binding sites immediately upstream
of the tk promoter. Extracts were prepared from transfected cells
and used to measure /3-galactosidase and CAT activities as well
as to estimate the amount of Krox-20 protein by measuring its
DNA-binding activity by gel retardation analysis with the
oligonucleotide carrying the consensus Krox-20 binding site. CAT
activity, after normalization relative to /3-galactosidase activity,
was taken as a measure of the transcription of the CAT gene,
which allowed an estimation to be made of the capacities of the
different Krox-20 derivatives to promote transcription from the
reporter gene promoter.
As shown in Figure 4B, there were important variations in the
amounts of oligonucleotide-binding activity present in cells
transfected with the different derivatives. Nevertheless, these
levels were always equal or higher than the level observed with
the wild-type protein, which was sub-saturating for transcriptional
activation. The results obtained with proteins produced in bacteria
suggested that the deletions did not affect the DNA-binding
activity of the proteins (Figure 2). Therefore the differences in
DNA-binding activity observed in SL2 cells were likely to be
due to changes in the amount of Krox-20 derivatives. Increases
in the amount of some of the deleted proteins may reflect higher
stability compared to the wild-type protein, which has been shown
to have a very short half-life in eukaryotic cells, in the order
of 20 min (Chavrier, Zerial, Bravo and Charnay, unpublished
result). In any case, our data suggested that the amount of each
derivative present in transfected cells was at least equal to the
amount of wild-type Krox-20.
Measure of the expression of the reporter gene indicated that
wild-type Krox-20 transactivated the construct containing its
binding site by a factor of 20- to 30-fold, while it had no effect
on ptkCAT (Figure 4C). Analysis of the relative levels of
transactivation by the different derivatives (Figure 5) revealed
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Transcriptional activation
For the determination of the transcriptional activities of the
different Krox-20 derivatives, we used a transient cotransfection
assay developed in SL2 cells by Courey and Tjian (31). The
Krox-20 expression constructs were cotransfected with reporter
plasmids and the plasmid pPAdh-|3gal, which contained the E.
coli lacZgene under the control of the Drosophila Adh promoter
and was used to normalize the experiments (7). The reporter
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Figure 5. Quantification of transactivation. The ratio of CAT activities from the
p6E-tkCAT reporter construct obtained with each Krox-20 derivative and with
the wild-type Krox-20 was taken as a measure of the transcriptional activity of
the derivative. The bars correspond to the means of the values obtained in at
least three independent experiments. The standard deviations are indicated.
2490 Nucleic Acids Research, Vol. 20, No. 10
the presence of two important regions located in the N-terminal
half of the protein. Elimination of the first 50 N-terminal amino
acids led to a 4- to 5-fold reduction in transactivation (Figures
4C and 5). This deleted protein was particularly interesting since
it corresponded precisely to the shorter Krox-20 protein that is
expected to be synthesized from a splice variant mRNA observed
in NIH3T3 cells (2). Further deletion of N-terminal sequences
up to position 143 did not produce additional significant changes
(Figure 4C). In contrast, elimination of 41 additional amino acids,
up to position 184, completely abolished transactivation, although
the amount of protein present in the cells was dramatically
increased, as judged from the DNA-binding activity (Figures 4B
and C). Proteins with larger N-terminal deletions behaved
similarly. In contrast to the N-terminal external deletions, the
internal deletion A186—331, which eliminated almost half of the
sequence upstream of the zinc fingers including the conserved
basic region (Figure 1), affected only marginally transactivation
(85% of the wild-type level, Figure 5). However, DNA-binding
was also increased (Figure 4B). Analysis of dose-response curves
indicated that the transcriptional activity of the A186-331 mutant
is at least 40% of the wild-type level (data not shown). Finally,
we tested the importance of the C-terminal tail of the protein by
specifically deleting it. Complete elimination, including the
downstream basic region, had no effect on DNA-binding activity
or on transcriptional activation (Figures 4 and 5).
In conclusion, our data pointed to the existence of two regions
required for transactivation that are located, respectively, between
positions 1 and 51 and 143 and 184. These regions did not appear
to be involved in DNA-binding (Figure 1). They did not affect
nuclear localization, since the proteins N184 and N238, although
inactive in the transactivation assay (Figures 4 and 5), were
located in the nucleus (Figure 1). Therefore, our results suggested
that these two regions were involved directly in transcriptional
activation.
Evolutionary conservation of the functional organization
As indicated above, Krox-20 belongs to a small subfamily of zinc
finger proteins with closely related DNA-binding domains.
Comparative analysis of the amino acid sequences indicated that
the degree of similarity is much lower outside of the zinc finger
domain (data not shown). The family members closest to Krox-20
in these regions are EGR-3 (13) and Krox-24 (8). In these cases,
some similarity is observed throughout the region upstream of
the zinc fingers (Figure 6), while no significant similarity is
detected downstream (data not shown). Since the two Krox-20
transactivation domains are located in the N-terminal region and
since they overlap with the acidic regions, we compared the
distribution of charges within the sequences of the three proteins
(Figure 6). Although the overall sequence conservation is low
(24% identity between Krox-20 and Krox-24, from position 1
to position 318, with numerous insertions and deletions), there
is a striking conservation of the charge distribution. In particular,
the two acidic regions that correlate with the transcriptional
activation domains in Krox-20 are also found in the two other
proteins, although in the case of Krox-24 the N-terminal acidic
region is interrupted by the insertion of a sequence rich in serine
and glycine. We believe that this conservation is indicative of
a functional significance of the acidic regions and suggests that,
like in the case of Krox-20, the two acidic regions of EGR-3
and of Krox-24 may constitute transactivation domains. A limited
deletional analysis of Krox-24 has been performed and the results
are consistent with these evolutionary considerations: while the
wild-type Krox-24 protein was able to activate transcription from
the p6E-tkCAT plasmid in SL2 cells by a factor of approximately
20-fold, deletion of the entire region upstream of the zinc fingers,
including the two acidic regions, completely abolished
transactivation without affecting DNA-binding (Nardelli and
Charnay, unpublished results). In addition, deletion of the region
located downstream to the zinc fingers also slightly reduced
transcriptional activation (2—3-fold effect). These data indicate
that, like in the case of Krox-20, the N-terminal part of Krox-24
contains a transactivation domain. They are consistent with an
analysis of hybrid proteins that suggested that the N-terminal part
of Krox-24 was involved in transcriptional activation (38).
DISCUSSION
Our analysis of the regions of the Krox-20 protein responsible
for transcriptional activation has first involved the identification
of the requirements for DNA binding. The data eliminated the
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338
277
337
Figure 6. Alignment of the amino acid sequences of the N-terminal regions of three members of the Krox-20 subfamily of zinc finger proteins: Krox-20 (2), EGR-3
(13) and Krox-24 (8). The sequences were aligned using the program CLUSTAL (57). Gaps are marked by dashes. Amino acids identical in the three sequences
are indicated by stars. Acidic amino acids are shown in reversed characters and basic amino acids are boxed. The two acidic regions of Krox-20 are overlined.
Nucleic Acids Research, Vol. 20, No. JO 2491
possibility that both basic regions were required simultaneously
for high affinity binding. Indeed, they suggested that only the
zinc fingers were necessary for DNA recognition. Nevertheless,
our data did not exclude an influence of regions outside the zinc
fingers on aspects of DNA-binding like association rate or salt
stability of DNA-protein complexes. The next question concerned
the regions required for nuclear localization. Proteins are targeted
to the nucleus by specific signals, encoded within their primary
sequence (39). The nuclear localization signals (NLS) have been
delineated in a large number of cases (40). No single, strict
consensus NLS has been derived, but these sequences are
generally short (8 — 10 amino acids) and contain a high proportion
of positively charged amino acids. The two basic sequences
flanking the Krox-20 zinc fingers fulfill these conditions and they
are the only highly basic sequences in the protein, apart from
the zinc fingers. They could therefore constitute an NLS. Our
deletion analysis does not allow an identification of the Krox-20
NLS. Nevertheless, it indicates that elimination of all sequences
upstream or downstream of the zinc fingers does not affect
nuclear localization, at least as judged by our immunofluorescence
assay. These data are consistent with the following possibilities:
each of the flanking basic regions constitutes a NLS and both
signals are not required simultaneously; the zinc fingers
themselves contain an NLS. Confirmation of one of these
hypotheses will require the analysis of the behavior of mutant
proteins consisting of only the zinc fingers or specifically mutated
within both basic regions.
Different types of transactivation domains have been identified
through the functional analysis of several transcription factors:
acidic regions, which may be the most common (41 -43), and
proline-, glutamine- or serine/threonine-rich regions (31, 44, 45).
Other types certainly await to be discovered. These regions are
thought to be involved in protein-protein interactions with other
elements of the transcription complex (46,47) and distinct classes
may function by different mechanisms (48,49). Their properties
are not well defined and their primary structures are poorly
conserved through evolution. Krox-20 does not contain any
glutamine-rich region, but several acidic or proline-rich regions
can be identified (Figure 1). Analysis of the transcriptional
activation of a reporter gene carrying multiple Krox-20 binding
sites, revealed the existence of at least two transactivation domains
located in the N-terminal half of the protein and overlapping with
the regions 1 - 5 0 and 143—183. Examination of Krox-20 amino
acid sequence indicated that the region 1-50 overlapped with
the first acidic region while the region 143-183 contained the
second acidic region and overlapped with a portion of a prolinerich region (Figure 1). Comparison of the mouse and Xenopus
laevis (D.Wilkinson, personal communication) Krox-20 amino
acid sequences over these regions indicates that, although there
is an about 50% divergence between these sequences, all the
negative charges except one are conserved (data not shown). In
contrast, the 7 proline residue stretch present in region 143-183
in the mouse sequence is reduced to 3 residues in the frog
sequence. These observations are consistent with the two major
transcriptional activation domains of Krox-20 being of the acidic
type. In addition, the limited effect (less than 2-fold decrease)
of the internal deletion A186-331, which eliminates the two
major proline-rich regions of the protein (Figure 1), suggests that
these latter regions are not important for transcriptional activation,
at least in the present assay system.
The comparison of the amino acid sequence of Krox-20 with
those of the closest members of the subfamily, EGR-3 and
Krox-24, also supports the involvement of the two acid regions
of the N-terminal half in transcriptional activation. These regions
are maintained in these proteins, despite a low degree of sequence
conservation. Furthermore, this suggests that the acidic regions
may also constitute transcription activation domains in EGR-3
and Krox-24. The demonstration that the major activation region
of Krox-24 is located upstream of the zinc fingers is consistent
with this idea. The definitive demonstration of the involvement
of the acidic regions in transcriptional activation will require
modification of the charge distribution by site-directed
mutagenesis.
In contrast to the N-terminal half, no function was found for
the C-terminal part of Krox-20 downstream of the DNA-binding
domain. This is again consistent with the outcome of the
comparative analysis of mouse and frog amino acid sequences
(D.Wilkinson, personal communication), which indicates no
significant similarity between the C-terminal parts of the two
proteins. This rapid divergence may reflect a less important role
of this region of the Krox-20 molecule. The sequence of the Cterminal region is also not conserved between Krox-20, EGR-3
and Krox-24. However, unlike Krox-20, elimination of this
region in Krox-24 leads to low but significant reduction in
transcriptional activation. This difference may indicate that the
highly divergent C-terminal regions of the members of the
Krox-20 subfamily have acquired distinct functions in the course
of evolution. In this respect, it is interesting to note that the Cterminal region of Krox-24 contains a series of short tandemly
repeated sequences rich in proline, serine, threonine and tyrosine
and which are similar to the repeats present in the C-terminal
region of the largest subunit of eukaryotic RNA polymerase II
(50, 51). These repeats could play a role in transcriptional
activation.
An heterologous system has been used in this study for the
evaluation of transcriptional activity. Originally, Drosophila cells
were chosen because, unlike mammalian cells, they are devoid
of Spl activity (31). Although Spl recognizes a DNA sequence
slightly different from the Krox-20 target sequence, there is some
cross-reactivity that might have interfered with our assay. In
addition, using the p6E-tkCAT reporter construct, we have
observed much lower levels of transactivation by Krox-20 in the
mammalian cell lines tested so far than in SL2 cells (only
3—4-fold in Cos7 cells, data not shown). We do not understand
presently the basis of this difference, which may be due to the
presence of a specific repressor or the absence of a necessary
activator or coactivator in the latter cells. In any case, this low
level of transactivation has prevented a quantitative analysis of
the effect of the different mutations in mammalian cells.
Nevertheless, the conclusions that have been reached in term of
nuclear localization and identification of transcription activation
domains are likely to stand in an homologous system: posttranslational processing (except N-glycosylation) and cellular
localization of recombinant proteins are very similar in insect
and mammalian cells (52, 53); the general mechanisms of
transcription control have appeared to be conserved and a number
of mammalian transcription factors have been shown to function
correctly in insect cells and vice versa (31, 54-56).
The present work suggests additional investigations and
provides some tools for further analysis of the physiological
functions of Krox-20. It is interesting that a protein encoded by
an alternatively spliced version of Krox-20 mRNA (2) has a
reduced transcriptional activity. This effect raises the possibility
that alternative splicing is physiologically involved in the
2492 Nucleic Acids Research, Vol. 20, No. 10
regulation of Krox-20 activity. The distribution of the two forms
of the mRNA during development will therefore deserve further
investigation. Deletions which increase DNA-binding and abolish
transactivation might act as dominant negative mutations.
Expression of such mutant genes in specific tissues or at specific
stages of development in transgenic mice might create phenotypes
informative on the role of Krox-20 or of Krox-20-related
molecules. Similarly, mutations reducing but not abolishing
transactivation might be useful as substitutes for gene inactivation
in a strategy of targeted mutagenesis by homologous
recombination.
ACKNOWLEDGEMENTS
We thank D.Wilkinson for communication of the amino acid
sequence of Xenopus laevis Krox-20 before publication and
R.Tjian for plasmids. This work was supported by grants from
CNRS, INSERM, ARC, LNFCC, MRT, FRM and AFM.
REFERENCES
1. Chavrier, P., Zerial, M., Lemaire, P., Almendral, J., Bravo, R. and Chamay,
P. (1988) EMBO ]., 7, 2 9 - 3 5 .
2. Chavrier, P.Janssen-Timmen, U., Mattel, M.-G., Zerial, M., Bravo, R. and
Chamay, P. (1989) Mol. Cell. Biol., 9, 787-797.
3. Brown, R.S., Sander, C. and Argos, P. (1985) FEBSLett., 186, 271-274.
4. Miller, J., McLachlan, A.D. and Klug, A. (1985) EMBOJ., 4, 1609-1614.
5. Chavrier, P., Lemaire, P., Revelant, O., Bravo, R. and Charnay, P. (1988)
Mol. Cell. Biol., 8, 1319-1326.
6. Kadonaga, J., Carner, K.R., Maslarz, F.R. and Tjian, R. (1987) Cell, 51,
1079-1090
7. Chavrier, P., Vesque, C.Galliot, B., Vigneron, M., Dolle, P., Duboule,
D. and Charnay, P. (1990) EMBOJ., 9, 1209-1218.
8. Lemaire, P., Revelant, O., Bravo, R. and Charnay, P. (1988) Proc. Nail.
Acad. Sci. USA, 85, 4691-4695.
9. Cao, X.M., Koski, R.A., Gashler, A., Mckiernan, M., Morris, C.F.,
Gaffney, R., Hay, R.V. and Sukhatme, V.P. (1990) Mol. Cell. Biol., 10,
1931-1939.
10. Christy, B.A. and Nathans, D. (1988) Proc. Nail. Acad. Sci. USA., 85,
7857-7861.
11. Milbrandt, J. (1987) Science, 238, 797-799.
12. Lim, R.W., Vanum, B.C. and Herschman, H.R. (1987) Oncogene, 1,
263-270.
13. Patwardhan, S., Gashler, A., Siegel, M. G., Chang, L. C , Joseph, L. J.,
Shows, T. B., Le Beau, M. M. and Sukhatme, V.P. (1991) Oncogene, 6,
917-928.
14. Crosby, S.D., Puetz, J.J.,Simburger, K. S., Fahrner, T. J. and Milbrandt,
J. (1991) Mol. Cell. Biol., 11, 3835-3841.
15. Call, M., Glaser, T., Ito, C. Y., Buckler, A. J., Pelletier, J., Haber, D.
A., Rose, E. A., Krai, A., Yeger, H., Lewis, W.H., Jones, C. and Housman,
D.E. (1990) Cell, 60, 509-520.
16. Dynan, W.S. and Tjian, R. (1983) Cell, 35, 79-87.
17. Nardelli, J., Gibson, T., Vesque, C. and Chamay, P. (1991) Nature, 349,
175-178.
18. Pavletich, N.P. and Pabo, C O . (1991) Science, 252, 809-817.
19. Wilkinson, D., Bhatt, S., Chavrier, P., Bravo, R. and Chamay, P. (1989)
Nature, 337, 461 -464.
20. Lumsden, A. and Keynes, R. (1989) Nature, 337, 424-428.
21. Keynes, R. and Lumsden, A. (1990) Neuron, 2, 1-9.
22. Fraser, S., Keynes, R. and Lumsden, A. (1990) Nature, 344, 431-435.
23. Wilkinson, D.G., Bhatt, S., Cook, M., Boncinelli, E. and Krumlauf, R.
(1989) Nature, 341, 405-409.
24. Gilardi, P., Schneider-Maunoury, S. and Charnay, P. (1991) Biochimie, 73,
85-91.
25. Lanfear, J., Jower, T. and Holland, P.W.H. (1991) Bioch. Biophys. Res.
Comm., 179, 1220-1224.
26. Studier, F.W. and Moffatt, B.A. (1986) / Mol. Biol., 189, 113-130.
27. Rosenberg, A.H., Lade, B.N., Chui, D.S., Lin, S.W., Dunn, J.J. and Studier,
F.W. (1987) Gene, 56, 125-135.
28. Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Nail. Acad. Sci.
USA, 76, 4350-4354.
29. Schneider, I. (1972) J. Embryol. Exp. Morphol., 27, 253-265.
30. Di Nocera, P.P. and Dawid, l.B. (1983) Proc. Nail. Acad. Sci. USA, 80,
7095-7098.
31. Courey, A.J. and Tjian, R. (1988) Cell, 55, 887-898.
32. Herbomel, P., Bourachot, B. and Yaniv, M. (1984) Cell, 39, 653-662.
33. Gorman, C M . , Moffat, L.F. and Howard, B.H. (1982) Mol. Cell. Biol.,
2, 1044-1051.
34. Fried, A. and Crothers, D.M. (1981) Nucl. Acids Res., 9, 6505-6525.
35. Gamer, M.M. and Revzin, A. (1981) Nucl. Acids Res., 9, 3047-3059.
36. Strauss, F. and Varshavsky, A. (1984) Cell, 37, 889-901.
37. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) In Molecular Cloning,
A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York).
38. Madden, S.L., Cook, D.M., Morris, J. F., Gashler, A., Sukhatme, V. P.
and Rauscher, F.J. (1991) Science, 253, 1550-1553.
39. De Robertis, E.M., Longthome, R.F. and Gurdon, J.B. (1978) Nature, 272,
254-256.
40. Garcia-Bustos, J., Heitman, J. and Hall, M.N. (1991) Biochim. Biophys.
Acta, 1071, 83-101.
41. Hope, I.A., Mahadevan, S. and Struhl, K. (1988) Nature, 333, 635-640.
42. Ma, J. and Ptashne, M. (1987) Cell, 51, 113-119.
43. Mitchell, P.J. and Tjian, R. (1989) Science, 245, 371-378.
44. Mermod, N.O'Neil, E.A., Kelly, T. and Tjian, R. (1989) Cell, 58,741 -753.
45. Tanaka, M. and Herr, W. (1990) Cell, 60, 375-386.
46. Ptashne, M. (1988) Nature, 335, 683-689.
47. Ptashne, M. and Gann, A.F. (1990) Nature, 346, 329-331.
48. Tasset, D., Tora, L., Fromental, C , Scheer, E. and Chambon, P (1990)
Cell, 62, 1177-1187.
49. Martin, K.J., Lillie, J.W. and Green, M.R. (1990) Nature, 346, 147-152.
50. Allison, L.A., Moyle, M., Shales, M. and Ingles, C.J. (1985) Cell, 42,
599-610.
51. Corden, J.L., Cadena, D.L., Ahearn, J. M. and Dahmus, M.E. (1985) Proc.
nail. Acad. Sci. U. S. A., 82, 7934-7938.
52. Luckow, V.A. and Summers, M.D. (1988) Biotechnology, 6, 47-52.
53. Tratner, I., De Togni, P., Sassone-Corsi, P. and Verma, I.M. (1990) J.
Virol., 64, 499-508.
54. Mermod, N., O'Neill, E.A., Kelly, T.J. and Tjian, R. (1989) Cell, 58,
741-753.
55. Williams, T. and Tjian, R. (1991) Genes Dev., 5, 670-682.
56. Driever, W., Ma, J.Nusslein-Volhard, C. and Ptashne, M. (1989) Nature,
342, 149-154.
57. Dessen, P., Fondrat, C , Valencien, C. and Mugnier, C. (1990) Comp. Appl.
in Biosciences, 6, 355-356.